Transfer RNA Modification
Module
4.6.2
GLENN R. BJÖRK* AND TORD G. HAGERVALL
[SECTION EDITOR: GLENN R. BJÖRK]
Posted July 25, 2005
Department of Molecular Biology, Umeå University, S-90187 Umeå University, Sweden
*Corresponding author. E-mail:
This e-mail address is being protected from spambots. You need JavaScript enabled to view it
Transfer RNA interacts with many different proteins, such as aminoacyl-tRNA ligases, elongation factors, and ribosomal proteins, and also directly with RNAs by interaction between the 3' end and 23S rRNA (in the peptidyltransfer center) and between the anticodon and 16S rRNA (in the decoding center). The ability to interact with diverse molecules may be a reason that, of all RNAs in the cell, tRNA contains the highest frequency of modified nucleosides, which are derivatives of the four major nucleosides. Their presence in tRNA is of ancient origin and, according to some theories, the early tRNA might have been a small hairpin-RNA (169) with an amino acid bound to it (88). A relic of this primordial tRNA may be the presence of some modified nucleosides, which are today formed by the addition of an intact amino acid (e.g., t6A, k2C, an GluQ; see Abbreviations below) or parts of amino acids (methylated and thiolated nucleosides and acp3U). This suggests an evolution of the tRNA modification concomitant with the evolution of translation.
Modified nucleosides were found in DNA as early as 1948 (174) and a few years later in RNA (74). Ψ was identified in 1957 independently by Cohn (71, 72, 73) and Davis and coworkers ("fifth nucleoside") (85, 323, 415). Soon after tRNA was discovered (164) the presence of other modified nucleosides was also established (98, 343). The synthesis of the modified nucleosides was an enigma for several years, and at the time they were discovered it was assumed that RNA polymerase incorporated them into the growing polynucleotide. However, a few years later, Svensson et al. (360) and Fleissner and Borek (117) independently showed that the synthesis of the methylated nucleosides occurs on the polynucleotide level, that is, after the primary transcript has been synthesized. We know now that in Escherichia coli and Salmonella enterica serovar Typhimurium all modified nucleosides, except Q, are synthesized on the polynucleotide level. Thus, after transcription, certain nucleotides are modified by specific enzymes during maturation and, therefore, these enzymes can be considered tRNA biosynthetic enzymes. At present 96 different modified nucleosides have been characterized in RNA from various organisms (http://medstat.med.utah.edu/RNAmods/) and structures of a few are shown in Fig. 1. Transfer RNA from all three phylogenetic domains, Archaea, Bacteria, and Eukarya (402) contains modified nucleosides (351). In tRNA from Bacteria 10% of the nucleosides are modified, whereas up to 25% of the nucleosides in tRNA from Eukarya are modified. Some of these modified nucleosides (D, Ψ, Um, m5U, ac4C, Cm, m1G, m7G, Gm, m1A, m6A, t6A, ms2t6A, and I) are present in tRNAs from organisms belonging to all three domains (15, 250). Moreover, some (Ψ13, Cm32, m1G37, t6A37, Ψ38, Ψ39, Ψ55, m1A58) are even present in comparable positions in the same subset of tRNAs (29). Unless convergent evolution has occurred, this modification pattern suggests a common evolutionary origin for at least some of these modified nucleosides (29, 64). Indeed this seems to be true for m1A and Ψ (165, 251, 310, 339) but not for m1G37, since the yeast (TRM5) and archaeal (MJ883) orthologs belong to the Rossman fold methyltransferases (10), whereas the bacterial ortholog (TrmD) belongs to the evolutionary and structurally distinct TREFOIL family (also called SPOUT family) (7, 106). Thus, modified nucleosides, which are present in all three domains, have evolved before the emergence of the three domains by divergent evolution from a common ancestor (e.g., m1A58, Ψ) or by convergent evolution (e.g., m1G37) following the separation of the domains. However, most modified nucleosides are domain specific or have an even narrower taxonomic distribution, demonstrating that the presence of these modified nucleosides evolved after the three phylogenetic domains diverged (29, 104, 243). Thus, the tRNA modification system is diverse and complex with different evolutionary traits. Detailed functional, biochemical, and evolutionary studies are required for many modifying enzymes and modified nucleosides before a complete knowledge of the tRNA modification machinery and of the function of the modified nucleosides themselves that are present in the three domains of life can be obtained.
Structures of different modified nucleosides as well as their abbreviations were initially described by Limbach et al. (232); the updated version can be found at http://medstat.med.utah.edu/RNAmods/. A compilation can also be found in reference 250. An index and an exponent indicate the number and the position of the substitution, respectively, for example, 6-dimethyladenosine is abbreviated m62A. c-, i-, k-, m-, n-, o-, r-, s-, and t- are abbreviations of carbonyl-, isopentenyl-, lysyl-, methyl-, amino-, oxy-, ribosyl-, thio-, and threonyl- groups, respectively. acp- denotes 3-amino-3-carboxypropyl. An abbreviation to the left or to the right of the nucleoside symbol denotes modifications of the base and the ribose, respectively. Other abbreviations are as follows: Ψ, pseudouridine; I, inosine; Q, queuosine; oQ, epoxyqueuosine; GluQ, glutamylqueuosine, R, purine; Y, pyrimidine, and N, any of the four major nucleosides. A number following an abbreviation for a modified nucleoside denotes the location in the tRNA sequence. An enzyme catalyzing the formation of m5U at position 54 in the tRNA is denoted tRNA(m5U54)methyltransferase and likewise for other tRNA-modifying enzymes. Methionyl-tRNA ligase is denoted MetRS, an unspecified aminoacyl-tRNA ligase is denoted AaRS, and the peptide encoded by the trmA gene is denoted TrmA and likewise for other gene products. Transfer RNA species are identified by their anticodon sequence. N34 denotes the nucleoside in position 34 (wobble position) of the tRNA and N(III) denotes the third nucleoside of the codon. ASL denotes the anticodon-stem-loop domain of a tRNA usually consisting of 17 nucleotides. [Fe-S] denotes an iron-cluster protein. TREFOIL (also called SPOUT) is a family of methyltransferases in which significant portion of the C-terminal forms a "trefoil knot." TRAM is a proposed RNA-binding domain found in 
(9).
The 86 tRNA genes (including one pseudogene) present in E. coli and in serovar Typhimurium (242) encode 46 different tRNA species, of which 41 have been sequenced (351). (These numbers are relevant only for E. coli (strain MG1655) and serovar Typhimurium because other strains of these species may have additional or fewer numbers of tRNA genes). Thirty-one different modified nucleosides have been located in tRNAs from E. coli and serovar Typhimurium and the identities of all have been established (Fig. 2). Therefore, at present, we have a complete picture of the tRNA modification pattern in these organisms. The modified nucleosides are mainly located in single-stranded regions of the tRNA and all tRNA species contain m5U54 and Ψ55 (Fig. 2). Positions 34 and 37 are not only frequently modified (45% in position 34 and 78% in position 37), but there is also a large variety of modified nucleosides in these two positions.
Some modified nucleosides (Ψ, s4U; D, Cm) are found in more than one position of the tRNA (Fig. 2), for example, Ψ is present in the anticodon region (positions 38, 39, and 40) and in positions 13, 32, 55, and 65. In 10 cases, a certain modified nucleoside is found in only one tRNA species (Ψ13 in 
, D21 in 
, m6A37 in 
, m6t6A37 in 
, ac4C34 in elongator 
; k2C34 in 
, I34 in 
, cmnm5Um34 in 
, Cm34 in 
, and i6A37 in 
; see Fig. 2). Of these the synthesis of seven requires, most likely, a specific modifying enzyme. (A few other nucleosides, like i6A37, mnm5U34, and cmnm5Um34, are present in only one or two tRNA species, but their synthesis may utilize enzymes also involved in the synthesis of other nucleosides, like ms2io6A37, mnm5s2U34.) Thus, bacteria have several tRNA-modifying enzymes acting on only one tRNA species.
An unmodified A in position 34 is not found in any tRNA and an unmodified U in the same position is only present in
, which also has an unusual structure in many respects (326). Thus, of the four major nucleosides, only unmodified G or C is found as wobble nucleoside. A certain modification pattern can be seen in position 34, for example, Q34 and (c)mnm5s2U34 are found in tRNAs reading codons of the type NAY and NAR, respectively, which are codons that are only found in mixed codon boxes (Fig. 3). This suggests a role for xm5U and Q derivatives in preventing missense errors within a codon box. A specific pattern is also found for modification in position 37 (Fig. 3). Codons that start with U or A are usually read by tRNAs having i6A or t6A derivatives in position 37, respectively, whereas codons starting with C or G are read by tRNAs having m1G, m2A, m6A, or an unmodified A in position 37 (Fig. 3). Unmodified A37 is found in 10 tRNA species but G37 is always converted to m1G, implying that an unmodified G is detrimental for the tRNA. This is also supported by the fact that m1G37 prevents tRNA from frameshifting, and lack of it reduces the growth rate considerably in serovar Typhimurium and yeast, and it is in fact essential in Streptococcus pneumoniae (see "Function of Modified Nucleosides in Translation," below). Although a good correlation exists between a specific modification at position 34 or 37 and a specific decoding capacity of the tRNA, it does not follow that the function of a particular modification is essential for all the tRNA species in which it is present. The presence of the modification is also determined by the specificity of the corresponding tRNA-modifying enzyme. For example, the presence of t6A derivatives in the subset of tRNAs reading codons starting with A may impose a function that is qualitatively or quantitatively different among the various tRNAs having the positive recognition determinants (e.g., U36-A37) for the tRNA(t6A37)synthase. In some tRNA species this modification may be neutral and is present simply because the tRNA fulfills the threonylation requirement (see also "Function of cmo5U34," below). Thus, the functional impact of a specific modification is not necessarily the same in all tRNA species it is part of (227).
Depending on growth conditions, tRNA from E. coli and serovar Typhimurium may also have selenium-containing modified nucleosides of which mnm5Se2U is the most prominent and likely to be present in tRNAs specific for lysine and glutamate in position 34 (398, 399, 401) and possibly 
.
There are 31 different modified nucleosides in the 41 sequenced tRNAs from E. coli and serovar Typhimurium, but the number of tRNA-modifying enzymes required for their synthesis is considerably higher. Some modified nucleosides, like Ψ, are present in several positions and their syntheses are catalyzed by position-specific enzymes. Furthermore, some modified nucleosides are complex, like mnm5s2U34 and ms2io6A37, and more than one enzyme is involved in their synthesis. From such considerations one can infer that about 45 different tRNA-modifying enzymes are required for tRNA modification. Assuming that the average size of a structural gene is about 1 kb, approximately 1% of the bacterial genome is devoted to the synthesis of these enzymes. The 46 tRNA species present in E. coli are coded for by 85 tRNA genes (and they constitute about 0.26 to 0.28% of the serovar Typhimurium or the E. coli genome (each tRNA gene is estimated to be 150 nucleotides and the genome is 4,879 kb in serovar Typhimurium [242] and 4,639 kb in E. coli [39]). Thus, in bacteria about 4-fold more genetic information is devoted to the synthesis of the tRNA-modifying enzymes than to the synthesis of their substrate, the tRNA.
The genes involved in the synthesis of modified nucleosides are given in Tables 1, 2, 3, and 4. Of the 45 putative tRNA modification genes, 27 are identified. Some gene products that are not tRNA-modifying enzymes in the sense that they are not interacting with the tRNA are, however, required for the synthesis of a substrate for the tRNA-modifying enzymes; for example, mutations in either aroA, aroB, or aroC genes, which block the synthesis of chorismic acid, also block the synthesis of cmo5U, suggesting that chorismic acid or a derivative of it is required for the synthesis of these modified nucleosides (28). The IscS protein participates in the synthesis of all thiolated nucleosides by mobilizing sulfur to the thiolating enzymes interacting with tRNAs (219, 263). Moreover, the IscU, IscA, and HscA proteins are required for the synthesis of s2C32 and ms2io6A37 but not for the synthesis of s4U8 and mnm5s2U34 (224). In addition, several genes are involved in the synthesis of isoprenoids, which are precursors to the synthesis of i6A37 and at at least four genes are involved in the synthesis of Que1, which is the precursor to Q34 in tRNA (304). Thus, modification of tRNA is intimately linked to various parts of the central and intermediary metabolism of the cell.
Table 1Genes influencing modifications in positions 8 to 32 in tRNA of E. coli and serovar Typhimurium |
Table 2Genes influencing modifications in position 34 in tRNA of E. coli and serovar Typhimurium |
Table 3Genes influencing modifications in position 37 (next to and 3' of the anticodon) in tRNA of E. coli and serovar Typhimurium |
Table 4Genes influencing modifications in positions 38 to 65 in tRNA of E. coli and serovar Typhimurium |
The amount of various tRNA species is correlated to the use of codons, such that abundant codons are read by major isoacceptor tRNAs, whereas the minor isoacceptor tRNAs decode the rare codons (177). Under various physiological conditions, such as growth in different media, the syntheses of different proteins are changing so that at higher growth rates proteins encoded by abundant codons are preferentially made, whereas at low growth rates proteins whose genes include rare codons increase. Accordingly, the major and minor tRNAs are regulated differently; the proportion of major tRNAs increases with growth rate whereas the amount of minor tRNAs decreases (108). Thus, groups of tRNAs are regulated differently and they are not regulated simply according to isoacceptor specificity. To ensure proper modification of the individual tRNAs, the tRNA-modifying enzymes may also be regulated in a way that should be correlated to the syntheses of tRNAs. Since some modified nucleosides may be present in several groups of differently regulated tRNAs, it is not clear which demand the syntheses of tRNA-modifying enzymes senses; in some tRNAs the presence of a particular modification may be very important for the function of the tRNA, whereas the same modification is less important in other tRNA species (see below). Therefore, response to various physiological conditions on the regulation of tRNA-modifying enzymes is difficult to predict, although it is clear that somehow the regulation must respond to the demand set by the amount of tRNA. Accordingly, all the tRNA-modifying enzymes are not regulated in a similar manner, and so far each tRNA-modifying enzyme studied seems to respond differently to, for example, growth rate. If the regulatory response for the synthesis of a specific tRNA-modifying enzyme is such that its synthesis is responding to a few tRNA species and not to all tRNA species it is acting on, the result may be undermodification of some tRNA species. This may be tolerated if such an undermodified tRNA is still performing its function in a way that is acceptable for the cell. Indeed, the presence of the thiolated nucleoside s4U in different tRNA species varies with growth rate such that the level of s4U in some tRNA species decreases sharply at increasing growth rate, whereas in others the level of s4U is independent of growth rate (109). The various genes encoding tRNA-modifying enzymes are not gathered in a few operons but are scattered all over the chromosome (Tables 1, 2, 3, and 4). Some of them are members of complex operons containing genes encoding proteins of the translational apparatus (e.g., trmD), whereas others are members of operons for which there is no apparent commonality (truA, miaA, and tgt). Below we review results concerning the regulation of the synthesis of some tRNA-modifying enzymes and the related gene organizations (see also earlier reviews [32, 397]). Tables 1, 2, 3, and 4 summarize the organization of genes for which only the DNA sequences were used to deduce the gene organization.
The truA (hisT) gene (Fig. 4) encodes the enzyme tRNA(Ψ38,39,40)synthase (78), which catalyzes the formation of Ψ in positions 38, 39, and 40 in at least 18 tRNA species of E. coli (351). The truA gene is part of an operon that has four (13), five (268), or perhaps seven (40) genes with unrelated products. In order of transcription, the first gene (pdxB) encodes an enzyme (erythronate-4-phosphate dehydrogenase) that catalyzes a step in the synthesis of pyridoxine (vitamin B6); the second gene encodes a protein that is similar to the Streptococcus mutans aspartate semialdehyde dehydrogenase (usg); the third gene is truA, and the fourth gene, dedA, encodes a 17-kDa integral membrane protein of unknown function (13, 325). The fifth gene, accD, encodes a 33.4-kDa protein of unknown function that was suggested to be part of the truA operon based on genetic and transcriptional analyses (268). The sixth gene, folC, encodes the folylpolyglutamate-dihydrofolate (41). Some genetic results suggest that dedA is the last gene of the operon (13), although some transcripts terminate further downstream of it (268). Because the stem-loop structure, present between accD and folC, does not act as a transcriptional terminator, the truA operon may extend downstream of the folC gene and include dedD (40). The entire truA operon seems to be transcribed from the Ppdx promoter, although transcriptional termination can occur just after the first structural gene, pdxB. The Ppdx promoter shares its –10 region with the divergently transcribed Pdiv promoter and these promoters are of equal strength in cells grown in rich medium at 37°C (325). The three last genes, accD, folC, and dedD, are also transcribed from PfolC, located upstream of accD. In addition, a rather strong internal promoter (Pint) of the truA operon is present in the C-terminal end of the pdxB gene. The Pint promoter has a "discriminator" region similar to stringently regulated promoters like the rrn P1, trmA, and the trmD promoters (58, 151, 370). The stop codon of the usg gene and the start codon of the truA gene overlaps, suggesting translational coupling. However, this coupling is not too strict, since the amount of TruA protein synthesized in minicells is 10 to 14 times lower than that of the Usg protein (13). The codon usage for the truA gene is similar to that of other genes encoding tRNA-modifying enzymes (see, e.g., trmA and trmD), also suggesting that the truA gene is expressed at low levels. Thus, the genes of the truA operon are differentially expressed and the level of the transcriptional and translational products of the truA gene is positively regulated by the growth rate of the cell in a manner similar to the syntheses of the trmA and rrn gene products (373).
The trmA gene (Fig. 5), which encodes the tRNA(m5U54)methyltransferase (TrmA), is transcribed from a weak promoter that shows extensive similarity to the strong and stringently controlled P1 promoter for the rRNA genes (rrn genes) (233). As for the rrn P1 promoter, the trmA promoter contains a GC-rich region located between the –10 and the +1 transcriptional start site. This region, which is called the "discriminator," is shared by stringently controlled promoters (370). The FIS protein (Factor for Inversion Stimulation) activates rRNA and tRNA promoters (313, 385). Although a putative FIS-binding site is centered on base pair –56 upstream of the trmA promoter, its impact on the expression of the trmA gene is not known. A TCCC sequence is located just upstream of the –10 region in the trmA promoter, in all seven rrn P1 promoters, and in some tRNA promoters, but not in any other σ70 promoters (151). The significance of the TCCC sequence in the trmA promoter is not known, but the same sequence in the rrnB P1 promoter is important for the expression and the growth-rate-dependent regulation of the rrnB P1 promoter (91, 124). These features of the trmA promoter explain the similarity between the rrn and trmA genes regarding growth-rate-dependent (151, 272) and stringent regulation (271). However, unlike the expression of the rrn genes, the expression of the trmA gene responds to gene dosage (273). Why the expression of the trmA and the rrn genes is regulated similarly is not clear. Although many tRNA species, which all contain m5U54, are differently regulated (108), regulation of TrmA with bulk tRNA, which is regulated as rrn, ensures that all tRNAs become properly modified even if TrmA will be in excess for the demand of some of the tRNA species at certain growth rates. One possible reason for the coregulation of the synthesis of TrmA with that of rRNA/tRNA could be that this enzyme exists in two forms: the native form and a form covalently bound to rRNA/tRNA (150). The divergently transcribed trmA and btuB promoters are separated only by 102 nucleotides, suggesting coordinate regulation of these two genes (22). Whereas the synthesis of the BtuB protein, which is the receptor for vitamin B12, is repressed by cobalamine (a precursor to vitamin B12 [187]), the synthesis of TrmA is not (151). Thus, only the expression of the btuB gene is regulated by cobalamine, although the btuB and trmA promoters are physically close to each other. Other notable features of the trmA operon are shown in Fig. 5.
The trmD operon (Fig. 6) consists of rpsP, which encodes ribosomal protein S16; rimM, which encodes a 21-kDa protein involved in ribosome maturation; trmD, which encodes the tRNA(m1G37)methyltransferase (TrmD); and rplS, which encodes ribosomal protein L19; and the operon is transcribed in that order (57) (Fig. 6). Just upstream of the promoter-proximal gene rpsP (S16), there is an attenuator-like structure, which causes transcription termination to about 70% in vitro. The trmD operon is transcribed into a single polycistronic mRNA species and the synthesis of the trmD operon mRNA is growth-rate-dependently and stringently controlled (58), but unlike other ribosomal protein operons, the trmD operon is not subjected to translational and transcriptional feedback regulation (393). The rate of synthesis of the four proteins is quite different, resulting in a 12-fold and 40-fold lower amount of RimM and TrmD, respectively, compared with the amount of the two ribosomal proteins, the genes that flank the rimM and trmD genes (392). This difference in expression is achieved by regulation at the translational level. A large stem-and-loop structure may be formed by mRNA sequences 100 nucleotides downstream of the start codon of rimM, folding back and base pairing with the translational start site of the rimM mRNA. Such a structure may prevent the entry of the ribosome and thus decrease the frequency of translation initiation. A similar stem-loop structure may also be formed in the beginning of the trmD gene and inhibit the initiation of translation of the trmD mRNA (394). The TrmD activity is invariant with growth rate, although the amount of mRNA transcript and TrmD polypeptide increases with growth rate (58, 392). Neither the enzymatic activity (275) nor the synthesis of the polypeptide (392) is stringently regulated, although accumulation of the trmD operon transcript is (58). Accordingly, the trmD promoter contains the "discriminator," a GC-rich region common to all stringently regulated genes (370). This discrepancy between the regulation of the synthesis of mRNA and proteins suggests that a regulatory device is operating at the translational level. Whether the complex mRNA structure at the beginning of the trmD gene is involved in this translational regulation is not known. Clearly, the trmD operon exhibits many complex regulatory features influencing the expression of these four genes and resulting in noncoordinate gene expression and regulation under different physiological conditions.
The synthesis of ms2i6A37 in E. coli and of ms2io6A37 in serovar Typhimurium involves at least two (miaA,miaB) and three genes (miaA,miaB and miaE), respectively. These genes are not present in the same operon but scattered around the chromosome. The tRNAs having ms2io6A37 in serovar Typhimurium, have ms2i6A37 in E. coli, since the miaE gene, which is involved in the last step in the synthesis of ms2io6A37, is not present in E. coli (286) (Tables 1, 2, 3, and 4). From DNA sequence it can be deduced that the miaB gene is monocistronic. The miaA gene (Fig. 7), the structural gene for the tRNA(i6A37)synthase, is part of a super operon consisting of perhaps nine genes. There are no intercistronic regions between the first five genes (yjeF, yjeE, amiB, mutL, and miaA), suggesting that the expression is translationally coupled. The first two genes encode proteins with unknown functions; the third gene, amiB, encodes a periplasmic N-acetyl-muramoyl-l-alanine amidase (376); the fourth gene is mutL, which encodes a protein involved in methyl-directed mismatch repair; the fifth gene is miaA (61, 76, 77), and the sixth gene is hfq, which encodes the host factor, HF-1, required for phage Qβ RNA-directed synthesis of complementary minus strand RNA (188). The expression of the operon is driven by at least four sigma-70 (σ70) promoters and three sigma-32 (σ32) heat-shock promoters (372). The miaA gene is transcribed from two promoters: one σ70 promoter (PmiaA) and one σ32 promoter (PmiaA-HS), which are located close to each other. Although the miaA gene is transcribed from the PmiaA-HS heat-shock promoter at 50°C, the transcription from this promoter contributes with only 10% of the total transcription at high temperature (372). Still, the total transcription of the miaA gene increases 3-fold at extreme heat schock (50°C). The decreased viability at high temperature of a miaA mutant compared with the wild type suggests that the heat-shock response of the miaA expression is of functional significance. In addition to these transcriptional regulatory features, there is a potential translational coupling between mutL and miaA (76, 77). The Hfq protein, which is encoded by the hfq gene downstream of the miaA gene, is an RNA chaperone and a bacterial member of the Sm family of RNA-binding proteins. It binds to at least 15 of the 46 known small RNAs present in E. coli (416) and many of the small RNAs require Hfq for activity (136). Hfq is also a positive regulator of RpoS expression and its regulatory effect may be on the translation of rpoS mRNA (50). Lack of Hfq increases the amount and the stability of the PmiaA and P1hfq transcripts, suggesting an autoregulation of the expression of these two transcripts, which results in a 2-fold increase of the MiaA protein in a mutant lacking Hfq (374). In stationary phase the PmiaAtranscript increases 12-fold and the level of MiaA protein increases 6-fold in a mutant lacking Hfq (374). Therefore, the amount of the MiaA protein is negatively regulated by the Hfq protein in logarithmically growing cells and in cells in stationary phase. The juxtaposition of the miaA and the hfq genes on the chromosome may facilitate this negative regulation of the miaA expression by Hfq.
The miaE gene, which is responsible for the hydroxylation of ms2i6A37 to ms2io6A37 in tRNA from serovar Typhimurium, is the second gene in a dicistronic operon. The transcription of the miaE gene is very low due to a strong (90 to 95%) transcriptional attenuator between the first gene of unknown function and the miaE gene. The miaE gene is absent in E. coli, consistent with the lack of the hydroxylated derivative of ms2i6A in its tRNA (286).
The genes queA and tgt are involved in the biosynthesis of Q and part of the same operon of five cistrons (307). The first gene (queA) in this operon is preceded by the main promoter, PM, but there are also two internal promoters (pi and PII) (341). The only ρ-independent transcriptional terminator present in the operon is located downstream of the fifth gene, secF, suggesting thatthe different transcripts initiated at these three promoters may all terminate after the secF gene. The PM promoter contains a "discriminator," which implies that the expression from this promoter, like the trmA, trmD, and truA promoters, is stringently regulated. A high-affinity FIS-binding site is centered on nucleotide –58, a location similar to the putative FIS-binding site in the trmA promoter. The FIS-binding site for the queA gene is located in an upstream activating sequence (UAS), the presence of which increases the transcription twofold (341).
The trmH(spoU) gene encodes the tRNA(Gm18)methyltransferase (289), and it is the fourth gene of a five-cistron operon containing, in order of transcription: gmk (guanylate kinase), rpoZ (the ω subunit of the RNA polymerase), spoT (ppGpp 3'-pyrophosphatase), trmH (spoU) [tRNA(Gm18)methyltransferase], and recG (junction-specific DNA helicase) (129, 189, 237, 289). Three promoters have been identified: P1 is upstream of the first gene, gmk, of the operon (322), P2 is located in the end of gmk and upstream of rpoZ (130, 322), and P3, which may be a weak promoter and which may confer independent transcription of trmH and recG, is located in the end of spoT (237). Apparently, SpoT is a low-abundant protein and limited overexpression is harmful to the bacteria, suggesting that it is under strict regulation (322). The expression of trmH may therefore also be low, consistent with a low expression of some other tRNA-modifying enzymes (e.g., TrmD).
The mnmC+ gene, which encodes an enzyme (MnmC) with dual activities in the synthesis of mnm5s2U, is monocistronic. The enzymatic activity is, as for the TrmD activity, invariant with growth rate, and the activity is not stringently regulated (272, 275).
S-Adenosyl-l-methionine (AdoMet) is one of the most widely used enzyme substrate (63) and it is the most commonly used cofactor for transfer of methyl groups. The methyl group of AdoMet is bound to a sulfonium ion and this CH3-S+-moiety of AdoMet has a high tendency of methyl group transfer, thereby losing its charge. The atomic targets for methyl transfer can be carbon, oxygen, nitrogen, or even halides, and the transfer can occur on DNA, RNA, proteins, polysaccharides, lipids, and various small molecules (for a review, see reference 67). In tRNA the target atom can be C, N, O, and perhaps S. [See below about the formation of the ms2-group of ms2i(o)6A37.] The catalytic requirements for the various methyl transfer reactions seem very flexible and at present five different folds of methyltransferases have been identified, providing an impressive example of functional evolutional convergence (327). The class IV methyltransferases or the "trefoil knot" family of methyltransferases (327) (the SPOUT [SpoU-TrmD] family of methyltransferases, here denoted TREFOIL family [10, 322]) is of special interest, since recently some RNA methyltransferases have been shown to belong to this class. The TREFOIL family is unique in three ways: (i) it has a six-stranded parallel β-sheet flanked by seven α-helices and the first three strands form half a Rossman fold, (ii) the active site is located in the interface of the homodimer, and the active site may be formed by residues from the two subunits, and (iii) a significant portion of the C terminus forms a "trefoil knot."
Nitrogen Methylation.
Synthesis of m1G37. The tRNA(m1G37)methyltransferase (TrmD) from E. coli is present in only 260 molecules per genome at a growth rate of k = 1.0 (392). The purified protein has a molecular mass of about 32 kDa in denatured form, which is consistent with the DNA sequence of its structural gene, but the molecular mass of the native enzyme is larger (46 kDa) as judged by molecular sieving (163). Although the expected molecular mass of a dimer should be 56 kDa, these data may be consistent with a dimer being the active enzyme, since molecular sieving is sensitive to the shape of the protein and therefore does not always result in accurate size determination (See "the structure of tRNA(m1G37)methyltransferase," below). The enzyme catalyzes a methylation at the N-1 position of G37 in 7 of the 46 different tRNA species present in E. coli. These seven tRNAs read leucine CUN, proline CCN, and arginine CGG codons, and consequently, the anticodons of these tRNAs all have G36. Analysis of mutations in 
in vivo and analysis of altered 
in vitro revealed that the entire structure of the tRNA is important for optimal activity (168, 297). The TrmD enzyme makes extensive contacts with the anticodon, the variable loop-stem structure, and the core region (126). In addition, the G36-G37 dinucleotide, which is present in all tRNAs in E. coli containing m1G37, is a crucial recognition element for the enzyme and insertion of a GpG nucleotide into several heterologous tRNA species at positions 36 and 37 made such tRNAs excellent substrates (305). Taken together, the GpG motif and a proper tertiary structure of the tRNA are pivotal recognition determinants for the TrmD enzyme.
The structures of TrmD from E. coli (106), from Haemophilus influenzae (7), from Aquifex aeolicus (235), and from Staphylococcus aureus (W. M. Holmes, personal communication) have recently been established. Whereas the TrmD from E. coli, H. influenzae, and S. aureus are very similar and contain a deep trefoil knot structure and belong to the TREFOIL family of proteins, the TrmD from A. aeolicus lacks such a structure. This would suggest that the evolution of the trefoil knot structure is a rather late event, since Aquifex is the most ancient kingdom of Bacteria. Gram-positive bacteria (e.g., S. aureus) and Proteobacteria (e.g., E. coli and H. influenzae) emerged much later. When more structures of bacterial TrmDs are available, it will be possible to estimate when the trefoil knot structure emerged among Bacteria. The TREFOIL family of methyltransferases comprises two distinct domains, a large N-terminal domain and a smaller C-terminal domain separated by a linker of 12 residues (E. coli) or 13 residues (H. influenzae). The N-terminal domain binds AdoMet and the anticodon loop and the C-terminal domain is not only important for tRNA binding but also for the catalytic activity (126). The structure of the knot is important for the AdoMet binding and the catalytic activity of the enzyme (106). The active form of TrmD is a homodimer with the interface between the two monomers building up a deep cleft in which the G36-G37 of the anticodon region is positioned. It is suggested that one tRNA is bound on each subunit with the anticodon loop in the cleft between the two subunits of the homodimer and the rest of the tRNA bound on the outside surface of each subunit (7), consistent with mutant analysis in vivo (297), in vitro (168), and with protection studies (126). The methylation of G37 may proceed by a deprotonation of the N1 of G37 assisted by a conserved Asp169, followed by a nucleophilic attack on the reactive methyl group of AdoMet by the negatively charged N1 of G37. The Glu116 may interact with the NH2 in position 2 of G37 and Arg154 with O6 and N7 of G37 and thereby discriminate G37 from other bases.
Carbon Methylation.
Synthesis of m5U54. TrmA from E. coli has been purified and is a 42-kDa polypeptide, which is also consistent with the nucleotide sequence of its structural gene, trmA (137, 146, 150, 274). However, Ny et al. (274) also recovered an additional TrmA-specific peptide that is associated with RNA. The RNA is bound covalently to the enzyme and consists of a piece of the 3'-end of 16S rRNA and a subset of undermodified tRNAs (150). As much as 50% of the TrmA molecules present in the bacterial cell are bound covalently to rRNA and/or tRNA. In logarithmically growing cells the enzyme is present in three forms: a 42-kDa native form, a 54-kDa TrmA-rRNA complex, and a 62-kDa TrmA-tRNA complex. Only RNA in the 54-kDa complex is accessible for phosphorylation by T4 polynucleotide kinase (150). Although the reason for the presence of these RNA-TrmA complexes is not understood, it may be related to an unknown second function of the TrmA peptide that is essential for cell viability (288).
A proposed mechanism for the catalytic formation of m5U54 by TrmA involves a covalent intermediate between the tRNA and a nucleophile in the enzyme (321), which has been identified as the cysteine at position 324 (198). The reaction catalyzed by TrmA is shown in Fig. 8 and proceeds as follows: (i) the SH group of Cys324 reacts with the 6-carbon of U54 of tRNA producing a nucleophilic center at the 5-carbon of U54 (enol or enolate; compound II in Fig. 8); (ii) a methyl transfer from AdoMet to the 5-carbon of U54 (compound III); (iii) a β-elimination produces m5U54 and a free enzyme (compound IV). (For more detailed discussion see reference 197.) The U54 is buried in the tRNA through stacking between G53 and Ψ55 and is involved in a reverse Hoogsteen hydrogen bond with A58. Therefore, before catalysis, TrmA must break open the T loop to gain access to U54, perhaps by disrupting the tertiary hydrogen bonds between the D and TΨC arms, which would also disrupt the U54-A58 interaction. A "flip-out" mechanism similar to the one used by the TruB enzyme in the formation of Ψ55 is plausible (165). This conformational change of the TΨC loop occurs before the formation of the Cys324-U54 covalent adduct (198, 409). The Cys324 counterpart in enzymes catalyzing the formation of m5C in tRNA or rRNA has the Cys in a conserved Thr-Cys motif, and the enzymes are also covalently bound to RNA in a way similar to that occurring in the formation of m5U54 in tRNA. In addition, these m5C methyltransferases have an upstream conserved Pro-Cys motif that is functioning as a general base and mediates RNA release from the RNA-Cys-Enzyme intermediate (121, 201, 236). Although TrmA contains four Cys in addition to Cys324, which is present in a Ser-Cys motif reminiscent of the Thr-Cys motif conserved in m5C methyltransferases, none of these four Cys is in a sequence similar to the Pro-Cys sequence conserved in the m5C methyltransferases. Thus, it is unlikely that any of these Cys acts as a general base in the formation of m5U54. Instead, an aspartic acid (D) in the conserved sequence D-P-P-R may act as a general base in the β-elimination step to release the TrmA peptide from the tRNA-TrmA complex (56).
A 17-bp oligonucleotide from position 49 to 65 of a tRNA representing the TΨC stem and loop accepts methyl groups in vitro with only a 3-fold reduction in kcat and a 6-fold increase in Km (147). In fact, an 11-mer, which only has two base pairs in the stem, is also a substrate for the enzyme, albeit at reduced efficiency as compared with the 17-mer. However, the 11-mer has the same Km as the 17-mer. Changes of bases in the 11-mer showed that the seven-base size of the TΨC loop was essential for the enzyme recognition, whereas base substitutions other than of U54 and the C56G change do not influence the recognition (144). Thus, the primary substrate recognition determinants for TrmA reside in the three-dimensional structure of the TΨC loop rather than in its primary sequence. However, local perturbations at or close to the methylation site influence methylation reaction (335).
Oxygen Methylation.
In E. coli, 2'-O-ribose-methylated nucleosides are present in tRNA at positions 18 (Gm), 32 (Cm, Um), and 34 (Cm, cmnm5Um). Methylation at the 2'-position of the ribose was established early in vitro when Gm18 was synthesized by using AdoMet as methyl donor, methyl-deficient tRNATyr as substrate, and cell extract from E. coli as enzyme source (127). The E. coli tRNA(Gm18)methyltransferase, which is encoded by the trmH (spoU) gene (289) has not been biochemically characterized, but it belongs to the TREFOIL superfamily (10) and would therefore be expected to be active as a dimer (327). Although the TrmH ortholog from Thermus thermophilus was suggested to be active as a monomer (214), recent elucidation of its structure shows that dimerization is essential for activity (270). This enzyme consists of only 194 amino acid residues compared with the E. coli enzyme of 229 amino acids (171). The core of the T. thermophilus TrmH enzyme, consisting of about 160 residues, which contains the three motifs (I, II, and III) present in all trefoil knot methyltransferases, binds AdoMet but is catalytically inactive. Only a small part of the two terminal regions are required for tRNA binding and thereby facilitating methyltransferase activity. Indeed, the D loop and stem are clamped between the N- and C-terminal helices from one subunit, while the Gm18 is formed by the other subunit in the homodimer (270). Unlike the E. coli enzyme, the enzyme from T. thermophilus has a broad substrate specificity, suggesting that the enzyme recognizes a common structure of the tRNA. However, some bases in certain positions, among them U8, activate the enzyme (172), explaining why thiolation of U8 to s4U8 decreases the efficiency of methylation by the T. thermophilus TrmH enzyme (170). The short regions in the N and C-termini required for tRNA binding may not impose a strong selectivity, explaining the broad substrate specificity of the T. thermophilus TrmH enzyme contrary to that of the E. coli counterpart. Thus, the E. coli enzyme may have, in addition to the trefoil knot methylation domain, a more complex tRNA-binding domain than the T. thermophilus enzyme consistent with it being larger than the T. thermophilus enzyme. Other TrmH family proteins may lack the RNA-binding domain and may therefore require a partner protein, which may be the case for the trefoil knot family protein from H. influenzae (229).
A novel RNA-dependent methylation mechanism was proposed for TrmH from T. thermophilus, in which the 5'-phosphate of G18 triggers the methylation by withdrawing a proton from an Arg residue, which in turn acts as a catalytic base and deprotonates the 2'-OH of G18. The activated oxygen then makes a nucleophilic attack on the reactive methyl group of AdoMet, resulting in the formation of Gm18 (270).
Five thiolated nucleosides are found in tRNA from serovar Typhimurium: 2-thiocytidine (s2C), 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A). In E. coli the same thiolated nucleosides are present except ms2io6A, which is substituted by the nonhydroxylated derivative ms2i6A. Two kinds of thiolation occur on tRNA: one is a conversion of a keto group (>C=O) to a thio group (>C=S) (s4U, s2C32, and s2U derivatives) and the other is an aromatic C—H to C—S bond conversion occurring when the ms2 group of ms2i(o)6A37 is formed. In the formation of all thiolated nucleosides, the sulfur originates from cysteine (8) and the IscS protein is required for the mobilization thereof (219, 263). The gene encoding IscS belongs to an operon (isc-operon) consisting of seven genes, transcribed in the order iscR-iscS-iscU-iscA-hscB-hscA-fdx, that are devoted to [Fe-S] cluster formation in various proteins. The IscR is a regulator of the operon and senses the [Fe-S] status of the cell (331). The desulfurase IscS is involved in the assembly of [Fe-S] clusters by mobilizing the sulfur from cysteine. IscU is a scaffold for [Fe-S] cluster assembly and accepts sulfur from IscS and delivers it to the target protein (342, 379). IscA is an alternative scaffold to IscU in IscS-directed [Fe-S] assembly and it interacts with Fdx (280). HscA, which binds to IscU, and HscB are specific chaperons for the ISC machinery (166). Following the action of IscS, sulfur for tRNA thiolation is mobilized into two principally distinct pathways: (i) sulfur is transferred from IscS to the tRNA-modifying enzymes ThiI and MnmA in the formation of s4U8 and s2U derivatives, respectively, without the participation of an [Fe-S] protein and (ii) sulfur is transferred with the participation of [Fe-S] proteins when forming s2C32 and ms2i(o)6A37 in tRNA (221, 224) (Fig. 9).
Synthesis of Thiolated Uridine Derivatives (s4U8, mnm5s2U34).
In some initial studies, Lipsett and coworkers identified two factors, A and C, required for the synthesis of s4U8 in E. coli (1). Accordingly, two genes, nuvA and nuvC, located at 9 and 44 min, respectively, on the chromosome, were suggested to be involved in the synthesis of this thiolated nucleoside (234). The nuvC mutant also requires thiamine for growth (315). The nuvA gene maps to a region where the thiI gene maps, which is why it is likely that the nuvA gene is identical with the thiI gene, although the original nuvA mutants have no thiamine requirement. It is still unclear which gene is mutated in the nuvC mutant (252).
The desulfurase IscS catalyzes the formation of alanine and sulfane sulfur (S0) from cysteine, resulting in a cysteine persulfide in the active site of IscS (118). The terminal sulfur of this persulfide is then transferred to ThiI, which also binds the tRNA substrate (190, 281). The latter protein has a PP-loop sequence (185SGGXDS190) , which is characteristic of the pyrophosphate synthase family of enzymes that catalyzes the adenylation and substitutions of carbonyl oxygens (42), consistent with the requirement of ATP in the formation of s4U8. Two cysteines (Cys344 and Cys456) of ThiI are essential for in vitro and in vivo thiolation of U8. A disulfide bond is formed between the Cys344 and Cys456, which is later reduced to generate ThiI in its resting state (253, 281, 405). Based on these results, two plausible mechanisms (A and B in Fig. 10) have been proposed for the formation of s4U8 (253). After the activation of oxygen by adenylation at position 4 of U8, a nucleophilic attack by the ThiI persulfide at Cys456 occurs, AMP is released, and the terminal sulfur of the ThiI persulfide binds to the 4-carbon of U8. A disulfide bond is formed between Cys456 and Cys344 that elicits the release of the s4U8-containing tRNA. After the reduction of the disulfide bond between Cys456 and Cys344, ThiI is again in its resting state and ready for a new round of catalysis. According to the alternative model (Fig. 10B), ThiI generates hydrogen sulfide, which subsequently serves as the nucleophile that displaces the AMP in the activated U8. In this model the ThiI is not covalently bound to U8 of tRNA during the catalysis. The ThiI enzyme recognizes no specific primary sequence outside the target nucleotide U8, which must, however, be the first nucleotide at the 5' end of at least a five-nucleotide-bulged loop. Deletion analysis of tRNAPhe reveals that a helix comprising the stacked amino acid acceptor and TΨC stems is a minisubstrate for ThiI (220).
Thiolation of U34 in the synthesis of (c)mnm5s2U34 does not depend on the (c)mnm5 side chain (107, 155, 191, 358). Similar to ThiI, MnmA also has a PP loop that binds ATP, and accordingly ATP but not GTP protects MnmA from proteolytic degradation (191). MnmA binds to the substrates tRNAGlu and tRNALys, which both have (c)mnm5s2U34 as the wobble nucleoside, but not to the nonsubstrate tRNAPhe, suggesting that MnmA is the tRNA-modifying enzyme (191). The in vitro formation of s2U requires the presence of IscS and ATP, suggesting that the thiolation reaction in the synthesis of s2U is similar to that in the synthesis of s4U8 catalyzed by ThiI (191). Since these experiments were done with purified MnmA in conjunction with crude extracts and the activity was low, participation of other protein(s) or factor(s) in the formation of s2U cannot be excluded. Indeed, four additional proteins (TusA to D) have been implicated to be required for this thiolation reaction (T. Suzuki, personal communication).
MnmE is likely to participate in the first step of the synthesis of the (c)mnm5 side chain, since a mutation in mnmE results in the presence of s2U in the tRNA (107, 155). MnmE consists of an N-terminal domain of about 220 amino acids, which is required for self-assembly to homodimers and homotrimers, a central GTPase domain, and a C-terminal domain containing Cys451, which is required for tRNA modification but not for GTP hydrolysis (412). The requirement for Cys451 in the formation of the (c)mnm5 side chain is reminiscent of the pivotal role of Cys324 in the formation of m5U54, in which a covalent intermediate between the Cys324 in TrmA and the C6 of U54 activates the C5 of U54 to accept a methyl group from AdoMet. If so, MnmE may be the tRNA-modifying enzyme, although an affinity between MnmE and tRNA has not been demonstrated. A small but significant part of the MnmE protein is associated with the inner membrane, and this localization or the ability to self-assemble does not require GTP hydrolysis nor Cys451 (412). A mutation in the gidA gene results in an induction of +2 frameshift similar to that induced by an mnmE mutant, suggesting that GidA is required for the formation of the mnm5 side chain (46). However, mutations in mnmE or gidA influence the frequency of +2 frameshifts differently and comparison of these frequencies with the frequency of +2 frameshifts induced by the mnmE, gidA double mutant suggests that MnmE participation precedes that of GidA in the synthesis of mnm5s2U34. The GidA protein, which is structurally conserved among Bacteria, has a dinucleotide-binding motif at the N terminus and purified GidA from Myxococcus xanthus complexes FAD, suggesting that GidA may be involved in an oxidation-reduction reaction or in sensing the redox state of the cell (391). The tripartite GTP-binding motif of MnmE is required for GTP hydrolysis in vitro and for tRNA modification in vivo (59, 412). Thus, it is not merely the GTP-binding ability that is required for tRNA modification but the ability to hydrolyze GTP (412). The MnmE from Thermotoga maritima has a tetrahydrofolate (THF)-binding site with an affinity for 5-formyl-THF in the micromolar range, suggesting that the first step in the synthesis of the cmnm5 side chain is a C1 group transfer from 5-formyl-THF to U34 in tRNA. Since the Cys451, which is pivotal for the covalent binding of U34 to MnmE, is too far from the THF-binding site, GTP hydrolysis may move these two sites closer together, which would be required for the addition of the formyl group to the 5C of U34 (332). After incorporation of glycine to the formylated intermediate (f5s2U34) and a reduction step catalyzed by GidA and FADH2, the final cmnm5 side chain is produced (332). Since the Saccharomyces cerevisiae nuclear-encoded mitochondrial homologs to MnmE (denoted Mss1p in S. cerevisiae) and GidA (Mto1p) form heterodimers (75), the formation of a heterodimer MnmE-GidA in the synthesis of the cmnm5 side chain is plausible. To obtain the final product mnm5s2U34, the dual-activity enzyme MnmC catalyzes two biosynthetic steps, which have been characterized in vitro by using a purified enzyme (155): (i) first the MnmC peptide catalyzes a cleavage of the cmnm5 side chain resulting in nm5s2U34. The MnmC peptide contains a FAD-dependent oxidoreductase domain and the purified MnmC protein contains FAD (56). Based on this information it was suggested that the cleavage of the cmnm5 side chain occurs by a FAD-mediated redox reaction.(ii) the nm5s2U34 moiety is then methylated by the MnmC peptide to mnm5s2U34 (155). In summary, the synthesis of (c)mnm5s2U34 proceeds accordingly:
where TusA, B, C, and D are proteins involved in the transfer of sulfur from IscS to MnmA (T. Suzuki, personal communication). The thiolation step is independent of the cmnm5 side chain formation and may therefore occur on any intermediate in the synthesis of cmnm5s2U34. 5-Formyl-tetrahydrofolate is denoted 5-f-THF. The Gly is suggested to be incorporated to the f5s2U34 to form an unsaturaed side chain, which is reduced by GidA-FADH2 to form the cmnm5 side chain (332). The MnmC1 denotes the domain of MnmC that is defective in the mnmC1 mutant and MnmC2 denotes the MnmC domain defective in the mnmC2 mutant (155).
Five genes [selA, encoding selenocysteinyl (Sec)-tRNASec synthase; selB, Sec-tRNASec-specific translation factor; selC, tRNASec; selD; and ybbB] are required for the incorporation of selenium into proteins and tRNA (223, 404) (reviewed in reference 353). Of these five genes two are required for selenation of tRNA: selD, which synthesizes the selenium donor, SePO3, and ybbB, which encodes the selenium phosphate-dependent tRNA 2-selenouridine synthase, here denoted tRNA(Se2)synthase (404). When selenium is available, up to 40 to 50% of (c)mnm5s2U34 in total tRNA is replaced by the selenium derivative (c)mnm5Se2U34 (209, 400). Only (c)mnm5s2U34 among the thiolated nucleoside is converted to a selenated derivative (401). About 50% of the Se in tRNA is associated with tRNAs specific for Glu and Lys (398) and the rest of Se might be associated with tRNA specific for Gln, since it also contains a (c)mnm5s2U34 derivative. The S to Se conversion is independent of the presence of the (c)mnm5 side chain (400) but requires the presence of the s2 group (209). The first step in the synthesis of a selenated nucleoside is the synthesis of the selenium donor (SePO3), which is catalyzed by SelD and requires ATP. It occurs as follows (133, 386):
The enzyme [selenophosphate-dependent tRNA(Se2U)synthase, YbbB], which catalyzes the substitution of sulfur for selenium in tRNA, has recently been purified and its structural gene ybbB has been identified (404). Deletion of ybbB abolishes all selenium incorporation into tRNA but does not influence the selenation of proteins. Purified YbbB contains two tightly bound tRNA molecules per enzyme monomer and uses SePO3 as selenium donor. The YbbB protein has the rhodanese sequence motif Cys-X-X-Gly within the sequence Cys96-Cys97-X-X-Gly. Conversion of Cys97 to Ser97 completely prevents the selenation of tRNA in vivo whereas a C96S alteration does not. However, in vitro the C97S alteration reduces the activity by only 50%. The formation of perselenide at Cys97 in the rhodanese domain during the enzymatic reaction is plausible. ATP may be required in this reaction, since the YbbB protein contains a P-loop motif in the C terminus end, although earlier work suggests that ATP is not required in the reaction catalyzed by the tRNA(Se2U)synthase (387). What happens with the sulfur after the selenation is not known. The reaction following the formation of the selenium donor may be:
Synthesis of s2C32 and ms2i(o)6A37.
Although sulfur mobilization through IscS is the major way of sulfur transfer for the formation of all thiolated nucleosides, including s2C32 and ms2i(o)6A37, an inefficient IscS-independent sulfur transfer system is also operating in the synthesis of only these two thiolated nucleosides (219, 263). However, little is known about what follows the action of IscS in the thiolation of C32 and i6A37. Recently, the ttcA gene product (TtcA) was identified as being involved only in the synthesis of s2C32 (180). TtcA shares similarities with ThiI and MnmA and has, as those proteins do, a PP-loop domain, suggesting that the catalytic activity requires ATP in a way similar to that of ThiI and it may also interact with tRNA as ThiI does. Unlike the syntheses of (c)mnm5s2U34 and s4U8, the synthesis of s2C32 requires active members of the ISC machinery, suggesting that an [Fe-S] cluster protein is involved and argues that the action of TtcA is mechanistically distinct in certain ways from that of ThiI and MnmA. Instead, TtcA may share the requirement for an [Fe-S] cluster assembly with MiaB (224). The TtcA is part of a broadly distributed protein family characterized by the presence of a central domain containing a PP-loop motif and a -C-X1-X2-C- motif. The latter feature is characteristic of the thioredoxin superfamily. Both cysteine residues in this conserved motif are required for the formation of s2C32 (180). The presence of TtcA homologs in organisms that do not have s2C in their tRNA suggests that such proteins may be involved in sulfur metabolism other than the thiolation of tRNA or involved in the synthesis of a thiolated nucleoside other than s2C32.
The first step in the synthesis of ms2i(o)6A37 is a transfer of the dimethylallyl moiety (earlier called isopentenyl; we are still abbreviating the nucleosides as i6A derivatives, since this is an established abbreviation in the tRNA modification field) from dimethylallyl diphosphate (DMAPP) to A37 and thereby forming i6A37 in a subset of tRNAs. Thereafter, the formation of the ms2 group of ms2io6A37 or ms2i6A37 occurs, because its formation depends on the presence of the i6-group (105, 110). The transfer of the dimethylallyl moiety is catalyzed by the miaA gene product (MiaA), which binds tRNA either as a monomer or as a multimer. The reaction is sequential with tRNA binding first before DMAPP (225, 248, 345, 346). The conserved GxxxGKT(S) in MiaA resembles the P-loop motif and it is part of the DMAPP-binding site, and the conserved RxxR sequence is part of the tRNA-binding site (346). Modification of a minihelix consisting of only the anticodon stem and loop of tRNAPhe proceeds with a kcat similar to that of the full-length undermodified tRNAPhe,suggesting that the enzyme primarily recognizes sequences near the target nucleoside (225, 345). Sequence comparison of tRNAs normally having ms2i6A37 indicates that the A36-A37-A38 sequence and a 5-bp anticodon stem are required for dimethylallylation (371). Consistent with this conclusion, two 
(I and V), which do not have ms2i6A37, have a mispair (G30-Ψ40) in the anticodon stem, suggesting that such mispairing proximal to the anticodon loop perturbs the enzymatic formation of i6A (142). By using crude extract, it was revealed that not only is such a mispair in the anticodon stem inhibiting the formation of i6A, but a G-C base pair at positions 29–41 and 30–40 and lack of G-C or C-G base pairs at position 31–39, are important for efficient formation of i6A37 (249). However, slightly different results were obtained by using a purified MiaA protein and a minihelix of tRNAPhe, since neither inversion from G30-C40 to C30-G40 nor a change to A30-U40 affected enzymatic activity (345). These and other results suggest that the enzyme does not make contacts with specific bases but rather recognizes an overall conformation of the helix in this part of the anticodon stem. Changing A36 or A38 in the sequence A36-A37-A38 abolishes modification, demonstrating that the enzyme makes specific base contacts in this region of the tRNA.
The next step in the synthesis of ms2i(o)6A37 is catalyzed by MiaB (112). The reaction is an aromatic C-H to C-S bond conversion, which is chemically intriguing. In contrast to the formation of thiolated uridines, which is not a redox process, the formation of the ms2 group on A37 is an oxidation reaction. Starvation of an E. coli ( relA, cys, met) mutant for methionine results in the accumulation of a precursor, which may be s2i6A (4), whereas starvation for cysteine (4) or iron (138, 311, 390) results in the accumulation i6A. These results suggest a sequential synthesis of ms2i(o)6A37 and a requirement for iron, cysteine, and AdoMet in the addition of the methylthio group. MiaB, which belongs to the Radical SAM superfamily with its characteristic CxxxCxxC motif (347), is an iron-sulfur protein containing most likely one [4Fe-4S] cluster under anaerobic conditions (291, 293). This explains the requirements for the ms2 formation for cysteine, iron, and AdoMet. The CxxxCxxC motif is pivotal for the activity of MiaB, because mutations in any of these three cysteines abolish the activity. The MiaB protein has the TRAM RNA-binding domain (proposed RNA-binding domain found in TRM2 and MiaB), suggesting that MiaB may interact with tRNA (9). The MiaB enzyme has a strict requirement for the presence of the dimethylallyl-group, since miaA mutants lack the ms2 group (105, 110). The sulfur mobilization is initiated by IscS, but how the sulfur is transferred to MiaB is not known. Since IscU, HscA, and Fdx are also required for the synthesis of ms2i(o)6A37, it is likely that the assembly of an [Fe-S] cluster is involved (224). The ISC machinery could be involved in de novo synthesis or repair of the [Fe-S] cluster in MiaB, although involvement of other [Fe-S] proteins in the methylthiolation reaction cannot be excluded. In addition, IscA also participates in an unknown way in the MiaB-mediated reaction, since a mutation in the iscA gene reduces the extent of methylthiolation but does not influence the synthesis of any other thiolated nucleosides (224). It is thought that a [4Fe-4S] cluster transfers an electron to the sulfonium ion of AdoMet producing methionine and a highly oxidizing 5'-deoxyadenosyl radical (119). One can hypothesize that such a 5'-dA radical abstracts the hydrogen atom at position 2 of A37 creating an A37 radical that is prepared to accept a sulfur from a sulfur donor. This donor is not yet identified but may be a persulfide and not a second [Fe-S] cluster, since MiaB has only one (293). Purified MiaB not only inserts sulfur at position 2 of i6A37 but also subsequently transfers a methyl group from a second AdoMet onto the possible intermediate s2i6A37 (292). Thus, MiaB is a bifunctional enzyme requiring AdoMet for both functions.
tRNA from serovar Typhimurium has the hydroxylated derivative ms2io6A37 in tRNAs that in E. coli have ms2i6A37 (54). The hydroxylation occurs only under aerobic conditions even though the tRNA(ms2io6A37)hydroxylase is also present anaerobically (51). The gene, miaE, encoding this enzyme is not present in E. coli (286). Overproduction of MiaE in a miaB mutant converts only 30% of i6A to io6A, suggesting that the MiaE is strongly dependent on the presence of the ms2-group (112). Thus, the synthesis of ms2io6A37 is sequential and proceeds as follows:
The most abundant modified nucleoside in E. coli is 5,6-dihydrouridine (D) and this nonaromatic modified nucleoside is found almost exclusively in conserved positions in the D loop. Transfer RNA from E. coli has D residues at five positions (16, 17, 20, 20:A, and 21; see Fig. 2) and one tRNA may have up to four of these modified nucleosides and most tRNA species contain at least one D. Only two tRNA species (specific for Tyr and Glu) do not contain D. Using a comparative genomic approach, three genes (dusA, dusB, and dusC) were identified as involved in D synthesis at five different positions. Deletions of all three genes result in tRNA devoid of D, suggesting that at least some of the three Dus proteins are not position specific, although they form D close to each other in the D loop. However, DusA is at least specific for position 21 in tRNAMet, but it must also be involved in the synthesis of D in other positions in tRNA, since deletion of the dusA gene results in the largest reduction of D in tRNA (27). The Dus proteins belong to a family of FMN binding proteins but they have no apparent RNA binding motif. However, DusB binds to the D-loop in vitro, suggesting that it is a tRNA modifying enzyme (27)
Pseudouridine (Ψ), which is a 5-ribosyl isomer of uridine (Fig. 1), was the first modified nucleoside ("fifth nucleoside") identified in tRNA in 1957 independently by Cohn (71) and Davis and Allen (85). It is frequently found in tRNA and present in seven positions (13, 32, 38, 39, 40, 55, and 65). The enzymes responsible for the synthesis of Ψ in all positions of tRNA have been identified [TruA (Ψ38,39,40), truB (Ψ55), TruC (Ψ65), TruD (Ψ13), and RluA (Ψ32)]. All enzymes are site specific, except TruA, which modifies three positions (38, 39, and 40), although these positions are near each other in the anticodon loop (position 38) and stem (positions 39 and 40). RluA is exceptional among bacterial tRNA-modifying enzymes, because it has a dual substrate specificity and modifies both tRNA and 23S rRNA (406).
The first four Ψ synthases to be cloned and sequenced were from E. coli (TruA, TruB, RluA, and RsuA, which is a 16S rRNA Ψ synthase) and they revealed no sequence similarities and were grouped into four separate families (152, 208). Recently, it was discovered that TruD, which catalyzes the formation of Ψ13 in only one tRNA in E. coli, constitutes an additional Ψ synthase family (196). Despite the low overall sequence similarity, five short sequence motifs (I, II, IIa, III, and IIIa) are present in all four families (87). Furthermore, the structures of TruA (120), TruB (165), RsuA (339), recently TruD (195), and RluD, which is a 23S rRNA(Ψ)synthase belonging to the RluA family (87), reveal a common fold containing five conserved residues, including the catalytic Asp. The presence of this common fold with the conserved residues in this protein family suggests that these enzymes arose by divergent evolution from a common ancestor (87, 165, 195, 251, 339).
The chemical mechanism of conversion of U to Ψ is most likely similar, if not identical, irrespective of position in the RNA or the particular RNA substrate. The formation of Ψ does not require any energy or any cofactor and it occurs on the polynucleotide level with a direct conversion of the U residue without any identified intermediate (69, 183). At least three chemical steps are required for its synthesis: (i) Cleavage of the N-glycosidic bond of the target U, (ii) rotation of the uracil ring to position C5 of U close to C1' of the ribose, and (iii) formation of a new C1'-C5 carbon-carbon bond. Two mechanisms are proposed for the formation of Ψ (145, 175, 192). According to the first mechanism (145, 192) a nucleophilic group, now suggested to be the conserved Asp in the protein, attacks C6 of uridine generating a cleavage of the N-glycosidic bond and a dihydropyrimidine bound via Asp to the protein (Michael addition mechanism). In the second mechanism (175), the nucleophilic attack on C1' of the ribose by Asp generates a cleavage of the N-glycosidic bond and an acylal intermediate bound to the protein via Asp. Both models then invoke a complete 180° rotation before the C1'-C5 bond is established. Existing experimental data do not distinguish between these mechanisms as reviewed by Mueller (251). Analysis of the RNA-TruB complex favors the first model in which the Ψ formation proceeds by an attack on the C6 of the pyrimidine base and not on the C1' of the sugar (283).
Recognition of the substrate is best understood for TruB. The target nucleoside is present in the highly conserved TΨC arm. Although this stem and loop is hydrogen bonded to the D loop, the recognition for catalysis is completely contained within the TΨC arm, because a 17-mer consisting of only the TΨC arm is as active as full-length tRNA (23, 148). Only the base pair (53–61) next to the TΨC-arm loop is essential for activity. The structure of TruB containing a 17-mer reveals that TruB recognizes the overall structure of the TΨC loop. The target nucleoside U55 is hidden in the structure by base pairing with G18 in the D loop and by stacking interactions with the m5U54-A58 base pair. The recognition of the m5U54-A58 base pair is important, since this base pair is not present in the initiator tRNAMet and it also lacks Ψ even though it has U55. To get access to the substrate, the enzyme flips out three bases U55, C56, and G57 in the loop to position U55 in the active site, whereas the structure of the stem of the TΨC arm is maintained (165). Upon binding of its substrate, TruB protein undergoes significant conformational changes such that it recognizes its substrate by tight binding followed by an induced fit to maximize the interaction (283). Since all growth defects induced by a deletion of the truB gene are reversed by a catalytically inactive TruB, the TruB protein may act as an RNA chaperon (153). The structure reveals that binding of TruB to the tRNA would disrupt the interaction between the TΨC loop and the D loop and thereby open up the tRNA structure. This would give the tRNA a second chance to fold correctly when the enzyme is released from the substrate, consistent with the suggestion that TruB may also act as an RNA chaperone (165). If so, the catalytic Asp is not required for opening up the tRNA structure but only for the catalytic activity.
TruA was the first Ψ synthase to be purified and characterized (11, 192). After addition of tRNA to the serovar Typhimurium TruA, a dimerization of the enzyme occurs and the structure of TruA also reveals a dimeric protein (120). The recognition determinants of yeast and E. coli TruA orthologs are similar but have subtle differences (222). Whereas the yeast enzyme catalyzes the formation of Ψ38 in both yeast tRNAAla and E. coli tRNALeu, albeit with different efficiencies, the E. coli enzyme can only use the E. coli tRNALeu as a substrate. The fact that all Ψ38-containing tRNAs in E. coli have G36, whereas other tRNAs having C36 and the potential target nucleoside U38 are not modified, suggests that G36 and C36 act as positive and negative determinants, respecitvely, for E. coli TruA. The yeast ortholog requires an intact tRNA structure and it is plausible that the E. coli enzyme has a similar requirement. Accordingly, the structure of the dimer suggests that TruA recognizes the shape and charge of the whole tRNA molecule and not just the anticodon stem to catalyze the formation of Ψ in the anticodon stem and loop (120). Presumably, TruA, like TruB, flips out its target U from the anticodon stem during the catalytic process.
RluA was identified as an enzyme catalyzing the formation of Ψ746 in 23S rRNA (406). During the characterization of the enzyme, it was discovered that tRNAPhe, which has Ψ32, is also a good substrate. The explanation is that Ψ746 is present in a loop of 23S rRNA that is identical with the anticodon loop of tRNAPhe. Three other tRNAs in E. coli have Ψ32 and the sequence of the anticodon loop of these tRNAs deviates by only one nucleoside from the sequence in the 23S rRNA and tRNAPhe, suggesting that the structure of the loop is the recognition determinant for RluA.
Both TruC, which synthesizes Ψ65 in only 
and 
, and TruD, which catalyzes the formation of Ψ13 in only 
, are site-specific enzymes, since deletion of the corresponding genes only affects the formation of Ψ at the respective positions (86, 196). The TruD folds into a V-shaped molecule with a catalytic domain with structural similarity to the catalytic domain of the other Ψ synthases despite their lack of similarities in the amino acid sequence (195). The likely tRNA-binding domain has an unusual structure with a fold not found in any other protein.
The modified nucleoside cmo5U34 or its methylester is found in one of the two or three tRNAs reading codons in the family codon boxes specific for val, ala, thr, pro, and ser (Fig. 3). Chorismic acid, which is a common metabolite in the synthesis of aromatic amino acids and aromatic vitamins, or a derivative of it, is required for the synthesis of cmo5U34 and its methylester (Fig. 11). One of the two carbon atoms of the side chain ―O―CH2―COOH originates from methionine. The role of chorismic acid or a derivative of it in the synthesis of the cmo5 side chain is not clear (28, 157). Recently two gene products were shown to be required for the synthesis of cmo5U34 (260). The CmoB, which has an AdoMet-binding site and therefore most likely is a methyltransferase, is required for the methylation of ho5U to form mo5U. Lack of CmoA, which also has an AdoMet-binding site, results in the accumulation of both ho5U and mo5U. Since the step(s) from mo5U to cmo5U apparently is not a simple methylation, it was suggested that CmoA may be part of a protein complex that is involved in the steps following the formation of mo5U in the formation of mcmo5U (Fig. 11). The tRNA(mcmo5U34)methyltransferase, which catalyzes the formation ofmcmo5U, has been partially purified and a UGA suppressor (supK) strain is deficient in the activity of this enzyme. Therefore, it was suggested that supK is the structural gene for this methyltransferase (295, 306). However, supK is the structural gene for release factor 2, which recognizes the UGA stop codon (194). Since the UGA stop codon of cmoA overlaps the start codon of the cmoB gene, deficiency of the release factor 2, which recognizes UGA, may induce readthrough at this UGA codon, resulting in an elongated CmoA peptide, which may be inactive. If the CmoA is the tRNA(mcmo5U34)methyltransferase, such an event could result in a lower activity of this enzyme in a strain having less release factor 2, consistent with the observation by Reeves and Roth (306).
The hypermodified nucleoside queuosine (Q; the base is denoted queuine, Que) consists of 7-aminomethyl-7-deazaguanine (Que1 also called base of preQ1) to which a cyclopentenediol is linked to the NH2 group (Fig. 1). It is present as the wobble nucleoside in tRNAs from most Bacteria and Eukarya (notable exceptions are yeast and Mycoplasma) but not from Archaea. However, a structural relative of Q called Archaeosine is present in archaeal tRNA in position 15, in contrast to Q, which is located in position 34. In some Eukarya, sugars like mannose or galactose can be linked to the cyclopentenediol. Unlike most modified nucleosides, the synthesis of a precursor to Q is independent of tRNA and is then inserted into tRNA by a base exchange catalyzed by the tRNA-guanine transglycosylase [Tgt] encoded by the tgt gene. The synthesis of Q starts with GTP and a GTP cyclohydrolase should be involved in its synthesis (213). The first intermediate identified in the synthesis was the base of preQ0 (7-cyano-7-deazaguanine, Que0), which may then be reduced to Que1 by an unidentified reductase (Fig. 12). Que1 is thought to be the natural substrate for Tgt, which catalyzes the exchange of G34 by Que1 in tRNA (278). Two mutants have been isolated that apparently are involved in the synthesis of precursors to Que1, although the genetic defects in these mutants were not identified (92, 267). These mutants may be defective in one of the four genes that were recently identified as required for the synthesis of Que1 (304); and one of these genes (queF) may be coding for a GTP cyclohydrolase and thus be involved before the synthesis of Que0 (304). One of the four recently identified gene products (QueE) in the synthesis of Q belongs to the Radical SAM family proteins and has an [Fe-S] cluster, which is consistent with the requirement for iron and methionine in the synthesis of Q (200, 279).
The exchange of Gua in position 34 by Que1 is catalyzed by Tgt and the structure of the enzyme from the bacterium Zymomonas mobilis is known (309, 407). Using an analog to Que1 (9-deazaguanine [9dzG]), a reaction intermediate was trapped in a ternary complex consisting of Tgt, a stem-loop RNA, and 9dzG. The structure of this ternary complex reveals that the RNA is tethered to the enzyme through the side chain of the conserved Asp280, suggesting that Asp280 mediates the nucleophilic attack on the C1' of the ribose (407). Earlier Asp102, the presence of which is essential for the enzymatic activity, and not Asp280 was suggested to be the amino acid mediating the nucleophilic attack on the C1' of ribose (207, 308). However, Asp102 may instead assist as a general base to deprotonate N9 of Que1 to enable it to form a covalent bond with C1' of the ribose (407). The conformation of the Tgt enzyme does not change on binding to the RNA. However, the RNA in the ternary complex adopts an unusual conformation with four of the seven nucleotides in the anticodon loop flipped out. The enzyme makes sequence-specific interactions with G34 and U35 consistent with the earlier conclusion that the major recognition determinants are U33-G34-U35 in a seven-base loop as determined by using a minihelix corresponding to the anticodon stem and loop (81, 258). The exchange of G34 by Que1 involves at least the following steps: (i) The enzyme binds and orients the tRNA and accommodates G34 of the tRNA into the Que1-binding pocket of the enzyme; (ii) a nucleophilic attack by Asp280 (E. coli Asp264) on C1' of the ribose occurs, generating a cleavage of the glycosidic bond and forming a covalent Tgt-tRNA intermediate; (iii) Que1 replaces the free Gua; (iv) Asp102 (E. coli Asp89) deprotonates N9 of Que1 allowing a nucleophilic attack on C1' of the ribose to form a new glycosidic bond. (See also a review by D. Iwata-Reuyl and references therein [179].)
The preQ1 in tRNA is then converted to Q by at least two enzymatic steps. QueA catalyzes an unprecedented reaction in biological systems, including transfer of the ribosyl moiety from AdoMet to the 7-amino group of preQ1 in the tRNA in conjunction with the elimination of Ade and Met (340, 384). In this reaction AdoMet serves as ribosyl group donor in contrast to its well established role as methyl donor or as generating a 5'-deoxyadenosyl radical. The QueA enzyme has substrate requirements similar to Tgt in that it also can use a minihelix as substrate. The resulting epoxy-Q is then converted to Q in an uncharacterized reaction that requires vitamin B12 (123). (See discussion about possible reaction mechanisms in reference 179).
Bioinformatic analysis of genome sequences has revealed the presence of "aa-tRNA synthase-like proteins," which contain the aminoacylation catalytic domain but lack the tRNA-binding domain (176, 324). One such "synthase-like" protein is GluQRS (glutamyl-Q-tRNA synthase), encoded by the yadB gene. This aaRS paralog contains the N-terminal catalytic domain homologous to GluRS but lacks the C-terminal anticodon-binding domain. In sharp contrast to GluRS, the GluQRS activates Glu in the absence of tRNA. Moreover, the activated Glu is transferred to the noncognate tRNAAsp and Glu is not attached to the 3'-CCA end of tRNAAsp but to Q present in the anticodon of tRNAAsp (38, 97, 317). The Glu bound to Q is base labile (97, 317), explaining why the glutamylqueosine (GluQ) has not been observed before. This lability of GluQ makes it a target for a regulatory function on the translation level provided that the presence of GluQ in tRNAAsp exerts an impact on the decoding capacity of this tRNA. Also in eukaryotes, the Q in tRNA is further modified by the addition of either a mannosyl group (in tRNAAsp) or a galactosyl group (in tRNATyr). Thus, the mannosylation of Q in tRNAAsp of higher eukaryotes is reminiscent of the glutamylation of the same Q in tRNAAsp of bacteria. In bacteria Q is further modified in tRNAAsp but not in tRNAHis and tRNAAsn, which also contain Q as a wobble nucleoside (reviewed in reference 141). The synthesis of Q is summarized in Fig. 12.
Bacteria universally contain no tRNA genes with an anticodon TAT that is cognate to the Ile AUA codon (240). Instead, a tDNA gene with the methionine anticodon CAT coding for a special tRNA that is able to translate the Ile AUA codon exists. Although this tRNA has C34 in the primary transcript, which would only read G, a lysine is added to the keto group at position 2, resulting in lysidine (k2C34), which reads A. This modification makes the 
able to decode AUA (256). The formation of k2C34 is catalyzed by the TilS enzyme (tRNA Ile lysidine synthase), which is encoded by the tilS (earlier called mesJ) gene (348). TilS belongs to a large family of PP-loop proteins, including the three tRNA-modifying enzymes TtcA (YdaO), ThiI, and MnmA. TilS has in its N terminus a PP-loop sequence characteristic of enzymes that bind ATP and generate PPi (PPi synthases). A recombinant TilS enzyme binds tRNA and catalyzes the formation of k2C34 in vitro in the presence of undermodified 
(isolated from a temperature-sensitive tilS strain), lysine, and ATP. How the ε-amino group of lysine reacts with the 2-keto group of C is not known. However, since TilS contains a PP loop, an initial activation of the 2-keto group by adenylation, followed by PPi release and by a nucleophilic attack on the activated C2 of cytidine by the ε-amino group of Lys, is plausible (348). This suggested enzymatic mechanism is reminiscent of that shown for ThiI, which also has a PP loop. The structure of TilS reveals that its N-terminal domain forms a crater, in the depth of which the PP loop is located, suggesting that the anticodon stem loop binds in this depression and thereby positions the wobble nucleoside such that ATP can attack the 2-keto group. An intriguing question is how the TilS enzyme discriminates between the unmodified 
and the 
with the same anticodons (CAU), although nucleosides at positions 5 and 44 may be critical in this discrimination (W. Mitchell, Genome Institute of Singapore, personal communication).
More than 35 years ago, inosine (I) was shown to be present in the wobble position of yeast 
(167), and this discovery strongly supported the predictions Crick made in his wobble hypothesis (80). Whereas organisms from the domain Eukarya have seven to eight tRNA species with I34, organisms from Bacteria have only one (
) reading the Arg codons CGU, CGC, and CGA. With a partly purified enyzme from yeast, it was shown that the conversion of A34 to I34 occurs by a hydrolytic deamination reaction in which water is the hydroxyl donor and free ammonia is probably the leaving group (18). Although not shown experimentally, the bacterial enzyme most likely uses the same mechanism. Whereas the yeast enzyme is a heterodimer, the bacterial enzyme apparently is a homodimer (131, 403). The bacterial enzyme (TadA) contains highly conserved residues shared with the yeast catalytic subunit Tad2, including the Zn2+-chelating residues (two Cys and one His) and a glutamate thought to mediate proton transfer (403). is the only tRNA able to read the A-ending codon CGA, and lack of TadA and consequently lack of I34 results in this tRNA being unable to read the CGA codon. Thus, TadA is an essential enzyme (403). The E. coli enzyme can efficiently use an anticodon-stem-loop (ASL) of as substrate and it has a strict requirement for the anticodon sequence ACG, which is the anticodon of its normal substrate (18). Further analysis using a purified enzyme and ASL as substrate suggests that, in addition to the importance of the anticodon sequence, the structure and the size of the loop, but not the sequence of the stem, are important positive determinants for TadA (403).This strict requirement for an anticodon sequence is consistent with the fact that in a mutant of serovar Typhimurium having a proline tRNA with an A34-G35-G36 anticodon instead of its normal anticodon G34-G35-G36, the wobble nucleoside A34 is not converted to I34 (66).
The identity elements for aminoacylation of most tRNAs are located at two distal extremities: the anticodon region and the amino-acid-accepting stem (132). Early on, it was shown that completely unmodified tRNA accepted amino acids almost as efficiently as the native, fully modified tRNA (reviewed in reference 30). However, a few unmodified tRNAs (E. coli
[269], [361], 
[365]) are severely affected in the aminoacylation reaction and the reason is that these tRNAs lack modified nucleosides in either position 34 (wobble position) or in position 37 (3' of and adjacent to the anticodon). Although most completely unmodified tRNAs are able to accept amino acids in vitro, under certain conditions such tRNAs show considerably changed kinetics of aminoacylation (318), because the unmodified tRNA does not adopt the native conformation (68, 159). Although the kinetic parameters of the unmodified 
are similar to those of the modified in the cognate interaction with AspRS, the unmodified is mischarged by ArgRS with considerable efficiency (285). The major effect is in the rate of mischarging (kcat), whereas the KM for unmodified in the noncognate interaction increases only 2-fold. Perret et al. (285) suggest that in yeast , a modified nucleoside(s) acts as an antideterminant. These results show that modifications change the structure of the tRNA in a way that may influence the efficiency of the aminoacylation reaction and counteract mischarging. However, the unmodified E. coli tRNAs, specific for methionine or valine, and the undermodified yeast 
are not mischarged (318, 328). Thus, modified nucleosides do not in general act as antideterminants for tRNA identity.
Position 34 (the Wobble Position).
Two isoleucine isoacceptors, (major) and (minor), are present in E. coli. These tRNAs contain G34 and the modified nucleoside lysidine (k2C34 or L34) as wobble nucleosides, respectively. Even though the chemical structures of G34 and k2C34 are quite different, IleRS aminoacylates both species, because G34 and k2C34 have common features recognized by the IleRS (334). When k2C34 is replaced in vitro by C34, such mutated (minor) is efficiently misacylated with Met (255), since C34 in this context is a positive identity determinant for MetRS (330, 355). Taken together, these results show that the modification of C34 to k2C34 in acts as a positive identity element for IleRS and as an antideterminant toward MetRS, thereby preventing a detrimental misacylation. In addition, variants of the major , which contain A34, U34, or C34 in place of G34, are not charged with isoleucine (254). Thus, the IleRS from E. coli must recognize as identity elements structural features that are common to the chemically different nucleosides G34 and k2C34.
Treatment with cyanogen bromide, which specifically attacks and modifies sulfur nucleosides in tRNA, among them mnm5s2U34, reduces the amino acid acceptance activity of tRNAGln, tRNAGlu, and tRNALys, which normally contain mnm5s2U34 (320). Accordingly, an in vitro made tRNALys, which lacks modified nucleosides (among them mnm5s2U34), has a 140-fold lower lysine-acceptance activity than native tRNALys, and absence of mnm5s2U34 in tRNAGlu reduces the specificity constant 15-fold (361, 365). Moreover, in the GluRS- complex, GluRS forms an H bond with mnm5s2U34 (395). These results imply a direct interaction between the mnm5 or the s2 group of mnm5s2U34 and the corresponding AaRS. Structural analysis of the interaction between GlnRS or LysRS and the respective anticodon reveals that the 2-thio group of mnm5s2U34 binds in the same way as the 2-keto group of C34, which is present in one of the two glutamine isoacceptors (82, 314). The size of the pocket in which the wobble base binds to GlnRS or to LysRS can not accommodate Q34, which is present in 
and 
, which are members of the same mixed codon box as the tRNAGln and 
, respectively. Thus, Q is an antideterminant for GlnRS and LysRS. Although earlier experiments suggest that the 2-thio group is not a positive determinant for and 
in the aminoacylation reaction (6, 336), recent results using purified components suggest that the thio group of mnm5s2U34 is the identity element of in the GluRS recognition process (239). Analysis of E. coli mutants defective in the synthesis of the mnm5 side chain (mnmE) or of the s2 group (mnmA) reveals that the 2-thio group is an important identity element for , , and , whereas the mnm5 side chain is not (212).
Position 37 (3' of and Adjacent to the Anticodon).
Various chemical treatments aimed to affect the structure of ms2i6A37 of E. coli
, 
, 
, and tRNASer do not influence the aminoacylation reaction (114, 134). Also m1G37 in serovar Typhimurium 
is not required for efficient charging in vivo (297). Tyr- and Phe-specific tRNAs, having unmodified A37, i6A37, or fully modified ms2i6A37, show the same aminoacylation kinetics (128, 396). Therefore, ms2i6A37 and m1G37 are not positive identity elements for their respective AaRS's. Specific replacement in vitro of t6A37 by A37 in the native drastically reduces the Ile-accepting activity, demonstrating that t6A37 is a strong positive identity element for IleRS (262). However, unmodified 
, which normally contains the t6A derivative m6t6A37, has kinetic characteristics similar to those of native 
(162, 329). Thus, a t6A derivative is a positive identity element for IleRS but not for ThrRS.
In summary, k2C34 of and mnm5s2U34 of 
or of act as positive elements for the corresponding aminoacyl-tRNA ligases and it is the 2-thio moiety of mnm5s2U34 that is the important element in the recognition process. Modification in position 37 seems not to be important for the aminoacylation reaction except for t6A37 of tRNAIle but not for tRNAThr. In a few cases, modified nucleosides (k2C34 for MetRS, an unidentified modified nucleoside in yeast tRNAAsp for ArgRS,and Q34 for GlnRS and LysRS) act as antideterminants and thereby reduce mischarging.
Figure 2 shows that positions 34 and 37 are frequently modified and, moreover, a large variety of modified nucleosides are found in these two positions, suggesting that their presence is important for the anticodon-codon interaction. The decoding information of the tRNA may in fact extend beyond the anticodon, especially toward the 3' side of the tRNA as far as to position 44 (143, 411). There is a pattern of modification in positions 34 and 37 that is related to the coding capacity of the tRNA (Fig. 3). In the family codon boxes, containing 4-fold degenerate codons, the U derivatives are cmo5U34 or mcmo5U34. In mixed codon boxes, containing 2-fold degenerate codons, the uridine derivatives are of the xnm5U, xnm5Um, or xnm5s2U types (x being m- or cm-). Thus, no unmodified U34, except in 
(Fig. 2), is present in tRNA from E. coli and serovar Typhimurium. Moreover, no A34 is found in any tRNA. These patterns of modification are true for cytoplasmic tRNAs in most organisms (one exception is tRNA from bacteria of the genus Mycoplasma). In the same mixed-codon boxes, the pyrimidine-reading tRNAs have Q34 or GluQ. Codons starting with U are read by tRNAs having ms2i(o)6A37 in position 37 (except 
), codons starting with A are read by tRNAs having t6A derivatives, and tRNAs specific for Leu, Pro, and Arg read codons starting with C, have m1G37 (except for , which has m2A).
Function of Modified Nucleosides in Position 32.
In general, it is assumed that the tRNA anticodon loop consists of seven nucleotides. However, since a bifurcated hydrogen bond is present between bases in positions 32 and 38, the loop consists of five nucleotides and the structure is surprisingly stable (16). All four tRNA species, , 
, 
, and 
, which have s2C32, have A38; and hence an S2(s2C32)―N6(A38) bifurcated hydrogen bond is present in these tRNAs. Thus, a change from an S2(s2C32)―N6(A38) hydrogen bond to an O2(C32)―N6(A38) bond would occur in the absence of the thio group of s2C32. Although it is difficult to evaluate how such a change would influence the structure of the anticodon loop (180), chemical conversion of s2C32 to C32 in changes the structure of its anticodon loop (21). Translation of MS2 RNA in vitro with cell extracts from E. coli results in the synthesis of the viral synthase and the coat protein but also in several other polypeptides, which are the results of +1 or –1 frameshifting events (14). Whereas the addition of native (s2C32) in 15-fold excess inhibits the endogenous –1 frameshifting, the s2C32-lacking (C32) does not (21). It was speculated that the native, but not the s2C32-lacking counterpart, competes efficiently with the frameshifting tRNA at the frameshifting site (30). If so, lack of s2C32 may lower the translational efficiency of in vitro. Accordingly, lack of s2C32 in reduces the rate of translation in vivo of the CGA codon (180). A mutant (ttcA) of serovar Typhimurium lacking s2C32 grows as wild type, and in an experiment in which mutant and wild-type cells were mixed, no disadvantage for the ttcA mutant was noticed (180). However, the presence of s2C32 increases the selection rate at the ribosomal aminoacyl-tRNA site (A site) at the AGG codon but not at any of the CGN cognate codons. Therefore, even if lack of s2C32 does not influence the physiology of the bacterium in a major way as monitored by growth rate, several steps in translation are defective because of the lack of s2C32.
Function of Modified Nucleosides in Position 34.
At the time Crick presented his wobble hypothesis (80) only a few modified nucleosides had been identified. Therefore, Crick considered the coding properties of only one modified nucleoside (inosine) in the wobble position. He suggested that unmodified U34 only base pairs with A and G (Table 5), since too-short base pairs would be formed between U34 and U(III) [U34 denotes U in position 34 of tRNA and U(III) denotes the third nucleoside of the codon]. However, not long thereafter, Nishimura and collaborators showed that some modified uridine derivatives have coding capacities that extend the wobble hypothesis of Crick (reviewed in reference 265). It was shown that cmo5U34 not only reads codons ending with A and G but also codons ending with U and that mnm5s2U34 reads G(III) less well than A(III). Later, stereochemical properties of nucleosides, nucleotides, or dinucleotides modified to various extents explained these decoding capacities (5, 344, 414). Whereas the C2'-endo and C3'-endo conformations are equally stable for unmodified pU, pcmo5U almost exclusively takes the C2'-endo conformation. In contrast, pmnm5s2U exclusively adopts the C3'-endo conformation. Although the mnm5 side chain contributes to the stability of the C3'-endo form of mnm5s2U34, the major stabilizing effect is caused by the 2-thio group (316, 359). Other theoretical considerations of the function of various modified nucleosides present in the wobble position have been presented and they are summarized in Table 5. Thus, the mnm5 side chain apparently should restrict pairing with G(III) but several experiments do not support this suggestion (reviewed in reference 364). Therefore, the function of the mnm5 side chain was revised, suggesting that a protonated form of mnm5s2U34 makes an unconventional base pairing with G (364). Note that such a protonation can not be formed for the mcm5 side chain, which is found in eukaryotic tRNAs, suggesting that mcm5s2U34 should not base pair with G(III) in contrast to mnm5s2U34.
Table 5Suggested wobble rules 1966 to 2004 |
According to the revised wobble rules, cmo5U34, which is present only in tRNAs reading family codon boxes (Fig. 3), should read A, G, and U but not C (Table 5). Triplet-dependent binding and in vitro protein synthesis have shown that this is indeed the case (178, 247, 277, 319). Presence of cmo5U34 in 
is required for the binding to the ribosomal A and P sites programmed with the A-, G-, or U-ending Val codons. Moreover, cmo5U34 is required for translocation of from the A to the P site at these codons (quoted in reference 3). An unmodified 
reads UCA, barely UCU, and not at all UCC and UCG. Introduction of only mo5U34 in such an unmodifiedenhances the reading of UCU and UCG, but still no reading of UCC was observed (363). Thus, all in vitro experiments with unmodified E. coli tRNAs or ASLs support the theoretical considerations that xmo5U34 derivatives enhance the wobble of U, which only base pairs with A(III) and G(III), to base pair with U(III) but not with C(III). However, there is evidence in vivo that the cmo5U34-containing tRNA also base pairs with C(III). According to the model how modification deficiency may induce frameshifting (Fig. 13), a near-cognate tRNA may out-compete the cognate tRNA provided that the latter is defective; for example, by being hypomodified. After a three-nucleotide translocation this near-cognate tRNA resides in the P site. If the ribosome pauses at this step (e.g., caused by low concentration of the next aminoacyl-tRNA, a rare codon, or a stop codon in the A site), the near-cognate tRNA in the P site shifts into the +1 frame at low frequency. In the proline family box, three tRNAs, encoded by proK, proL, and proM genes, read the proline codons according to the theory above. The CCC codon is only read by the G34-containing proL, since the proM
, which has cmo5U34 as wobble nucleoside, should not read the CCC codon. However, a frameshifting event at a CCC codon is dependent on the presence of cmo5U34, suggesting that the reads the CCC codon (299). A strain deleted for the proL and proK genes and thereby lacking the corresponding tRNAs is viable, demonstrating that the proM with cmo5U34 as wobble nucleoside can read all four proline codons (260). Similarly, a strain having only the 
with cmo5U34 as a wobble nucleoside is also viable, demonstrating that the can read all four Ala codons (125). Thus, in vivo at least and are able to interact with C(III) contrary to the theory and to results obtained in vitro.
The revised wobble rules suggest that nucleosides with an xnm5-modification base pair with both A(III) and G(III). (The functional aspects of the xcm5 modification will not be considered further, since it is not found in bacteria.) Recent measurements of P- and A-site binding of 
having unmodified U34, or mnm5U34 reveal that the modified derivatives restore binding to both the AAG and AAA codons to a similar extent in the P and A sites (410). The crystal structure of an
bound to an AAG codon located in the ribosomal A site of the 30S subunit revealed that the mnm5U34 makes a bifurcated hydrogen bond between O2 of mnm5U34 and N1 and N2 of G(III), explaining the ability of the mnm5U34 to pair with G(III) (257). Such an mnm5U34-G(III) pairing requires the presence of t6A37. Note, however, that tRNAs specific for Gln or Glu do not have t6A in position 37, although they have mnm5s2U34 in the wobble position. The mnm5U34-G(III) base pair in is also in an unusual conformation, which is far from the geometry expected from a conventional G34-U(III) wobble pair, yet also far from the unconventional pairing between mnm5U34 and G(III) with two hydrogen bonds as proposed by Takai and Yokoyama (364).
If mnm5s2U34 had a restricting effect, one would predict that removal of either the s2 or xmn5 modifications should increase the interaction toward G(III). The ochre suppressor 
(supG or supL) reads UAA and UAG nonsense codons and has mnm5s2U34. This tRNA contains cmnm5s2U34, nm5s2U34, and s2U34 in mnmC1, mnmC2, and mnmE mutants, respectively (107, 154, 155). Each mutation reduces the reading of UAG more than the reading of UAA. Also, in triplet-dependent binding of 
the mnm5-side-chain deficiency reduces the binding to AAG more than to AAA (107). These results suggest that the modification at the 5-position promotes rather than restricts binding toward G, consistent with the formation of an mnm5U34-G(III) base pair with a bifurcated hydrogen bond (257) or with the unconventional base pair with two hydrogen bonds (364). Therefore, the primary function of the mnm5 side-chain may not be to impose the selective reading toward A, but to increase the efficiency of reading both codons and preferentially G-ending codons. Accordingly, the mnm5 side chain facilitates the reading in vivo of GAG 4-fold whereas it decreases 2-fold the reading of GAA (211).
All tRNAs reading NAA codons, as in the mixed codon boxes for His/Gln, Asn/Lys, and Asp/Glu (Fig. 3), have the unmodified anticodon sequence 5'-U34-U35-N36-3'. Because stacking interactions between uridines are poor (378), an anticodon with such a U-rich sequence should be unstable. Thiolation at the 2-position stabilizes a duplex by improving stacking interactions between uridines and by the ability of the s2 group to shift the equilibrium between the 2'-endo and 3'-endo sugar puckering to the 3'-endo conformation (2, 215, 408, 414). These considerations explain why tRNAs from all organisms (except Mycoplasma) reading the NAA codons have a thiolated uridine derivative as the wobble nucleoside (c.f. Fig 3 for E. coli and serovar Typhimurium). From the crystal structure of the bound to the A site it was predicted that the s2 modification would increase the stacking of mnm5U34 with U35 and weaken the bifurcated hydrogen bond from mnm5U to G(III) and have no influence on the conventional hydrogen bonding to A(III) (257). If so, the final outcome of the presence of both modifications would be an improved decoding of both AAA and AAG codons. The revised wobble rules suggest that the s2 group of mnm5s2U establishes an efficient base pair with A(III) and a poor base pair with G(III). Results from triplet-dependent binding and in vitro protein synthesis support this model, since mnm5s2U34 primarily recognizes A as the third letter of the codon (238, 333). Also , deficient of the s2 group, binds in vitro much better to GAG than to GAA, whereas the reverse is true for the fully modified (6). These results support the hypothesis that the s2 group of mnm5s2U34 enhances reading of A and imposes a restrictive wobbling toward G. Indeed, in vivo the s2 group enhances 4-fold the reading of GAA and has no or only a minor effect on the reading of GAG (211). Apparently, thiolation of U34 restricts wobble toward G(III) and enhances base pairing with A(III), whereas the mnm5 side chain enhances base pairing with G(III). Since the rate of translation of the GAA codon by wild-type , which contains both the mnm5 and s2 modifications, is two-fold faster than translation of the GAG codon (211), the impact of thiolation of the wobble nucleoside is epistatic over the effect mediated by the mnm5 side-chain.
The revised wobble model (Table 5) also predicts that misreading of U(III) and C(III) in mRNA should be reduced by the mnm5s2U34 modification. Only one report has been published addressing this question (158). The misreading by can be measured as an incorporation of Lys toward Asn codons in phage MS2 coat protein, since such amino-acid-substituted MS2 coat proteins have different charges and can be separated by two-dimensional gel electrophoresis (296). Measurements of misreading in the MS2 coat protein mRNA of near-cognate AAU/C Asn codons by mnm5- or s2-deficient, which reads AAA/G codons, revealed a reduced misreading by hypomodified , contrary to the expectation. Since the level of lysylated is not affected by the hypomodification, the reduced missense error cannot be caused by a low concentration of lys-. Several explanations may be considered for these surprising results. One explanation would be that if the function of these modifications is to increase the efficiency of reading of AAA and AAG, the observed effect of hypomodification should be more pronounced toward the more inefficient reading of AAG than of the efficiently read AAA codon. Accordingly, the misreading of the near-cognate Asn codons would be even less efficient and thereby less misreading would be observed (31). Alternatively, both the mnm5 and the s2 modifications of increase the order of the anticodon (359), and such an ordered anticodon loop may improve a "two out of three" decoding (218), resulting in higher misreading by the fully modified than that induced by the hypomodified tRNA (359). Finally, it has been suggested that these results need clarification with respect to possible frameshifting events that are induced by hypomodification of tRNA (3). Since amino acid starvation, for example, Asn, induces peptidyl-tRNA to detach from the P-site codon and slip either forward (+1 frameshifting) or backward (–1 frameshifting)) (19, 113), the severe asparagine starvation, which is part of the experimental design of Hagervall et al. (158), would induce frameshifting. Such translational errors were not monitored by Hagervall et al. (158), since only full-length coat protein variants were monitored. However, the extent of asparagine starvation was the same for the wild type as for the two hypomodification mutants, making a different degree of frameshifting less likely to explain the results by Hagervall et al. (158). Moreover, if the hypomodification of , as suggested by Yokoyama et al. (414), induces misreading of the asparagine codons, such hypomodified would compete better with the residual Asn-tRNAAsn in entering the A site, which would decrease frameshifting by peptidyl-tRNA. Therefore, frameshifting by poor reading of the A-site asparagine codon by hypomodified cannot explain the results by Hagervall et al. (158). The hypomodified is, however, more prone to frameshift when residing in the P site (see below), but such frameshifting is dependent on a pause induced by a shortness of the aa-tRNA reading the codon following the Lys codon. Since this aa-tRNA should be found in high concentrations as a result of the deprivation of asparagine, frameshifting by the hypomodified residing in the P site should be minimal. Although Hagervall et al. (158) did not monitor the frameshifting product(s), we find it unlikely that even a somewhat higher degree of frameshifting induced by the hypomodified would be able to mask a potentially higher misreading in such a way that it appears as such a dramatic decrease in misreading as was observed.
The Phe/Leu and Ser/Arg mixed codon boxes are decoded by tRNAs having xm5U derivatives as wobble nucleosides, but contrary to the tRNAs decoding the mixed codon boxes His/Gln, Asn/Lys, and Asp/Glu, the wobble nucleosides in the Leu and Arg tRNAs are not thiolated. However, in the Phe/Leu mixed codon box, the contains a 2'-O-ribose methylation (362) that stabilizes the C3'-endo conformation by a steric repulsion between the 2'-O-methyl group and the 3'-phosphate group in a way similar to that induced by an s2 group (193). The 
reads UUA and UUG with only a slight preference for UUA in vitro (135, 362), explaining why cells lacking an active
, which reads only the UUG codon, is viable. A mutation in gidA, which removes the cmnm5 modification of, making such cells dependent on the reading the UUG codon (259), demonstrates that the cmnm5 modification in 
is required for reading the UUG codon. It was proposed that the cmnm5 side chain in becomes protonated and thereby the cmnm5Um34 is able to efficiently base pair with G(III) besides its ability to base pair with A(III) (364). Alternatively, the cmnm5Um of pairs with G(III) by forming a bifurcated hydrogen bond to G(III) in a way similar to the mnm5U34 of 
makes with G(III) (257). Since the 2'-O- ribose methylation should drive the ribose-puckering equilibrium from C2'- toward C3'-endo conformation, thereby favoring the base pairing of Um34 with A(III), it seems that the most important function of the cmnm5 modification in is to enhance the pairing of cmnm5Um34 with G(III).
The mnm5 modification of stabilizes the C3'-endo form and this may be further stabilized by stacking with the nucleoside 3' of and next to it (C35) (316), but the protonation of the side chain should make the mnm5U34 also able to pair with G(III) (364) or the mnm5U may form a bifurcated hydrogen bond to G(III) as shown for the same side chain in (257). However, some experiments suggest that recognizes AGG much less efficiently than AGA (350), although these experiments may be misleading, since 
with C34 as wobble nucleoside is also present in the cell and this tRNA should efficiently read AGG.
An ochre suppressor derivative of contains only mnm5s2U34 in a selD mutant, whereas in the wild-type selD+ strain, up to 40% of the wobble nucleoside is converted to mnm5Se2U34 (209). The selD1 mutation imposes a 2-fold reduction of the ability of the suppressor derivative of to read UAG but does not affect the reading of UAA (209). Apparently, the presence of selenium instead of sulfur at position 2 of uridine improves the ability to wobble. This is consistent with results obtained in vitro in triplet-binding experiments (399). Transfer 
and containing an s2 group preferentially bind to A-ending codons, whereas for the Se2-containing tRNAs this preference is either reversed (tRNALys) or diminished (tRNAGlu). Therefore, replacement of sulfur by selenium in mnm5s2U34 increases the wobble interaction with G. Compared with sulfur, selenium equalizes the reading of the A- and G-ending codons.
The elongator 
is the only tRNA in E. coli and serovar Typhimurium that contains ac4C34. Upon chemical removal of the ac4 group, the tRNA misreads the isoleucine codon AUA (356) and it also more efficiently recognizes the cognate codon AUG. Note that the AUA (Ile) codon is normally read by a minor tRNAIle, which has the modified nucleoside k2C34 (L34) in the wobble position. Thus, the ac4 modification in the CAU anticodon of decreases the reading efficiency of both the complementary AUG codon and the noncomplementary AUA codon. The ability of C to read A most likely requires a specific conformation of the tRNA, since most amber suppressors, which normally have C34, are unable to read the ochre codon UAA. Thus, the function of the ac4 modification apparently is primarily to prevent misreading of AUA(Ile) and this is achieved by a slight reduction in reading the cognate AUG(Met) codon by the .
The AUA(Ile) codon is normally read by the minor species (161). This tRNA has lysidine (k2C34, also abbreviated L34) in the wobble position (256) and recognizes only AUA (161). Thus, the lysylation of C34 alters the wobble nucleoside such that it recognizes A instead of G. This is technically not a wobble, but a complete conversion of base-pairing specificity (256). Partial inactivation of TilS, which synthesizes k2C34, results in an AUA codon-reading defect (348). As expected TilS is essential for viability, since deficiency of this enzyme results in lack of a tRNA reading the AUA codon. The fidelity of AUA reading is improved by the presence of ac4C34 in , because ac4C prevents misreading of AUA by the (356). As discussed previously, k2C34 also prevents misacylation with methionine. Therefore, a single posttranscriptional modification converts both the aminoacylation and the codon specificities (for more details, see a recent review [140]).
The tgt mutant lacks Q in tRNAs specific for Tyr, His, Asn, and Asp (266). The his operon is regulated by an attenuation mechanism, which is sensitive to the charging and translational efficiency of tRNAHis (184). A reduced rate of cognate decoding of the seven His(CAU) codons in the attenuator results in derepression of the his operon. No difference in the expression of the his operon was observed in tgt+ versus tgt cells, suggesting that the Q-deficient tRNAHis is as efficient as the wild-type tRNA in translating the His codons in the leader region of the his attenuator. However, the binding efficiency of G34-
to triplet-programmed ribosomes is 2-fold lower than that of Q34-, although the preference for UAU over UAC is the same in both tRNAs (266). Thus, whereas Q34 does not influence the decoding efficiency of tRNAHis, the binding efficiency of E. coli tRNATyr to ribosomes is reduced 2-fold without change in the codon choice. This suggests that the influence of Q34 on individual tRNA species may be different and that the Q-containing tRNAs prefer the U-ending codons when decoding occurs in the A site. However, the reverse is true when the Q-containing tRNA is residing in the P site, although the influence of Q per se in the P-site decoding was not addressed (55).Translation of specific mRNAs seems to be influenced by the presence of Q, since translation of the virF mRNA in a tgt mutant of Shigella flexneri is reduced 2-fold in rich medium and almost abolished when cells are grown in minimal medium (99, 100, 101). Although the molecular mechanism of this translational defect is not established, one interpretation is that the activity of the Q34-deficient tRNAs is severely reduced in reading some codon(s) in the virF mRNA from Shigella. Consistent with this suggestion is that Q influences the codon choice and induces missense errors (24, 26, 122, 244). However, Q34 deficiency does not provoke a major change in growth rate of the tgt mutant (266).
In summary, modifications of U in position 34 may increase (cmo5U and mcmo5U) or decrease (mnm5s2U, cmnm5Um, mnm5U) the wobble capacity. Although in vitro results are consistent with the hypothesis that xmo5 modifications extend the wobble capacity to include base pairing with U, surprisingly, some in vivo results suggest that such a modified U, at least in some tRNA species, may base pair with all four major nucleosides. The hypothesis that the xnm5-modified nucleosides should base pair with A and G is supported by in vitro and in vivo experiments, and the xnm5 side chain is interacting with G via a bifurcated hydrogen bond. On the other hand, the s2 group may be the major contributing factor in the postulated restrictiveness of mnm5s2U34, although for some tRNAs (Leu and Arg) ribose methylation and/or the mnm5 side chain may induce restrictive wobbling. The acetylation of C (ac4C) prevents misreading at the expense of a reduced ability to read the cognate codon, and the lysylation of C (k2C) completely changes the coding capacity from pairing with G to pairing with A. The presence of the hypermodified Q is strongly required for translation of some specific mRNAs but does not affect translation in general. This specific requirement for Q by some mRNAs may be related to its influence on codon choice and fidelity.
Function of Modified Nucleosides in Position 37 (3' of and Next to the Anticodon).
The anticodon loop contains several conserved and semiconserved nucleotides that form an extended structural signature (17). At position 32, which is the first nucleotide in the seven-member anticodon loop, there is always a pyrimidine followed by the universal U in position 33. As stated earlier various modified nucleosides are found in position 34 but none at positions 35 and 36 in E. coli, and indeed it is a very limited number of modified nucleosides in these two positions of the anticodon considering all sequenced tRNAs. In E. coli and serovar Typhimurium unmodified A37 is found in 10 tRNA species but no unmodified G37 is present, since G37 is always modified to m1G37. In general, this is also true for all organisms, since out of more than 500 sequenced tRNAs only three have an unmodified G37 (discussed in reference 33). Of the 10 tRNA species with unmodified A37, eight have C36, that is, they read codons starting with G and therefore the base pair next to position 37 is a strong C36-G(I). Transfer RNAs reading codons starting with U or A (having A36 or U36, respectively) and making a weak A/U36-U/A(I) base pair have a hydrophobic modification (i6 derivatives) or a bulky charged derivative (t6 derivatives), respectively, in position 37. There are only two exceptions (and initiator) to this rule. Apparently, a weak A-U interaction in the first position of the codon requires a stabilizing modification [ms2i(o)6A37 or t6A37] next to it, whereas a more stable G-C interaction in the same position does not and these tRNAs have m2A37, m6A37, m1G37, or unmodified A37. There are also some conserved interactions between various nucleotides in the anticodon loop. A bifurcated hydrogen bond is present between the pyrimidine in position 32 and the base in position 38 (Y32-N38) as well as in interaction between U33 and the nucleotides in positions 35 and 36 (17). All these interactions, which are phylogenetically conserved, are building up the characteristic structure of the anticodon loop. By using as a model system the duplex formation between the anticodons of two tRNAs, it was shown that they are orders of magnitude more stable than a 3-bp double helix and that the modified nucleosides contribute with up to an order of magnitude to the stability of the complex (143). Structural analyses of anticodon stem and loops (ASLs ) of yeast and E. coli, of E. coli, or of human tRNALys3 reveal that introduction of modifications in position 37 (m1G37 in yeast tRNAPhe, i6A37 in E. coli tRNAPhe, t6A37 in the two tRNALys) negates interaction with U33 and thereby destabilizes the closed anticodon loop; in addition, introduction of these modifications orders the structure of the loop by improving the stacking of its 3' side (60, 357, 359). The t6A37 of base pairing with the AAA/G codons stabilizes the base pair between U36 and A(I) not by improving the stacking with U36 but rather by forming an interstrand stack with A(I) (257). Thus, modifications in position 37 create the open, structured loop required for efficient and correct codon binding.
Presence of t6A stabilizes the interactions between two complementary anticodons by increasing the stacking interaction and t6A-deficient E. coli or yeast tRNAArgIII binds less well to cognate polynucleotide- or triplet-programmed ribosomes (246, 389). Only one tRNA,, has m6t6A37. This tRNA has t6A37 instead of m6t6A in the tsaA1 mutant. This hypomodified tRNA reads the cognate codon ACC less efficiently than wild-type tRNA (300). Apparently, the t6 derivatives, which are present in a subset of tRNAs from all organisms, improve the efficiency of the tRNA.
The hypermodified nucleosides ms2io6A37 (serovar Typhimurium) and ms2i6A37 (E. coli) are present in tRNAs that read codons starting with U (the only exceptions are , which has A37, and 
,which has i6A37). The lack of the ms2i6 modification reduces the codon-reading efficiency of the tRNA in vitro (89, 128, 327, 383). Since these modified nucleosides are present in amber suppressor tRNAs, the efficiency of such tRNAs in vivo only differing in the degree of this modification has been determined. The influence of the codon context can also be analyzed by placing the amber codon in different contexts. Such analyses have revealed that a complete lack of the modification, as in a miaA mutant, results not only in a dramatic reduction (up to 99%) of the efficiency of the suppressor tRNA, but also induces an increased codon context sensitivity of the unmodified tRNA (37, 44, 111, 290, 383). The ms2io6 modification specifically counteracts a C on the 3' side of the codon, which seems to be particularly unfavorable (111). The major effect on the efficiency of the tRNA is imposed by the i6 group of ms2i6A37, since a miaB mutation, which results in a tRNA having i6A37 instead of ms2io6A37, has much less effect than a mutation in the miaA gene (112). This result is also consistent with results obtained in vitro (53, 128). The hydroxyl group present in serovar Typhimurium tRNA has no effect on the efficiency of a suppressor derivative of (286). Therefore, both the i6 and the ms2 groups contribute to the function of ms2io6A37, whereas the hydroxylation, which is only occurring in serovar Typhimurium, does not influence the anticodon-codon interaction. Thus, ms2i6A37 enhances the activity of the tRNA and makes it less sensitive to the codon context. The physiological consequences of ms2 i6 deficiency are a drastic reduction (up to 50%) of the growth rate and the polypeptide chain-elongation rate, resulting in a pleiotropic effect on cell physiology (90, 110, 245). Thus, ms2i6A37 has a profound influence on the physiology of the bacterial cell and plays a fundamental role in the efficiency and fidelity of translation.
Transfer RNAs from all organisms that read codons of the type CUN(Leu), CCN(Pro), and CGG (Arg) (Fig. 3) contain m1G37, the presence of which apparently is due to a convergent evolution. A strong evolutionary pressure must have been maintained against an unmodified G37, since only three of the more than 500 sequenced tRNAs contain unmodified G37. The function of m1G37 may thus be the same irrespective of the origin of the tRNA. Although an undetectable level of m1G37 or lack of it in serovar Typhimurium, E. coli, or yeast results in a severe growth defect, the mutants are viable (33, 34, 287). The presence of m1G37 in tRNA from Streptococcus pneumoniae, however, is essential for growth (276). Thus, the presence of m1G37 in tRNA exerts a strong beneficial effect on the function of tRNA. This also explains the convergent evolution resulting in the presence of this modified nucleoside in the same subpopulation of tRNA from all organisms (29, 64). Hence, an effect by m1G37 on the efficiency of tRNA in the cognate interaction is suggested and, indeed, deficiency of m1G37 results in a slower polypeptide elongation rate (226). Since m1G37 deficiency of 
and 
(the identity of the nucleoside in position 34 of has not been determined, but it is a U or a U derivative based on DNA sequence) does not influence reading of the cognate codons CUA and CUC, the reduced polypeptide elongation rate is caused by either the m1G37-deficient
, the three proline tRNAs, 
, or several or all of them, suggesting the functional impact of m1G37 in a tRNA-dependent manner (226). The translation elongation cycle consists of several steps, of which the first is the selection of the ternary complex, consisting of aa-tRNA*GTP*EF-Tu. The presence of m1G37 strongly affects the rate with which three proline and one arginine tRNA species enters the ribosomal A site as a ternary complex, whereas the presence of m1G37 does not influence the rate with which leucine tRNAs are accepted. Thus, consistent with the results obtained by measuring the elongation rate of protein synthesis, m1G37 influences the binding of the ternary complex to the cognate codon in a tRNA-dependent manner (227).
In summary, position 37 is frequently modified and modifications create the open structured loop required for efficient and correct codon binding. The hypermodified nucleoside ms2i(o)6A37 improves the efficiency of the tRNA up to 10-fold and makes the tRNA less sensitive to the codon context. All organisms have m1G37 in tRNAs reading codons of the type CUN(Leu), CCN(Pro), and CGG(Arg) and its presence is apparently due to convergent evolution. Presence of m1G37 prevents frameshifting and dramatically improves the growth; in fact, in some bacteria it is essential for viability. Presence of this modified nucleoside improves the rate by which the ternary complex binds to the cognate codon in a tRNA-dependent manner. Modifications at position 37 have a profound influence on cell physiology and play a fundamental role in the efficiency of translation.
Function of Ψ in the Anticodon Region.
The TruA (HisT) enzyme synthesizes Ψ in positions 38 (anticodon loop), 39 and 40 (anticodon stem) in 17 tRNA species, among them tRNAHis (351). Mutants defective in the synthesis of these Ψs have a derepressed his operon, since the histidine leader mRNA contains seven consecutive histidine codons that are read inefficiently by tRNAHis lacking Ψ38,39 (184). The growth rate of the truA mutant and its polypeptide chain-elongation rate are reduced by 30% in glucose minimal medium (282). Ψ deficiency in position 38 in an amber suppressor derivative of tRNAGln affects the activity of the tRNA more than what Ψ deficiency in position 39 in an amber suppressor derivative of tRNATyr does (43, 156). Moreover, Ψ38 deficiency in tRNALeu reduces the aa-tRNA selection step more than Ψ39 deficiency of tRNAPro does (227). Also, misincorporation by a near-cognate tRNA is correlated to Ψ modification at position 38 (284). Thus, Ψ in the anticodon region has a strong impact on the activity of the tRNA. Presence of Ψ in the stem increases the activity, even though this effect is less than the one imposed by Ψ in the loop (position 38). Presence of Ψ in position 39 stabilizes the base pair A31-Ψ39 in the stem and promotes stacking interaction on the 3' side of the loop in E. coli and in an anticodon stem and loop (ASL) of E. coli
(84, 103). Transfer RNAs having Ψ38 belong to a small family of tRNAs (7% of total) that has a single hydrogen bond between a U derivative (U/Um/Ψ) in position 32 and a U derivative (U38/Ψ38) in position 38 (16). Even though the structure of the Um32-Ψ38 is similar to that of U32-U38, lack of Ψ38 still strongly influences the activity of several tRNAs as described above, perhaps because the Ψ modification creates an extra imino hydrogen at the nitrogen that is engaged in the glycosyl bond of the unmodified U (see Fig. 1). This imino group has the potential to form a hydrogen-water bridge to an oxygen of the 5'-phosphate of Ψ, resulting in a more stable structure (84). Therefore, the Ψ38 modification may exert its strong influence on the activity of the tRNA by creating a water or a magnesium bridge within the anticodon loop. Such a bridge may stabilize and adapt the anticodon to interact more efficiently with the mRNA.
In summary, the anticodon loop may be stabilized by a hydrogen-water bridge between the imino group (see Fig. 1) and one of the oxygens of the 5'-phosphate of Ψ. Such a stabilized anticodon loop improves the efficiency of cognate codon interactions as revealed by a faster polypeptide chain-elongation rate, faster growth rate, and a more efficient tRNAHis as measured by derepression of the his operon in a mutant lacking Ψ38,39. The influence of Ψ38, which is present in the loop, on the activity of tRNA is more pronounced than Ψ39, which is present in the anticodon stem.
Function of Modified Nucleosides outside the Anticodon Region.
Little knowledge of the function of modified nucleosides outside the anticodon region has been obtained. The reason may be that their effects on the anticodon-codon interaction are minor and that the methods used are too insensitive to reveal certain functions of these modified nucleosides. A mutant, nuvA, lacking s4U in the tRNA grows normally (368), but this nucleoside is directly involved in photoprotection. Only a minor reduction (4%) in growth rate is observed for a mutant (trmA) completely lacking m5U54 in its tRNA; still such undermodified tRNA has a lower stability, increased error level, and decreased A-site binding in vitro (83, 199). Although the presence of m5U54 in the tRNA is not essential for cellular growth, the structural gene (trmA) for the tRNA(m5U54)methyltransferase is (288). It was suggested, therefore, that the TrmA peptide has two functions—one catalyzing the formation of m5U54 in tRNA and the other involved in an unknown but essential function. The unknown function may be related to the observation that 50% of tRNA(m5U54)methyltransferase is covalently bound to 16S rRNA and undermodified tRNA (150).
Absence of the TruB protein, but not its catalytic product Ψ55 in tRNA, results in a disadvantage in a mixed population experiment (153, 202). This may indicate that the TruB protein has another function than to synthesize Ψ55 and it was suggested that TruB may also act as an RNA chaperon (153). Even though Ψ55 deficiency does not influence the growth rate, suggesting an efficient translation, lack of Ψ55 reduces the efficiency of reading the Arg codon CGA but not the reading of codon CGU (380). Moreover, Ψ55 contributes to thermal stress tolerance (202). Pseudouridine 55 and Gm18 interact in the tRNA tertiary structure by hydrogen bonding, thereby stabilizing the L-shaped structure of tRNA (217, 302), and m5U54 is close in space to the Ψ55-Gm18 interaction. In one genetic background, deletion of truB in conjunction with an insertion in trmA enhances the effect induced by a mutation only in truB (202), whereas this does not occur in conjunction with a point mutation in trmA and in another genetic background (380). Lack of Gm18 reduces the growth rate in a medium-dependent manner especially at high temperature (380). In conjunction with lack of Ψ55 this reduction in growth rate is enhanced, supporting the view that the tertiary interaction between Gm18 and Ψ55 is pivotal for the stability of the tRNA.
In summary, analysis in vivo of the influence of modifications (s4U8, Gm18, m5U54, Ψ55) located in the body of the tRNA has revealed minor effects on growth rate and, in a few cases, a minor effect on reading specific codons. The modifications of Gm18 and Ψ55 are involved in stabilizing the 3D structure of tRNA.
tRNA Modification and Translational Fidelity.
Missense Errors. It was suggested that the presence of ms2i6A37 in a tRNA making an A36-U base pair with the first nucleoside (U) in the codon would stabilize this base pairing and inhibit the wobble capacity and thus reduce a fatal first-position misreading (96, 186, 264, 389). Indeed, the presence of the ms2i6 modification reduces in vitro a first position error of reading the leucine codon CUU (255, 396). However, first-position miscoding of the arginine codon CGU by 
, which has ms2i6A37 and should read UGU/C codons, is not influenced by the presence of ms2i6A37 (44). Thus, whereas ms2i6A37 prevents first-position errors by 
, it has no effect on the same kind of errors by 
. On the other hand, third-position errors are increased by the presence of the modification both in vivo (37, 44, 290, 383) and in vitro (89), most likely by decreased proofreading (37, 89). Thus, when a mismatch occurs in the wobble position, the presence of the ms2i6 modification increases such near-cognate misreading by decreased proofreading, whereas it is neutral or decreases the first-position misreading by noncognate tRNAs.
Frameshift Errors. Since many amino acids can be substituted without influencing the stability or the activity of the protein, most missense errors are not harmful to a protein. In sharp contrast to the missense errors, frameshift errors are usually detrimental to the synthesis and the activity of a protein, since after such an event the amino acid sequence is completely different. Moreover, most often the ribosome encounters a stop codon in the +1 or –1 reading frame and releases a truncated protein (+1 or –1 frameshift indicates that the ribosome moves one base forward in the 3' direction or one base backward in the 5' direction, respectively). Accordingly, the frequency of frameshift errors is at least one order of magnitude lower than the frequency of missense errors (216). There are numerous examples of how peptidyl-tRNA slippage is induced when the ribosome pauses at the A site (reviewed in reference 113). We have presented a model (Fig. 13) (381) describing how frameshifting may be promoted by hypomodified tRNA, and the model has features in common with other frameshifting models (19, 160, 173, 354, 388).
Hypomodified tRNA may affect reading-frame maintenance either by altering the rate by which the ternary complex, which consists of aa-tRNA-GTP-EF-Tu, binds to the ribosome or by altering the dissociation of the bound tRNA in the A site. (Below we will specify only the aa-tRNA in the ternary complex when discussing codon binding in the ribosomal A site.) The binding of the cognate aa-tRNA to the in-frame codon in the A site occurs in competition with near-cognate and noncognate tRNAs and also with those aa-tRNAs that bind out of frame. Figure 13 shows our model predicting how a hypomodified tRNA will induce frameshifting. According to this model the frameshifting event always occurs by peptidyl-tRNA slippage, although the peptidyl-tRNA is different in each case. In the first case (Fig. 13A) a hypomodified cognate tRNA has a reduced affinity for the codon in the ribosomal A-site allowing a wild-type near-cognate tRNA to outcompete it and be accepted into the A site (A-site effect by hypomodified tRNA). After a normal three-nucleotide translocation, this near-cognate peptidyl-tRNA might not interact optimally with the surroundings in the ribosomal P site. Such nonoptimal P-site interaction reduces the efficiency of in-frame binding and results in +1 frameshift errors (25, 160, 381). These errors are thought to result from slippage of peptidyl-tRNA before the reqruitment of the next aa-tRNA into the A site. Although in principle the slippage may occur in both the +1 and –1 direction, the –1 direction apparently is not frequent (see below). In the second case (Fig. 13B) the hypomodification causes a slow entry of the hypomodified cognate tRNA into the A site and induces a pause that allows the wild-type cognate peptidyl-tRNA to slip into the +1 frame (A-site effect by hypomodifed tRNA). Alternatively (Fig. 13C), the defect of the hypomodified cognate tRNA may not be in the A-site selection step but rather the undermodification disrupts the interaction of the tRNA in the P site after the translocation step. Modification deficiency makes the anticodon-codon interaction in the P site less optimal, similar to the interaction by a near-cognate tRNA (Fig. 13A), resulting in an increased frequency of frameshifting (P-site effect by undermodified tRNA). Of course, undermodfied tRNAs may cause frameshifting by mediating both A- and P-site effects.
Based on this model several mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB, and miaC] defective in the presence of a specific tRNA modification were tested for the ability to maintain the reading frame. Specific assays for either an A-site or a P-site effect by the hypomodified tRNA were used to study the influence of specific modified nucleosides in tRNA (381). In general, if an A-site effect is observed, a P-site effect is also observed by the same tRNA (e.g., tRNALys and mnm5s2U34; tRNATyr and ms2io6A37; tRNAPro and m1G37; tRNAArg and m1G37). In two cases (tRNAPhe deficient in ms2io6A37 and tRNAGln deficient in mnm5s2U34), no A-site effect occurs although there is a strong P-site effect. Thus, several modified nucleosides improve reading-frame maintenance by promoting efficient A-site selection, preventing peptidyl-tRNA slippage, or both. This function of modified nucleosides is codon and tRNA dependent.
A model, different from the one presented here, was proposed earlier, explaining how m1G37 deficiency suppresses +1 frameshift mutations (36). According to this model absence of m1G37 creates a possible four-base anticodon allowing the unmodified G37 to make a Watson-Crick base pair with an extra C at the frameshifting site. Following such a four-base interaction in the A site, a four-base translocation positions the ribosome in the correct reading frame, explaining how lack of m1G37 suppresses a +1 frameshift mutation. This hypothesis is similar to the model generally accepted at that time ("four-base translocation model"), which explains how a classical frameshift suppressor tRNA with an extra base in the anticodon loop suppresses +1 frameshift mutations. Two of these classical frameshift suppressors, sufB2 and sufA6, are derivatives of proL and proK
, respectively, which both contain m1G37. According to the "four-base-translocation" model it was thought that the extra base in the anticodon loop created a four-base anticodon instead of the normal three-base anticodon. However, this is not the case, since m1G37, which blocks base pairing with C, is present in the sufB2 and sufA6 tRNAPro's in a position that results in a three-base anticodon bordered by U33 on the 5' side and by m1G37 on the 3'-side (299). Moreover, the frameshift event occurs in the P site (298). Thus, the four-base translocation model is invalidated for these classical frameshift suppressors (299) and it is unlikely that m1G37 prevents frameshifting by such mechanism but rather it prevents frameshifting according to the model presented in Fig. 13.
In principle, according to the model presented in Fig. 13, the peptidyl tRNA may also slip in the –1 direction. However, the above-mentioned mutants defective in tRNA modification that induce +1 frameshift, rarely induce –1 frameshift (382). In one case the presence of the mnm5 modification of mnm5s2U34 in tRNALysincreases the frequency of –1 frameshifting. Similarly, presence of either mnm5 or the s2 modifications increases –1 frameshifting at AAA, GAA, or UAA codons (228), whereas the mnm5 modification but not the s2 modificaiton of mnm5s2U34 in decreases frameshifting at the slippery site U-UUA-AAA (49). Q34 in orhas no influence on –1 frameshifting (49, 241). Thus, contrary to the pivotal role of many modified nucleosides in preventing +1 frameshift errors, the presence of modified nucleosides does not or only sligthly influences –1 frameshifting, suggesting that the mechanism by which +1 and –1 frameshift errors are induced may not be the same (382).
In summary, presence of ms2i(o)6A37 increases third-position errors but decreases first-position errors. Although many modified nucleosides have vastly different structures, are present in different tRNAs, and in different positions of the tRNA, many (m1G37, ms2i(o)6A37, mnm5s2U34, Q34, Ψ38,39) have a common function, they improve the reading-frame maintenance.
Deficiency of methionine, cysteine, or threonine results in tRNAs lacking methylated, thiolated, or threonylated nucleosides, respectively. Metabolic limitations such as starvation for leucine, histidine, arginine, or phosphate or the addition of the protein synthesis inhibitors, puromycin or chloramphenicol, result in the accumulation of uniquely undermodified tRNAs (204, 205, 206). Unbalanced metabolism, as such, is probably not the primary reason for the appearance of undermodified tRNA, since amino acid limitation during balanced growth also induces the appearance of undermodified tRNA (366). Whereas the level of mnm5s2U34 in E. coli is invariant with growth rate, the level of s4U8 in some but not all tRNAs decreases with increasing growth rate (109). Furthermore, the formation of Q34 and ms2i6A37 is growth phase dependent (20, 338). Thus, at least a few tRNA modifications seem to be dependent on the growth rate, the growth phase of the bacteria, or on other physiological stress conditions. Therefore, it is likely that all tRNAs are not always fully modified and that the proportion of hypomodified tRNAs in the population changes in a manner dependent on the environment.
Three aromatic amino acids (Tyr, Trp, and Phe) and four vitamins (ubiquinone, folate, menaquinone, and enterochelin) all originate from the same precursor, chorismic acid. The four vitamins are pivotal in the aerobic (ubiquinone) and anaerobic (menaquinone) electron transport, iron uptake (enterochelin), and C1-metabolism (folate). The synthesis of the ms2 group of ms2io6A37 (138, 311, 390) and the conversion of GTP to Que1 requires iron (200). Thus, the level of enterochelin and consequently iron transport determines the level of these two modified nucleosides in tRNA. The deficiency of the ms2 group of the ms2io6A37 modification stimulates the transport of aromatic amino acids and lack of the i(o)6 group (as in miaA) increases under certain stress conditions the frequency of specific mutations (GC to TA transversions) (52, 76, 77). Therefore, undermodification of ms2io6A37 and Q34 may be part of an important adaptive mechanism for bacteria growing at iron-limited conditions. Indeed, it has been suggested that such an adaptation is important for the pathogenicity of bacteria (139). An early step in the synthesis of cmo5U34/mcmo5U34 requires chorismic acid or a metabolite in an hitherto unknown biosynthetic branch from chorismic acid (28, 157). Thus, the level of chorismic acid is critical for the synthesis of several modified nucleosides (ms2io6A37, cmo5U, mcmo5U, Q). The degree of tRNA modification set by the level of chorismic acid may therefore regulate several parts of intermediary metabolism in a manner similar to how ms2io6A37 deficiency affects the transport of aromatic amino acids and the frequency of mutations.
Unlike most modified nucleosides, the thiolated derivatives, like s4U, have spectra that extend into the near-UV light (300 to 400 nm; λmax of s4U is 334 nm). These wavelengths are part of the sunlight and induce growth delay and killing of bacteria. When bacteria are illuminated by near-UV light, the cessation of growth occurs well before cell death (181) and this growth delay requires the presence of s4U8 in tRNA (210, 303, 368, 369). Upon irradiation either in vivo or in vitro, s4U8 is cross-linked to the nearby nucleoside C13 (116, 367) and such cross-linked tRNA triggers the accumulation of ppGpp, which inhibits cell growth by its effect on rRNA synthesis (303). Moreover, s4U8-C13 cross-linking mediates the induction of some oxidative stress proteins, ppGpp-inducible proteins, and the dinucleotide ApppGpp (210). Thus, s4U8 acts as a sensor of near-UV irradiation by mediating, through the synthesis of ppGpp and ApppGpp, the induction of specific proteins as a cellular response to this stress (210). Prior exposure of cells to near-UV light (300 to 400 nm) antagonizes the mutagenic effect of UV light at 254 nm and inhibits subsequent induction of the SOS response by irradiation with UV light (reviewed in reference 115). The SOS response inhibits cell division and induces several DNA-repair systems. This photoprotection by near-UV light requires the presence of s4U in tRNA (62, 115). Although s4U8 is the most prevalent thiolated nucleoside in tRNA, several others also exist (e.g., mnm5s2U34). Those thiolated nucleosides can also be part of the sensing mechanism for near-UV light, since ppGpp accumulates in a mutant lacking s4U8 although not to the same extent as in a wild-type strain (369). Taken together, the results strongly support the hypothesis that s4U8 in tRNA and perhaps some other thiolated nucleosides act as sensors for near-UV light and mediate the cellular responses to protect the cell from this type of stress.
Growth under oxygen limitation affects the synthesis of at least two modified nucleosides, Q and ms2io6A. The conversion of oQ (epoxy-Q) to Q requires vitamin B12, which is only synthesized under anaerobic conditions (123, 182). Consequently, tRNA from bacteria, grown aerobically in a medium lacking vitamin B12, has oQ34 in place of Q34. The hydroxylation of ms2i6A37 requires molecular oxygen, and accordingly ms2io6A37 is only synthesized under aerobic conditions (51). Thus, when bacteria are shifted between growth conditions, which differ in the presence or absence of oxygen, changes in the modification of tRNA occur. Such changes may sense aerobiosis in serovar Typhimurium (51).
A mutation in truA of E. coli or serovar Typhimurium induces pleiotropic effects of which many, but not all, can be explained by its influence on tRNA-mediated transcriptional attenuation (45, 47, 79, 377). One such pleiotropic effect not easily reconciled with a tRNA-mediated attenuation mechanism is how Ψ deficiency influences the synthesis of ppGpp during histidine starvation (349). Starvation of a stringent (relA+) truA mutant for histidine does not provoke ppGpp synthesis. In contrast, in the same truA mutant, synthesis of ppGpp occurs upon starvation for serine, threonine, or arginine whose cognate tRNAs do not contain Ψ38, 39, or 40. This anomaly may be because Ψ-deficient uncharged tRNAHis, which normally contains Ψ38 and Ψ39, can either not bind to the ribosomal A site, a prerequisite for the relA+-mediated ppGpp synthesis, or if bound is unable to activate the RelA-dependent synthesis of ppGpp. Nevertheless, such histidine starvation of a truA mutant leads to an abrupt cessation of stable RNA synthesis in a relA+ strain even if ppGpp is not synthesized. Thus, histidine starvation of a truA mutant provokes stringent response without ppGpp accumulation (349). However, starvation of a relA+, truA mutant for lysine, for which cognate tRNA contains Ψ39, results in accumulation of ppGpp (261). Apparently, presence and absence of Ψ38, which is normally present in tRNAHis, determines whether synthesis of ppGpp occurs and why lack of Ψ38 in makes this tRNA unable to signal RelA to make ppGpp. How synthesis of rRNA is inhibited is not clear. The presence of Ψ38 influences the activity of suppressor tRNAs (43, 156) and the degree of misreading (284) more than the presence of Ψ39. Therefore, these observations suggest that Ψ located in the anticodon loop (position 38) influences the activity of the tRNA in the decoding process more than Ψ located in the anticodon stem (positions 39 and 40). A mutation in the truA gene also reduces the capacity to derepress the ilv and leu operons either by a mechanism which bypasses the attenuation control or by altering it (48).
As discussed above, the level of tRNA modification is sensitive to various metabolic stress conditions and developmental stages. Thus, the degree of tRNA modification may act as a regulatory device by influencing translation of specific mRNAs as a response to some environmental changes. However, a meaningful regulatory device must also show some specificity; that is, it should target a specific part of the metabolism and affect the translation of some specific mRNA(s) more than translation in general. There are several ways one may envision how hypomodification may implement the required specificity (35). First, the deficiency of a specific modified nucleoside may occur in a tRNA-dependent manner. Second, the functional impact by such hypomodification may be tRNA dependent. Third, hypomodification may exert its specific effect by its influence on the sensitivity to codon context, on codon choice by the tRNA, and on which site frameshifting occurs. Finally, the presence of some modified nucleosides is important for the recognition of aminoacyl-tRNAs and hypomodification may change the charging level of a tRNA which in turn influences the efficiency of translation.
Although the regulatory effect of hypomodification may occur at several places in an mRNA (presence of specific structures, sequences prone to frameshifting, etc.), the 5' end seems to be especially sensitive to small translational aberrations. Ribosomes stalled early on an mRNA due to modification deficiency will indirectly influence translation initiation of that mRNA, resulting in a lower degree of translation. There is a preferential usage of rare codons within the 25 first codons of mRNAs. This codon bias early in the mRNA is critical for the efficiency of translation (65). Therefore, some mRNAs may have sites in the beginning that are sensitive to the modification status of the tRNA reading these sites. There are a few examples of how a specific hypomodification influences the translation of a certain mRNA more than translation in general.
The pathogen S. flexneri shares extensive similarities to E. coli aside from the presence of the virulence plasmid, which is required for its pathogenicity. Encoded from the virulence plasmid is the regulator protein VirF, which belongs to the araC protein family and its activity is required for the virulence of the bacterium. The VirF protein positively regulates the expression of the transcriptional activator VirG, which is required for the intracellular propagation of the bacterium. VirF also positively regulates another transcriptional activator VirB, which in turn activates the expression of the ipa, mxi, and spa virulence operons. These operons encode the invasins IpaBCD and the Mxi-Spa type III secretion apparatus. Thus, VirF seems to be the key regulator protein in the network that regulates the virulence of Shigella. A tgt mutant of Shigella lacks Q34 in its tRNA and grows similarly to wild type, indicating that most mRNAs are efficiently translated independently of the presence of Q. Thus, as for E. coli, lack of Q in Shigella has only minor effects on the growth and physiology of the bacterium. However, in a tgt mutant grown in rich medium the virulence, as judged by the expression of hemolytic activity, which is a measurement of the production of Ipa proteins, is decreased by 50%. Concomitantly, the level of VirF is reduced 2-fold in the tgt mutant, although the level of virF mRNA is the same as in wild type (101). The expression of VirF and thereby the expression of the other downstream virulence genes are completely abolished in a tgt mutant grown in minimal medium although the level of virF mRNA is the same as in the wild type, which in wild type expresses full virulent phenotype (100). Clearly, translation of virF mRNA is sensitive to the presence of Q34 in tRNA and, since VirF is a key regulator in the cascade inducing virulence gene expression, the result is a reduced virulence phenotype of the bacterium. Also, lack of ms2i6A37 in tRNA results in a poor translation of the virF mRNA and thereby an avirulent phenotype (102). Translational regulation of protein synthesis from mRNAs, whose products are important for virulence of a bacterium have also been established for two other pathogens. A mutation in gidA, which mediates mnm5 deficiency of the wobble nucleoside mnm5s2U34, reduces translation of act mRNA, which codes for a potent virulence factor of Aeromonas hydrophila, resulting in a reduced virulence of the bacterium (337). Similarly, a mutation in gidA of Pseudomonas syringae reduces the translation of salA mRNA, which codes for a protein in a two-component system important for the establishment of virulence of this bacterium (203). Thus, in at least three pathogens the presence of a specific modified nucleoside in the tRNA is pivotal for the virulence of the bacteria and the explanation is poor translation of a specific mRNA, whose product is pivotal for virulence gene expression.
The synthesis of thiamine and purines in serovar Typhimurium shares a common intermediate, 4-aminoimidazole ribotide (AIR). Deletion of the purF gene, which encodes the first enzyme in the purine pathway, blocks the synthesis of both purines and thiamine, explaining why a purF mutant requires both when grown on glucose. However, under certain conditions such as growth on other carbon sources or anaerobiosis, a purF mutant does not require thiamine (93, 95). The thiamine-independent growth under these conditions is due to the activation of the alternative pyrimidine biosynthetic pathway (94). This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the common intermediate AIR. Mutations in the trmD gene, which result in m1G37 deficiency, also activate the alternative pyrimidine biosynthetic pathway, inducing a thiamine-independent growth on glucose (34). This trmD-mediated thiamine-independent phenotype is specific for lack of m1G37, since many other mutations inducing hypomodification of tRNA or other translational aberrations do not induce this phenotype. It was suggested that a slow decoding event at a codon(s) early in an unidentified mRNA(s) read by a tRNA(s) normally containing m1G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the alternative pyrimidine biosynthetic pathway.
Lack of Ψ in the anticodon region, as in a truA mutant, reduces the growth rate in minimal medium with ammonium as nitrogen source and glucose as carbon source. However, on some nitrogen sources (Arg or Pro) other than ammonium, truA mutants grow faster than wild type (312). When grown on arginine as nitrogen source, the activity of the ammonium-assimilatory enzyme glutamate synthase is lower, but the activity of the glutamate dehydrogenase is higher in the truA mutant than in the congenic wild-type strain. Furthermore, the transport of arginine is elevated in the truA strain, which partly explains the more rapid growth on glucose-arginine medium by the mutant. Similarly, derepression of Leu transport is reduced in a truA mutant (301). Although the molecular mechanism for these phenomenons has not been established, the results suggest that lack of Ψ in the anticodon stem and loop influences the efficiency of translation of some specific mRNAs.
The zwf and pgl gene products convert glucose 6-phosphate to 6-phosphogluconate, which in turn is converted to pentose 5-phosphate by the gnd gene product. In a miaA mutant, which lacks ms2i6A37 (E. coli) or ms2io6A37 (serovar Typhimurium) in its tRNAs, the specific activity of Gnd is twice that in miaA+ cells. This effect is specific for the gnd gene expression, since a miaA mutation does not influence the expression of the zwf gene (185). Moreover, a mutation in miaA or truA increases the oxidation of various carbon compounds (226, 375). Thus, although the mechanism(s) is not known, the level of tRNA modification influences specifically the level of enzymes in central metabolic pathways, thereby exerting a regulatory role in cellular metabolism.
In summary, unbalanced metabolism may induce hypomodification of tRNA, which in turn may influence the translation of some mRNAs. If this mRNA codes for a regulatory protein belonging to a regulatory network, as in the case of Shigella, it will have a strong influence on the expression of genes in this network. Perhaps mRNAs encoding regulatory proteins, which are normally present in low amounts in the cell, are especially sensitive to the modification status of the tRNA necessary for their translation. Similarly, enzymes involved in the synthesis of vitamins may also be found in low amounts in the cell and their syntheses may also be sensitive to hypomodification of tRNA. These mRNAs may therefore be the targets that link metabolism to translation and constitute the regulatory targets upon which the hypomodified tRNAs act.
This work was supported by grants from the Swedish Cancer Foundation (project 680) and the Swedish Science Research Council (project BU-2930).
We are grateful for the critical reading of the manuscript by A. Byström, P. Chen, R. Leipuviene, H. Lundgren, K. Nilsson, J. Näsvall, and M. Pollard. We thank R. Leipuviene, K. Nilsson, and J. Näsvall for help in drawing some of the figures (Fig. 1, 8, 9, 10, 11, and 12) and G. Jäger and K. Nilsson for their help in compiling genetic data of tRNA modification as shown in Tables 1 to 4.
References
1. Abrell, J. W., E. E. Kaufman, and M. N. Lipsett. 1971. The biosynthesis of 4-thiouridylate. Separation and purification of two enzymes in the transfer ribonucleic acid-sulfurtransferase system. J. Biol. Chem. 246:294–301. [PubMed]
2. Agris, P. F. 1991. Wobble position modified nucleosides evolved to select transfer RNA codon recognition: a modified-wobble hypothesis. Biochimie 73:1345–1349. [PubMed] [CrossRef]
3. Agris, P. F. 2004. Decoding the genome: a modified view. Nucleic Acids Res. 32:223–238. [PubMed] [CrossRef]
4. Agris, P. F., D. J. Armstrong, K. P. Schäfer, and D. Söll. 1975. Maturation of a hypermodified nucleoside in transfer RNA. Nucleic Acids Res. 2:691–698. [PubMed] [CrossRef]
5. Agris, P. F., H. Sierzputowska-Gracz, W. Smith, A. Malkiewicz, E. Sochacka, and B. Nawrot. 1992. Thiolation of uridine carbon-2 restricts the motional dynamics of the transfer RNA wobble position nucleoside. J. Am. Chem. Soc. 114:2652–2656. [CrossRef]
6. Agris, P. F., D. Söll, and T. Seno. 1973. Biological function of 2-thiouridine in Escherichia coli glutamic acid transfer ribonucleic acid. Biochemistry 12:4331–4337. [PubMed] [CrossRef]
7. Ahn, H. J., H. W. Kim, H. J. Yoon, B. I. Lee, S. W. Suh, and J. K. Yang. 2003. Crystal structure of tRNA(m1G37)methyltransferase: insights into tRNA recognition. EMBO J. 22:2593–2603. [PubMed] [CrossRef]
8. Ajitkumar, P., and J. D. Cherayil. 1988. Thionucleosides in transfer ribonucleic acid: diversity, structure, biosynthesis, and function. Microbiol. Rev. 52:103–113. [PubMed]
9. Anantharaman, V., E. V. Koonin, and L. Aravind. 2001. TRAM, a predicted RNA-binding domain, common to tRNA uracil methylation and adenine thiolation enzymes. FEMS Microbiol. Lett. 197:215–221. [PubMed] [CrossRef]
10. Anantharaman, V., E. V. Koonin, and L. Aravind. 2002. SPOUT: a class of methyltransferases that includes spoU and trmD RNA methylase superfamilies, and novel superfamilies of predicted prokaryotic RNA methylases. J. Mol. Microbiol. Biotechnol. 4:71–75. [PubMed]
11. Arena, F., G. Ciliberto, S. Ciampi, and R. Cortese. 1978. Purification of pseudouridylate synthetase I from Salmonella typhimurium. Nucleic Acids Res. 5:4523–4536. [PubMed] [CrossRef]
12. Arps, P. J., C. C. Marvel, B. C. Rubin, D. A. Tolan, E. E. Penhoet, and M. E. Winkler. 1985. Structural features of the hisT operon of Escherichia coli K-12. Nucleic Acids Res. 13:5297–5315. [PubMed] [CrossRef]
13. Arps, P. J., and M. E. Winkler. 1987. Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette. J. Bacteriol. 169:1061–1070. [PubMed]
14. Atkins, J. F., R. F. Gesteland, B. R. Reid, and C. W. Anderson. 1979. Normal tRNAs promote ribosomal frameshifting. Cell 18:1119–1131. [PubMed] [CrossRef]
15. Auffinger P., and E. Westhof. 1998. Location and distribution of modified nucleotides in tRNA, p. 569–576. In H. Grosjean and R. Benne (ed.), Modification and Editing of RNA. ASM Press, Washington, D.C.
16. Auffinger, P., and E. Westhof. 1999. Singly and bifurcated hydrogen-bonded base-pairs in tRNA anticodon hairpins and ribozymes. J. Mol. Biol. 292:467–483. [PubMed] [CrossRef]
17. Auffinger, P., and E. Westhof. 2001. An extended structural signature for the tRNA anticodon loop. RNA 7:334–341. [PubMed] [CrossRef]
18. Auxilien, S., P. F. Crain, R. W. Trewyn, and H. Grosjean. 1996. Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA. J. Mol. Biol. 262:437–458. [PubMed] [CrossRef]
19. Barak, Z., D. Lindsley, and J. Gallant. 1996. On the mechanism of leftward frameshifting at several hungry codons. J. Mol. Biol. 256:676–684. [PubMed] [CrossRef]
20. Bartz, J., D. Söll, W. J. Burrows, and F. Skoog. 1970. Identification of the cytokinin-active ribonucleosides in pure Escherichia coli tRNA species. Proc. Natl. Acad. Sci. USA 67:1448–1453. [PubMed] [CrossRef]
21. Baumann, U., W. Fischer, and M. Sprinzl. 1985. Analysis of modification-dependent structural alterations in the anticodon loop of Escherichia coli tRNAArg and their effects on the translation of MS2 RNA. Eur. J. Biochem. 152:645–649. [PubMed] [CrossRef]
22. Beck, C. F., and R. A. Warren. 1988. Divergent promoters, a common form of gene organization. Microbiol. Rev. 52:318–326. [PubMed]
23. Becker, H. F., Y. Motorin, M. Sissler, C. Florentz, and H. Grosjean. 1997. Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T Psi-loop of yeast tRNAs. J. Mol. Biol. 274:505–518. [PubMed] [CrossRef]
24. Beier, H., M. Barciszewska, G. Krupp, R. Mitnacht, and H. J. Gross. 1984. UAG readthrough during TMV RNA translation: isolation and sequence of two tRNAsTyr with suppressor activity from tobacco plants. EMBO J. 3:351–356. [PubMed]
25. Belcourt, M. F., and P. J. Farabaugh. 1990. Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site. Cell 62:339–352. [PubMed] [CrossRef]
26. Bienz, M., and E. Kubli. 1981. Wild-type tRNATyrG reads the TMV RNA stop codon, but Q base-modified tRNATyrQ does not. Nature 294:188–190. [CrossRef]
27. Bishop, A. C., J. M. Xu, R. C. Johnson, P. Schimmel, and V. deCrecyLagard. 2002. Identification of the tRNA-dihydrouridine synthase family. J. Biol. Chem. 277:25090–25095. [PubMed] [CrossRef]
28. Björk, G. R. 1980. A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli. J. Mol. Biol. 140:391–410. [PubMed] [CrossRef]
29. Björk, G. R. 1986. Transfer RNA modification in different organisms. Chem. Scr. 26B:91–95.
30. Björk, G. R. 1992. The role of modified nucleosides in tRNA interactions, p. 23–85. In D. L. Hatfield, B. J. Lee, and R. M. Pirtle (ed.), Transfer RNA in Protein Synthesis. CRC Press, Boca Raton, Fla.
31. Björk, G. R. 1995. Genetic dissection of synthesis and function of modified nucleosides in bacterial transfer RNA. Prog. Nucleic Acid Res. Mol. Biol. 50:263–338. [PubMed] [CrossRef]
32. Björk, G. R. 1995. Biosynthesis and function of modified nucleosides in tRNA, p. 165–205. In D. Söll and U. L. RajBhandary (ed.), tRNA: Structure, Biosynthesis, and Function. ASM Press, Washington, D.C.
33. Björk, G. R., K. Jacobsson, K. Nilsson, M. J. O. Johansson, A. S. Byström, and O. P. Persson. 2001. A primordial tRNA modification required for the evolution of life? EMBO J. 20:231–239. [PubMed] [CrossRef]
34. Björk, G. R., and K. Nilsson. 2003. 1-Methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism. J. Bacteriol. 185:750–759. [PubMed] [CrossRef]
35. Björk, G. R., and T. Rasmuson. 1998. Links between tRNA modification and metabolism and modified nucleosides as tumor marker, p. 471–491. In H. Grosjean and B. Benne (ed.), Modification and Editing of RNA. ASM Press, Washington, D.C.
36. Björk, G. R., P. M. Wikström, and A. S. Byström. 1989. Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine. Science 244:986–989. [PubMed] [CrossRef]
37. Björnsson, A., and L. A. Isaksson. 1993. UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin. J. Mol. Biol. 232:1017–1029. [PubMed] [CrossRef]
38. Blaise, M., H. D. Becker, G. Keith, C. Cambillau, J. Lapointe, R. Giege, and D. Kern. 2004. A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon. Nucleic Acids Res. 32:2768–2775. [PubMed] [CrossRef]
39. Blattner, F. R., G. I. Plunkett, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, and G. Mayhew. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462. [PubMed] [CrossRef]
40. Bognar, A., C. Pyne, M. Yu, and G. Basi. 1989. Transcription of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli. J. Bacteriol. 171:1854–1861. [PubMed]
41. Bognar, A. L., C. Osborne, and B. Shane. 1987. Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene. J. Biol. Chem. 262:12337–12343. [PubMed]
42. Bork, P., and E. V. Koonin. 1994. A P-loop-like motif in a widespread ATP pyrophosphatase domain: implications for the evolution of sequence motifs and enzyme activity. Proteins 20:347–355. [CrossRef]
43. Bossi, L., and J. R. Roth. 1980. The influence of codon context on genetic code translation. Nature 286:123–127. [PubMed] [CrossRef]
44. Bouadloun, F., T. Srichaiyo, L. A. Isaksson, and G. R. Björk. 1986. Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy. J. Bacteriol. 166:1022–1027. [PubMed]
45. Boy, E., F. Borne, and J. C. Patte. 1978. Effect of mutations affecting lysyl-tRNALys on the regulation of lysine biosynthesis in Escherichia coli. Mol. Gen. Genet. 159:33–38. [PubMed] [CrossRef]
46. Bregeon, D., V. Colot, M. Radman, and F. Taddei. 2001. Translational misreading: a tRNA modification counteracts a +2 ribosomal frameshift. Genes Dev. 15:2295–2306. [PubMed] [CrossRef]
47. Brenchley, J. E., and L. S. Williams. 1975. Transfer RNA involvment in the regulation of enzyme synthesis. Annu. Rev. Microbiol. 29:251–274. [CrossRef]
48. Bresalier, R. S., A. A. Rizzino, and M. Freundlich. 1975. Reduced maximal levels of derepression of the isoleucine-valine and leucine enzymes in hisT mutants of Salmonella typhimurium. Nature 253:279–280. [PubMed] [CrossRef]
49. Brierley, I., M. R. Meredith, A. J. Bloys, and T. G. Hagervall. 1997. Expression of a coronavirus ribosomal frameshift signal in Escherichia coli: influence of tRNA anticodon modification on frameshifting. J. Mol. Biol. 270:360–373. [PubMed] [CrossRef]
50. Brown, L., and T. Elliott. 1996. Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene. J. Bacteriol. 178:3763–3770. [PubMed]
51. Buck, M., and B. N. Ames. 1984. A modified nucleotide in tRNA as a possible regulator of aerobiosis: synthesis of cis-2-methyl-thioribosylzeatin in the tRNA of Salmonella. Cell 36:523–531. [PubMed] [CrossRef]
52. Buck, M., and E. Griffiths. 1981. Regulation of aromatic amino acid transport by tRNA: role of 2-methylthio-N6-(delta2-isopentenyl)-adenosine. Nucleic Acids Res. 9:401–414. [PubMed] [CrossRef]
53. Buck, M., and E. Griffiths. 1982. Iron mediated methylthiolation of tRNA as a regulator of operon expression in Escherichia coli. Nucleic Acids Res. 10:2609–2624. [PubMed] [CrossRef]
54. Buck, M., J. A. McCloskey, B. Basile, and B. N. Ames. 1982. cis 2-Methylthio-ribosylzeatin (ms2io6A) is present in the transfer RNA of Salmonella typhimurium, but not Escherichia coli. Nucleic Acids Res. 10:5649–5662. [PubMed] [CrossRef]
55. Bucklin, D. J., N. M. Wills, R. F. Gesteland, and J. F. Atkins. 2005. P-site pairing subtleties revealed by the effects of different tRNAs on programmed translational bypassing where anticodon re-pairing to mRNA is separated from dissociation. J. Mol. Biol. 345:39–49. [PubMed] [CrossRef]
56. Bujnicki, J. M., Y. Oudjama, M. Roovers, S. Owczarek, J. Caillet, and L. Droogmans. 2004. Identification of a bifunctional enzyme MnmC involved in the biosynthesis of a hypermodified uridine in the wobble position of tRNA. RNA 10:1236–1242. [PubMed] [CrossRef]
57. Byström, A. S., K. J. Hjalmarsson, P. M. Wikström, and G. R. Björk. 1983. The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide. EMBO J. 2:899–905. [PubMed]
58. Byström, A. S., A. von Gabain, and G. R. Björk. 1989. Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species. J. Mol. Biol. 208:575–586. [PubMed] [CrossRef]
59. Cabedo, H., F. Macian, M. Villarroya, J. C. Escudero, M. MartinezVicente, E. Knecht, and M. E. Armengod. 1999. The Escherichia coli trmE (MnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties. EMBO J. 18:7063–7076. [PubMed] [CrossRef]
60. Cabello-Villegas, J., M. E. Winkler, and E. P. Nikonowicz. 2002. Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe). J. Mol. Biol. 319:1015–1034. [PubMed] [CrossRef]
61. Caillet, J., and L. Droogmans. 1988. Molecular cloning of the Escherichia coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in tRNA. J. Bacteriol. 170:4147–4152. [PubMed]
62. Caldeira de Araujo, A., and A. Favre. 1986. Near ultraviolet DNA damage induces the SOS responses in Escherichia coli. EMBO J. 5:175–179. [PubMed]
63. Cantoni, G. L. 1975. Biological methylation: selected aspects. Annu. Rev. Biochem. 44:435–451. [PubMed] [CrossRef]
64. Cermakian, N., and R. Cedergren. 1998. Modified nucleosides always were: an evolutionary model, p. 535–541. In H. Grosjean and R. Benne (ed.), Modification and Editing of RNA. ASM Press, Washington, D.C.
65. Chen, G. F., and M. Inouye. 1990. Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes. Nucleic Acids Res. 18:1465–1473. [PubMed] [CrossRef]
66. Chen, P., Q. Qian, S. Zhang, L. A. Isaksson, and G. R. Björk. 2002. A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine. J. Mol. Biol. 317:481–492. [PubMed] [CrossRef]
67. Cheng, X. D., and R. J. Roberts. 2001. AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res. 29:3784–3795. [PubMed] [CrossRef]
68. Chow, C. S., L. S. Behlen, O. C. Uhlenbeck, and J. K. Barton. 1992. Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping. Biochemistry 31:972–982. [PubMed] [CrossRef]
69. Ciampi, M. S., F. Arena, and R. Cortese. 1977. Biosynthesis of pseudouridine in the in vitro transcribed tRNATyr precursor. FEBS Lett. 77:75–82. [PubMed] [CrossRef]
70. Claesson, C., T. Samuelsson, F. Lustig, and T. Borén. 1990. Codon reading properties of an unmodified transfer RNA. FEBS Lett. 273:173–176. [PubMed] [CrossRef]
71. Cohn, W. E. 1957. Minor constituents of ribonucleic acids. Fed. Proc. 16:166.
72. Cohn, W. E. 1959. 5-Ribosyl uracil, a carbon-carbon ribofuranosyl nucleoside in ribonucleic acids. Biochim. Biophys. Acta 32:570–571. [CrossRef]
73. Cohn, W. E. 1960. Pseudouridine, a cabon-carbon linked ribonucleoside in ribonucleic acids: Isolation, structure, and chemical characteristics. J. Biol. Chem. 235:1488–1498. [PubMed]
74. Cohn, W. E., and E. Volkin. 1951. Nucleoside-5'-phosphates from ribonucleic acid. Nature 167:483–484. [CrossRef]
75. Colby, G., M. Wu, and A. Tzagoloff. 1998. MTO1 codes for a mitochondrial protein required for respiration in paromomycin-resistant mutants of Saccharomyces cerevisiae. J. Biol. Chem. 273:27945–27952. [PubMed] [CrossRef]
76. Connolly, D. M., and M. E. Winkler. 1989. Genetic and physiological relationships among the miaA gene, 2-methylthio-N6-(delta 2-isopentenyl)-adenosine tRNA modification, and spontaneous mutagenesis in Escherichia coli K-12. J. Bacteriol. 171:3233–3246. [PubMed]
77. Connolly, D. M., and M. E. Winkler. 1991. Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations. J. Bacteriol. 173:1711–1721. [PubMed]
78. Cortese, R., H. O. Kammen, S. J. Spengler, and B. N. Ames. 1974. Biosynthesis of pseudouridine in transfer ribonucleic acid. J. Biol. Chem. 249:1103–1108. [PubMed]
79. Cortese, R., R. Landsberg, R. A. Haar, H. E. Umbarger, and B. N. Ames. 1974. Pleiotropy of hisT mutants blocked in pseudouridine synthesis in tRNA: leucine and isoleucine-valine operons. Proc. Natl. Acad. Sci. USA 71:1857–1861. [PubMed] [CrossRef]
80. Crick, F. H. C. 1966. Codon-anticodon pairing. The wobble hypothesis. J. Mol. Biol. 19:548–555. [PubMed] [CrossRef]
81. Curnow, A. W., and G. A. Garcia. 1995. tRNA-guanine transglycosylase from Escherichia coli - minimal tRNA structure and sequence requirements for recognition. J. Biol. Chem. 270:17264–17267. [PubMed] [CrossRef]
82. Cusack, S., A. Yaremchuk, and M. Tukalo. 1996. The crystal structures of T-thermophilus lysyl-tRNA synthetase complexed with E-coli tRNA(Lys) and a T-thermophilus tRNA(Lys) transcript: anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue. EMBO J. 15:6321–6334. [PubMed]
83. Davanloo, P., M. Sprinzl, K. Watanabe, M. Albani, and H. Kersten. 1979. Role of ribothymidine in the thermal stability of transfer RNA as monitored by proton magnetic resonance. Nucleic Acids Res. 6:1571–1581. [PubMed] [CrossRef]
84. Davis, D. R., and C. D. Poulter. 1991. 1H-15N NMR studies of Escherichia coli tRNAPhe from hisT mutants: a structural role for pseudouridine. Biochemistry 30:4223–4231. [PubMed] [CrossRef]
85. Davis, F. F., and F. W. Allen. 1957. Ribonucleic acids from yeast which contain a fifth nucleotide. J. Biol. Chem. 227:907–915. [PubMed]
86. Del Campo, M., Y. Kaya, and J. Ofengand. 2001. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli. RNA 7:1603–1615. [PubMed]
87. Del Campo, M., J. Ofengand, and A. Malhotra. 2004. Crystal structure of the catalytic domain of RluD, the only rRNA pseudouridine synthase required for normal growth of Escherichia coli. RNA 10:231–239. [PubMed] [CrossRef]
88. Di Giulio, M. 1998. Reflections on the origin of the genetic code: a hypothesis. J. Theor. Biol. 191:191–196. [PubMed] [CrossRef]
89. Diaz, I., and M. Ehrenberg. 1991. ms2i6A deficiency enhances proofreading in translation. J. Mol. Biol. 222:1161–1171. [PubMed] [CrossRef]
90. Diaz, I., S. Pedersen, and C. G. Kurland. 1987. Effects of miaA on translation and growth rates. Mol. Gen. Genet. 208:373–376. [PubMed] [CrossRef]
91. Dickson, R. R., T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse. 1989. Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli. J. Bacteriol. 171:4862–4870. [PubMed]
92. Dineshkumar, T. K., S. Thanedar, C. Subbulakshmi, and U. Varshney. 2002. An unexpected absence of queuosine modification in the tRNAs of an Escherichia coli B strain. Microbiology 148:3779–3787. [PubMed]
93. Downs, D. M. 1992. Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium. J. Bacteriol. 174:1515–1521. [PubMed]
94. Downs, D. M., and L. Petersen. 1994. apbA, a new genetic locus involved in thiamine biosynthesis in Salmonella typhimurium. J. Bacteriol. 176:4858–4864. [PubMed]
95. Downs, D. M., and J. R. Roth. 1991. Synthesis of thiamine in Salmonella typhimurium independent of the purF function. J. Bacteriol. 173:6597–6604. [PubMed]
96. Dube, S. K., K. A. Marcker, B. F. Clark, and S. Cory. 1968. Nucleotide sequence of N-formyl-methionyl-transfer RNA. Nature 218:232–233. [PubMed] [CrossRef]
97. Dubois, D. Y., M. Blaise, H. D. Becker, V. Campanacci, G. Keith, R. Giegé, C. Cambillau, J. Lapointe, and D. Kern. 2004. An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNAAsp. Proc. Natl. Acad. Sci. USA 102:7530–7535. [CrossRef]
98. Dunn, D. B. 1959. Additional components in ribonucleic acid of rat-liver fractions. Biochim. Biophys. Acta 34:286–288. [PubMed] [CrossRef]
99. Durand, J. M., and G. R. Björk. 2003. Putrescine or a combination of methionine and arginine restores virulence gene expression in a tRNA modification-deficient mutant of Shigella flexneri: a possible role in adaptation of virulence. Mol. Microbiol. 47:519–527. [PubMed] [CrossRef]
100. Durand, J. M., B. Dagberg, B. E. Uhlin, and G. R. Björk. 2000. Transfer RNA modification, temperature and DNA superhelicity have a common target in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene. Mol. Microbiol. 35:924–935. [PubMed] [CrossRef]
101. Durand, J. M., N. Okada, T. Tobe, M. Watarai, I. Fukuda, T. Suzuki, N. Nakata, K. Komatsu, M. Yoshikawa, and C. Sasakawa. 1994. vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (tgt) of Escherichia coli K-12. J. Bacteriol. 176:4627–4634. [PubMed]
102. Durand, J. M. B., G. R. Björk, A. Kuwae, M. Yoshikawa, and C. Sasakawa. 1997. The modified nucleoside 2-methylthio-N-6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes. J. Bacteriol. 179:5777–5782. [PubMed]
103. Durant, P. C., and D. R. Davis. 1999. Stabilization of the anticodon stem-loop of tRNA(LyS,3) by an A(+)-C base-pair and by pseudouridine. J. Mol. Biol. 285:115–131. [PubMed] [CrossRef]
104. Edmonds, C. G., P. F. Crain, R. Gupta, T. Hashizume, C. H. Hocart, J. A. Kowalak, S. C. Pomerantz, K. O. Stetter, and J. A. McCloskey. 1991. Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria). J. Bacteriol. 173:3138–3148. [PubMed]
105. Eisenberg, S. P., M. Yarus, and L. Soll. 1979. The effect of an Escherichia coli regulatory mutation on transfer RNA structure. J. Mol. Biol. 135:111–126. [PubMed] [CrossRef]
106. Elkins, P. A., J. M. Watts, M. Zalacain, A. van Thiel, P. R. Vitazka, M. Redlak, C. Andraos-Selim, F. Rastinejad, and W. M. Holmes. 2003. Insights into catalysis by a knotted TrmD tRNA methyltransferase. J. Mol. Biol. 333:931–949. [PubMed] [CrossRef]
107. Elseviers, D., L. A. Petrullo, and P. J. Gallagher. 1984. Novel E. coli mutants deficient in biosynthesis of 5-methylaminomethyl-2-thiouridine. Nucleic Acids Res. 12:3521–3534. [PubMed] [CrossRef]
108. Emilsson, V., and C. G. Kurland. 1990. Growth rate dependence of transfer RNA abundance in Escherichia coli. EMBO J. 9:4359–4366. [PubMed]
109. Emilsson, V., A. K. Näslund, and C. G. Kurland. 1992. Thiolation of transfer RNA in Escherichia coli varies with growth rate. Nucleic Acids Res. 20:4499–4505. [PubMed] [CrossRef]
110. Ericson, J. U., and G. R. Björk. 1986. Pleiotropic effects induced by modification deficiency next to the anticodon of tRNA from Salmonella typhimurium LT2. J. Bacteriol. 166:1013–1021. [PubMed]
111. Ericson, J. U., and G. R. Björk. 1991. tRNA anticodons with the modified nucleoside 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine distinguish between bases 3' of the codon. J. Mol. Biol. 218:509–516. [PubMed] [CrossRef]
112. Esberg, B., and G. R. Björk. 1995. The methylthio group (ms2) of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA. J. Bacteriol. 177:1967–1975. [PubMed]
113. Farabaugh, P. J., and G. R. Björk. 1999. How translational accuracy influences reading frame maintenance. EMBO J. 18:1427–1434. [PubMed] [CrossRef]
114. Faulkner, R. D., and M. Uziel. 1971. Iodine modification of E. coli tRNAPhe : reversible modification of 2-methylthio-N6-isopentenyladenosine and lack of disulfide formation. Biochim. Biophys. Acta 238:464–474. [PubMed]
115. Favre, A., E. Hajnsdorf, K. Thiam, and A. Caldeira de Araujo. 1985. Mutagenesis and growth delay induced in Escherichia coli by near-ultraviolet radiations. Biochimie 67:335–342. [CrossRef]
116. Favre, A., M. Yaniv, and A. M. Michelson. 1969. The photochemistry of 4-thiouridine in Escherichia coli t-RNAVal1. Biochem. Biophys. Res. Commun. 37:266–271. [PubMed] [CrossRef]
117. Fleissner, E., and E. Borek. 1962. A new enzyme of RNA synthesis: RNA methylase. Proc. Natl. Acad. Sci. USA 48:1199–1203. [PubMed] [CrossRef]
118. Flint, D. H. 1996. Escherichia coli contains a protein that is homologous in function and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vinelandii and that can participate in the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase. J. Biol. Chem. 271:16068–16074. [PubMed]
119. Fontecave, M., E. Mulliez, and S. Ollagnier-de-Choudens. 2001. Adenosylmethionine as a source of 5'-deoxyadenosyl radicals. Curr. Opin. Chem. Biol. 5:506–511. [PubMed] [CrossRef]
120. Foster, P. G., L. Huang, D. V. Santi, and R. M. Stroud. 2000. The structural basis for tRNA recognition and pseudouridine formation by pseudouridine synthase I. Nat. Struct. Biol. 7:23–27. [PubMed] [CrossRef]
121. Foster, P. G., C. R. Nunes, P. Greene, D. Moustakas, and R. M. Stroud. 2003. The first structure of an RNA m(5)C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate. Structure (Cambridge) 11:1609–1620. [PubMed] [CrossRef]
122. Frey, B., G. Jänel, U. Michelsen, and H. Kersten. 1989. Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr. J. Bacteriol. 171:1524–1530. [PubMed]
123. Frey, B., J. McCloskey, W. Kersten, and H. Kersten. 1988. New function of vitamin B12: cobamide-dependent reduction of epoxyqueuosine to queuosine in tRNAs of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 170:2078–2082. [PubMed]
124. Gaal, T., J. Barkei, R. R. Dickson, H. A. deBoer, P. L. deHaseth, H. Alavi, and R. L. Gourse. 1989. Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants. J. Bacteriol. 171:4852–4861. [PubMed]
125. Gabriel, K., J. Schneider, and W. H. McClain. 1996. Functional evidence for indirect recognition of G.U in tRNA(Ala) by alanyl-tRNA synthetase. Science 271:195–197. [PubMed] [CrossRef]
126. Gabryszuk, J., and W. M. Holmes. 1997. tRNA recognition for modification: solution probing of tRNA complexed with Escherichia coli tRNA (guanosine-1) methyltransferase. RNA 3:1327–1336. [PubMed]
127. Gefter, M. L. 1969. The in vitro synthesis of 2'-omethylguanosine and 2-methylthio 6N (gamma,gamma, dimethylallyl) adenosine in transfer RNA of Escherichia coli. Biochem. Biophys. Res. Commun. 36:435–441. [PubMed] [CrossRef]
128. Gefter, M. L., and R. L. Russell. 1969. Role of modifications in tyrosine transfer RNA: a modified base affecting ribosome binding. J. Mol. Biol. 39:145–157. [PubMed] [CrossRef]
129. Gentry, D., C. Bengra, K. Ikehara, and M. Cashel. 1993. Guanylate kinase of Escherichia coli K-12. J. Biol. Chem. 268:14316–14321. [PubMed]
130. Gentry, D. R., and R. R. Burgess. 1989. rpoZ, encoding the omega subunit of Escherichia coli RNA polymerase, is in the same operon as spoT. J. Bacteriol. 171:1271–1277. [PubMed]
131. Gerber, A. P., and W. Keller. 1999. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science 286:1146–1149. [PubMed] [CrossRef]
132. Giege, R., M. Sissler, and C. Florentz. 1998. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 26:5017–5035. [PubMed] [CrossRef]
133. Glass, R. S., W. P. Singh, W. Jung, Z. Veres, T. D. Scholz, and T. C. Stadtman. 1993. Monoselenophosphate - synthesis, characterization, and identity with the prokaryotic biological selenium donor, compound sepx. Biochemistry 32:12555–12559. [PubMed] [CrossRef]
134. Goddard, J. P., and M. Lowdon. 1981. The effect upon aminoacylation of bisulphite addition to 2-methylthio-N6-isopentenyl adenosine of Escherichia coli phenylalanine tRNA. FEBS Lett. 130:221–222. [PubMed] [CrossRef]
135. Goldman, E., W. M. Holmes, and G. W. Hatfield. 1979. Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA. J. Mol. Biol. 129:567–585. [PubMed] [CrossRef]
136. Gottesman, S. 2004. The small RNA regulators of Escherichia coli: roles and mechanisms. Annu. Rev. Microbiol. 58:303–328. [PubMed] [CrossRef]
137. Greenberg, R., and B. Dudock. 1980. Isolation and chracterization of m5U-methyltransferase from Escherichia coli. J. Biol. Chem. 255:8296–8302. [PubMed]
138. Griffiths, E., and J. Humphreys. 1978. Alterations in tRNAs containing 2-methylthio-N6-(delta2-isopentenyl)-adenosine during growth of enteropathogenic Escherichia coli in the presence of iron-binding proteins. Eur. J. Biochem. 82:503–513. [PubMed] [CrossRef]
139. Griffiths, E., J. Humphreys, A. Leach, and L. Scanlon. 1978. Alterations in the tRNA's of Escherichia coli recovered from lethally infected animals. Infect. Immun. 22:312–317. [PubMed]
140. Grosjean, H., and G. R. Björk. 2004. Enzymatic conversion of cytidine to lysidine in anticodon of bacterial tRNAIle. Trends Biochem. Sci. 29:165–168. [PubMed] [CrossRef]
141. Grosjean, H., V. de Crécy-Lagard, and G. R. Björk. 2004. Aminoacylation of the anticodon stem by a tRNA-synthetase paralog: relic of an ancient code? Trends Biochem. Sci., 29: 519-522 [CrossRef]
142. Grosjean, H., K. Nicoghosian, E. Haumont, D. Söll, and R. Cedergren. 1985. Nucleotide sequences of two serine tRNAs with a GGA anticodon: the structure-function relationships in the serine family of E. coli tRNAs. Nucleic Acids Res. 13:5697–5706. [PubMed] [CrossRef]
143. Grosjean, H. J., S. de Henau, and D. M. Crothers. 1978. On the physical basis for ambiguity in genetic coding interactions. Proc. Natl. Acad. Sci. USA 75:610–614. [PubMed] [CrossRef]
144. Gu, X. R., K. M. Ivanetich, and D. V. Santi. 1996. Recognition of the T-arm of tRNA by tRNA (m(5)U54)-methyltransferase is not sequence specific. Biochemistry 35:11652–11659. [PubMed] [CrossRef]
145. Gu, X. R., Y. Q. Liu, and D. V. Santi. 1999. The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA. Proc. Natl. Acad. Sci. USA 96:14270–14275. [PubMed] [CrossRef]
146. Gu, X. R., and D. V. Santi. 1990. High-level expression of Escherichia coli tRNA (m5U54)-methyltransferase. DNA Cell Biol. 9:273–278. [PubMed] [CrossRef]
147. Gu, X. R., and D. V. Santi. 1991. The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase. Biochemistry 30:2999–3002. [PubMed] [CrossRef]
148. Gu, X. R., M. Yu, K. M. Ivanetich, and D. V. Santi. 1998. Molecular recognition of tRNA by tRNA pseudouridine 55 synthase. Biochemistry 37:339–343. [PubMed] [CrossRef]
149. Gustafsson, C. 1992. The tRNA modifying enzyme tRNA(m5U54)methyltransferase and its structural gene trmA. Ph.D. thesis. Umeå University, Umeå, Sweden.
150. Gustafsson, C., and G. R. Björk. 1993. The tRNA-(m5U54)-methyltransferase of Escherichia coli is present in two forms in vivo, one of which is present as bound to tRNA and to a 3'-end fragment of 16 S rRNA. J. Biol. Chem. 268:1326–1331. [PubMed]
151. Gustafsson, C., P. H. Lindström, T. G. Hagervall, K. B. Esberg, and G. R. Björk. 1991. The trmA promoter has regulatory features and sequence elements in common with the rRNA P1 promoter family of Escherichia coli. J. Bacteriol. 173:1757–1764. [PubMed]
152. Gustafsson, C., R. Reid, P. J. Greene, and D. V. Santi. 1996. Identification of new RNA modifying enzymes by iterative genome search using known modifying enzymes as probes. Nucleic Acids Res. 24:3756–3762. [PubMed] [CrossRef]
153. Gutgsell, N., N. Englund, L. H. Niu, Y. Kaya, B. G. Lane, and J. Ofengand. 2000. Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells. RNA 6:1870–1881. [PubMed] [CrossRef]
154. Hagervall, T. G., and G. R. Björk. 1984. Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency. Mol. Gen. Genet. 196:194–200. [PubMed] [CrossRef]
155. Hagervall, T. G., C. G. Edmonds, J. A. McCloskey, and G. R. Björk. 1987. Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities. J. Biol. Chem. 262:8488–8495. [PubMed]
156. Hagervall, T. G., J. U. Ericson, K. B. Esberg, J. N. Li, and G. R. Björk. 1990. Role of tRNA modification in translational fidelity. Biochim. Biophys. Acta 1050:263–266. [PubMed]
157. Hagervall, T. G., Y. H. Jönsson, C. G. Edmonds, J. A. McCloskey, and G. R. Björk. 1990. Chorismic acid, a key metabolite in modification of tRNA. J. Bacteriol. 172:252–259. [PubMed]
158. Hagervall, T. G., S. C. Pomerantz, and J. A. McCloskey. 1998. Reduced misreading of asparagine codons by Escherichia coli tRNA(Lys) with hypomodified derivatives of 5-methylaminomethyl-2-thiouridine in the wobble position. J. Mol. Biol. 284:33–42. [PubMed] [CrossRef]
159. Hall, K. B., J. R. Sampson, O. C. Uhlenbeck, and A. G. Redfield. 1989. Structure of an unmodified tRNA molecule. Biochemistry 28:5794–5801. [PubMed] [CrossRef]
160. Hansen, T. M., P. V. Baranov, I. P. Ivanov, R. F. Gesteland, and J. F. Atkins. 2003. Maintenance of the correct open reading frame by the ribosome. EMBO Rep. 4:499–504. [PubMed] [CrossRef]
161. Harada, F., and S. Nishimura. 1974. Purification and characterization of AUA specific isoleucine transfer ribonucleic acid from Escherichia coli B. Biochemistry 13:300–307. [PubMed] [CrossRef]
162. Hasegawa, T., M. Miyano, H. Himeno, Y. Sano, K. Kimura, and M. Shimizu. 1992. Identity determinants of E. coli threonine tRNA. Biochem. Biophys. Res. Commun. 184:478–484. [PubMed] [CrossRef]
163. Hjalmarsson, K. J., A. S. Byström, and G. R. Björk. 1983. Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli. J. Biol. Chem. 258:1343–1351. [PubMed]
164. Hoagland, M. B., P. C. Zamecnik, and M. L. Stephenson. 1957. Intermediate reactions in protein synthesis. Biochim. Biophys. Acta 24:215–216. [PubMed] [CrossRef]
165. Hoang, C., and A. R. FerreDAmare. 2001. Cocrystal structure of a tRNA Psi 55 pseudouridine synthase: Nucleotide flipping by an RNA-modifying enzyme. Cell 107:929–939. [PubMed] [CrossRef]
166. Hoff, K. G., D. T. Ta, T. L. Tapley, J. J. Silberg, and L. E. Vickery. 2002. Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU. J. Biol. Chem. 277:27353–27359. [PubMed] [CrossRef]
167. Holley, R. W., G. A. Everett, J. T. Madison, and A. Zamir. 1965. Nucleotide sequences in the yeast alanine transfer ribonucleic acid. J. Biol. Chem. 240:2122–2128. [PubMed]
168. Holmes, W. M., C. Andraos-Selim, I. Roberts, and S. Z. Wahab. 1992. Structural requirements for tRNA methylation. Action of Escherichia coli tRNA(guanosine-1)methyltransferase on tRNALeu1 structural variants. J. Biol. Chem. 267:13440–13445. [PubMed]
169. Hopfield, J. J. 1978. Origin of the genetic code: a testable hypothesis based on tRNA structure, sequence, and kinetic proofreading. Proc. Natl. Acad. Sci. USA 75:4334–4338. [PubMed] [CrossRef]
170. Hori, H., M. Saneyoshi, I. Kumagai, K. Miura, and K. Watanabe. 1989. Effects of modification of 4-thiouridine in E. coli tRNA(fMet) on its methyl acceptor activity by thermostable Gm-methylases. J. Biochem. (Tokyo) 106:798–802. [PubMed]
171. Hori, H., T. Suzuki, K. Sugawara, Y. Inoue, T. Shibata, S. Kuramitsu, S. Yokoyama, T. Oshima, and K. Watanabe. 2002. Identification and characterization of tRNA (Gm18) methyltransferase from Thermus thermophilus HB8: domain structure and conserved amino acid sequence motifs. Genes Cells 7:259–272. [PubMed] [CrossRef]
172. Hori, H., N. Yamazaki, T. Matsumoto, Y. Watanabe, T. Ueda, K. Nishikawa, I. Kumagai, and K. Watanabe. 1998. Substrate recognition of tRNA (guanosine-2'-)-methyltransferase from Thermus thermophilus HB27. J. Biol. Chem. 273:25721–25727. [PubMed] [CrossRef]
173. Horsfield, J. A., D. N. Wilson, S. A. Mannering, F. M. Adamski, and W. P. Tate. 1995. Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism. Nucleic Acids Res. 23:1487–1494. [PubMed] [CrossRef]
174. Hotchkiss, R. 1948. The quantitative separation of purines, pyrimidines, and nucleosides by paper chromatography. J. Biol. Chem. 175:315–332. [PubMed]
175. Huang, L. X., M. Pookanjanatavip, X. G. Gu, and D. V. Santi. 1998. A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst. Biochemistry 37:344–351. [PubMed] [CrossRef]
176. Ibba, M., and D. Söll. 2004. Aminoacyl-tRNAs: setting the limits of the genetic code. Genes Dev. 18:731–738. [PubMed] [CrossRef]
177. Ikemura, T. 1981. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes. J. Mol. Biol. 146:1–21. [PubMed] [CrossRef]
178. Ishikura, H., Y. Yamada, and S. Nishimura. 1971. Structure of serine tRNA from Escherichia coli. I. Purification of serine tRNA's with different codon responses. Biochim. Biophys. Acta 228:471–481. [PubMed]
179. Iwata-Reuyl, D. 2003. Biosynthesis of the 7-deazaguanosine hypermodified nucleosides of transfer RNA. Bioorg. Chem. 31:24–43. [PubMed] [CrossRef]
180. Jäger, G., R. Leipuviene, M. G. Pollard, Q. Qian, and G. R. Björk. 2004. The conserved Cys-X(1)-X(2)-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium. J. Bacteriol. 186:750–757. [PubMed] [CrossRef]
181. Jagger, J. 1983. Physiological effects of near-ultraviolet radiation on bacteria. Photochem. Photobiol. Rev. 7:1–73.
182. Jeter, R. M., B. M. Olivera, and J. R. Roth. 1984. Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions. J. Bacteriol. 159:206–213. [PubMed]
183. Johnson, L., and D. Söll. 1970. In vitro biosynthesis of pseudouridine at the polynucleotide level by an enzyme extract from Escherichia coli. Proc. Natl. Acad. Sci. USA 67:943–950. [PubMed] [CrossRef]
184. Johnston, H. M., W. M. Barnes, F. G. Chumley, L. Bossi, and J. R. Roth. 1980. Model for regulation of the histidine operon of Salmonella. Proc. Natl. Acad. Sci. USA 77:508–512. [PubMed] [CrossRef]
185. Jones, W. R., G. J. Barcak, and R. E. Wolf, Jr. 1990. Altered growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level in hisT mutants of Salmonella typhimurium and Escherichia coli. J. Bacteriol. 172:1197–1205. [PubMed]
186. Jukes, T. H. 1973. Possibilities for the evolution of the genetic code from a preceding form. Nature 246:22–26. [PubMed] [CrossRef]
187. Kadner, R. J. 1978. Repression of synthesis of the vitamin B12 receptor in Escherichia coli. J. Bacteriol. 136:1050–1057. [PubMed]
188. Kajitani, M., and A. Ishihama. 1991. Identification and sequence determination of the host factor gene for bacteriophage Q beta. Nucleic Acids Res. 19:1063–1066. [PubMed] [CrossRef]
189. Kalman, M., H. Murphy, and M. Cashel. 1992. The nucleotide sequence of recG, the distal spo operon gene in Escherichia coli K-12. Gene 110:95–99. [PubMed] [CrossRef]
190. Kambampati, R., and C. T. Lauhon. 2000. Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. J. Biol. Chem. 275:10727–10730. [PubMed] [CrossRef]
191. Kambampati, R., and C. T. Lauhon. 2003. MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry 42:1109–1117. [PubMed] [CrossRef]
192. Kammen, H. O., C. C. Marvel, L. Hardy, and E. E. Penhoet. 1988. Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I. J. Biol. Chem. 263:2255–2263. [PubMed]
193. Kawai, G., Y. Yamamoto, T. Kamimura, T. Masegi, M. Sekine, T. Hata, T. Iimori, T. Watanabe, T. Miyazawa, and S. Yokoyama. 1992. Conformational rigidity of specific pyrimidine residues in tRNA arises from posttranscriptional modifications that enhance steric interaction between the base and the 2'-hydroxyl group. Biochemistry 31:1040–1046. [PubMed] [CrossRef]
194. Kawakami, K., Y. H. Jönsson, G. R. Björk, H. Ikeda, and Y. Nakamura. 1988. Chromosomal location and structure of the operon encoding peptide-chain-release factor 2 of Escherichia coli. Proc. Natl. Acad. Sci. USA 85:5620–5624. [PubMed] [CrossRef]
195. Kaya, Y., M. Del Campo, J. Ofengand, and A. Malhotra. 2004. Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold. J. Biol. Chem. 279:18107-18110. [CrossRef]
196. Kaya, Y., and J. Ofengand. 2003. A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya. RNA 9:711–721. [PubMed] [CrossRef]
197. Kealey, J. T., X. Gu, and D. V. Santi. 1994. Enzymatic mechanism of tRNA (m(5)u54)methyltransferase. Biochimie 76:1133–1142. [PubMed] [CrossRef]
198. Kealey, J. T., and D. V. Santi. 1991. Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase. Biochemistry 30:9724–9728. [PubMed] [CrossRef]
199. Kersten, H., M. Albani, E. M:annlein, R. Praisler, P. Wurmbach, and K. H. Nierhaus. 1981. On the role of ribosylthymine in prokaryotic tRNA function. Eur. J. Biochem. 114:451–456. [PubMed] [CrossRef]
200. Kersten, H., and W. Kersten. 1990. Biosynthesis and function of queuine and queosine tRNAs, 45B: p. B69–B108. In C. W. Gehrke and K. C. T. Kuo (ed.), Chromatography and Modification of Nucleosides. Part B: Biological Roles and Function of Modification Journal Chromatography Library. Elsevier, Amsterdam, The Netherlands.
201. King, M. Y., and K. L. Redman. 2002. RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine. Biochemistry 41:11218–11225. [PubMed] [CrossRef]
202. Kinghorn, S. M., C. P. O'Byrne, I. R. Booth, and I. Stansfield. 2002. Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance. Microbiology 148:3511–3520. [PubMed]
203. Kinscherf, T. G., and D. K. Willis. 2002. Global regulation by gidA in Pseudomonas syringae. J. Bacteriol. 184:2281–2286. [PubMed] [CrossRef]
204. Kitchingman, G. R., and M. J. Fournier. 1975. Unbalanced growth and the production of unique transfer ribonucleic acids in relaxed-control Escherichia coli. J. Bacteriol. 124:1382–1394. [PubMed]
205. Kitchingman, G. R., and M. J. Fournier. 1977. Modification-deficient transfer ribonucleic acids from relaxed control Escherichia coli: structures of the major undermodified phenylalanine and leucine transfer RNAs produced during leucine starvation. Biochemistry 16:2213–2220. [PubMed] [CrossRef]
206. Kitchingman, G. R., E. Webb, and M. J. Fournier. 1976. Unique phenylalanine transfer ribonucleic acids in relaxed control Escherichia coli: genetic origin and some functional properties. Biochemistry 15:1848–1857. [PubMed] [CrossRef]
207. Kittendorf, J. D., L. M. Barcomb, S. T. Nonekowski, and G. A. Garcia. 2001. tRNA-guanine transglycosylase from Escherichia coli: molecular mechanism and role of aspartate 89. Biochemistry 40:14123–14133. [PubMed] [CrossRef]
208. Koonin, E. V. 1996. Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. Nucleic Acids Res. 24:2411–2415. [PubMed] [CrossRef]
209. Kramer, G. F., and B. N. Ames. 1988. Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium. J. Bacteriol. 170:736–743. [PubMed]
210. Kramer, G. F., J. C. Baker, and B. N. Ames. 1988. Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response. J. Bacteriol. 170:2344–2351. [PubMed]
211. Kruger, M. K., S. Pedersen, T. G. Hagervall, and M. A. Sorensen. 1998. The modification of the wobble base of tRNA(Glu) modulates the translation rate of glutamic acid codons in vivo. J. Mol. Biol. 284:621–631. [PubMed] [CrossRef]
212. Kruger, M. K., and M. A. Sorensen. 1998. Aminoacylation of hypomodified tRNA(Glu) in vivo. J. Mol. Biol. 284:609–620. [PubMed] [CrossRef]
213. Kuchino, Y., H. Kasai, K. Nihei, and S. Nishimura. 1976. Biosynthesis of the modified nucleoside Q in transfer RNA. Nucleic Acids Res. 3:393–398. [PubMed]
214. Kumagai, I., K. Watanabe, and T. Oshima. 1982. A thermostable tRNA (guanosine-2')-methyltransferase from Thermus thermophilus HB27 and the effect of ribose methylation on the conformational stability of tRNA. J. Biol. Chem. 257:7388–7395. [PubMed]
215. Kumar, R. K., and D. R. Davis. 1997. Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability. Nucleic Acids Res. 25:1272–1280. [PubMed] [CrossRef]
216. Kurland, C. G., D. Hughes, and M. Ehrenberg. 1996. Limitations of translation accuracy, p. 979–1004. In F. C.Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella. Cellular and Molecular Biology. ASM Press, Washington, D.C.
217. Ladner, J. E., A. Jack, J. D. Robertus, R. S. Brown, D. Rhodes, B. F. Clark, and A. Klug. 1975. Structure of yeast phenylalanine transfer RNA at 2.5 A resolution. Proc. Natl. Acad. Sci. USA 72:4414–4418. [PubMed] [CrossRef]
218. Lagerkvist, U. 1978. "Two out of three": an alternative method for codon reading. Proc. Natl. Acad. Sci. USA 75:1759–1762. [PubMed] [CrossRef]
219. Lauhon, C. T. 2002. Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli. J. Bacteriol. 184:6820–6829. [PubMed] [CrossRef]
220. Lauhon, C. T., W. M. Erwin, and G. N. Ton. 2004. Substrate specificity for 4-thiouridine modification in Escherichia coli. J. Biol. Chem. 279:23022-23029. [CrossRef]
221. Lauhon, C. T., E. Skovran, H. D. Urbina, D. M. Downs, and L. E. Vickery. 2004. Substitutions in an active site loop of escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo. J. Biol. Chem. 279:19551-19558. [CrossRef]
222. Lecointe, F., G. Simos, A. Sauer, E. C. Hurt, Y. Motorin, and H. Grosjean. 1998. Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of Psi(38) and Psi(39) in tRNA anticodon loop. J. Biol. Chem. 273:1316–1323. [PubMed] [CrossRef]
223. Leinfelder, W., K. Forchhammer, F. Zinoni, G. Sawers, M. A. Mandrand-Berthelot, and A. Böck. 1988. Escherichia coli genes whose products are involved in selenium metabolism. J. Bacteriol. 170:540–546. [PubMed]
224. Leipuviene, R., Q. Qian, and G. R. Björk. 2004. Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways. J. Bacteriol. 186:758–766. [PubMed] [CrossRef]
225. Leung, H. C. E., Y. Q. Chen, and M. E. Winkler. 1997. Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12. J. Biol. Chem. 272:13073–13083. [PubMed] [CrossRef]
226. Li, J. N., and G. R. Björk. 1995. 1-methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium. J. Bacteriol. 177:6593–6600. [PubMed]
227. Li, J. N., B. Esberg, J. F. Curran, and G. R. Björk. 1997. Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner. J. Mol. Biol. 271:209–221. [PubMed] [CrossRef]
228. Licznar, P., N. Mejlhede, M. F. Prère, N. Wills, R. F. Gesteland, J. F. Atkins, and O. Fayet. 2003. Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs. EMBO J. 22:4770–4778. [PubMed] [CrossRef]
229. Lim, K., H. Zhang, A. Tempczyk, W. Krajewski, N. Bonander, J. Toedt, A. Howard, E. Eisenstein, and O. Herzberg. 2003. Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): a cofactor bound at a site formed by a knot. Proteins 51:56–67. [PubMed] [CrossRef]
230. Lim, V. I. 1994. Analysis of action of wobble nucleoside modifications on codon-anticodon pairing within the ribosome. J. Mol. Biol. 240:8–19. [PubMed] [CrossRef]
231. Lim, V. I ., and J. F. Curran. 2001. Analysis of codon : anticodon interactions within the ribosome provides new insights into codon reading and the genetic code structure. RNA 7:942–957. [PubMed] [CrossRef]
232. Limbach, P. A., P. F. Crain, and J. A. McCloskey. 1994. Summary: the modified nucleosides of RNA. Nucleic Acids Res. 22:2183–2196. [PubMed] [CrossRef]
233. Lindström, P. H., D. Stüber, and G. R. Björk. 1985. Genetic organization and transcription from the gene (trmA) responsible for synthesis of tRNA (uracil-5)-methyltransferase by Escherichia coli. J. Bacteriol. 164:1117–1123. [PubMed]
234. Lipsett, M. N. 1978. Enzymes producing 4-thiouridine in Escherichia coli tRNA: approximate chromosomal locations of the genes and enzyme activities in a 4-thiouridine-deficient mutant. J. Bacteriol. 135:993–997. [PubMed]
235. Liu, J., W. Wang, D. H. Shin, H. Yokota, R. Kim, and S. H. Kim. 2003. Crystal structure of tRNA (m1G37) methyltransferase from Aquifex aeolicus at 2.6 A resolution: a novel methyltransferase fold. Proteins 53:326–328. [PubMed] [CrossRef]
236. Liu, Y., and D. V. Santi. 2000. m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts. Proc. Natl. Acad. Sci. USA 97:8263–8265. [PubMed] [CrossRef]
237. Lloyd, R. G., and G. J. Sharples. 1991. Molecular organization and nucleotide sequence of the recG locus of Escherichia coli K-12. J. Bacteriol. 173:6837–6843. [PubMed]
238. Lustig, F., P. Elias, T. Axberg, T. Samuelsson, I. Tittawella, and U. Lagerkvist. 1981. Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro. J. Biol. Chem. 256:2635–2643. [PubMed]
239. Madore, E., C. Florentz, R. Giege, S. Sekine, S. Yokoyama, and J. Lapointe. 1999. Effect of modified nucleotides on Escherichia coli tRNA(Glu) structure and on its aminoacylation by glutamyl-tRNA synthetase—predominant and distinct roles of the mnm(5) and s(2) modifications of U34. Eur. J. Biochem. 266:1128–1135. [PubMed] [CrossRef]
240. Marck, C., and H. Grosjean. 2002. tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features. RNA 8:1189–1232. [PubMed] [CrossRef]
241. Masucci, J. P., J. Gallant, D. Lindsley, and J. Atkinson. 2002. Influence of the relA gene on ribosome frameshifting. Mol. Genet. Genom. 268:81–86. [CrossRef]
242. McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S. Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan, H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852–856. [PubMed] [CrossRef]
243. McCloskey, J. 1986. Nucleoside modification in archaebacterial transfer RNA. Syst. Appl. Microbiol. 7:246–252.
244. Meier, F., B. Suter, H. Grosjean, G. Keith, and E. Kubli. 1985. Queuosine modification of the wobble base in tRNAHis influences 'in vivo' decoding properties. EMBO J. 4:823–827. [PubMed]
245. Mikkola, R., and C. G. Kurland. 1988. Media dependence of translational mutant phenotype. FEMS Microbiol. Lett. 56:265–270. [CrossRef]
246. Miller, J. P., Z. Hussain, and M. P. Schweizer. 1976. The involvement of the anticodon adjacent modified nucleoside N-(9-(ß-D-ribofuranosyl) purine-6-ylcarbamoyl)-threonine in the biological function of E. coli tRNAile. Nucleic Acids Res. 3:1185–1201. [PubMed]
247. Mitra, S. K., F. Lustig, B. Akesson, T. Axberg, P. Elias, and U. Lagerkvist. 1979. Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro. J. Biol. Chem. 254:6397–6401. [PubMed]
248. Moore, J. A., and C. D. Poulter. 1997. Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: A binding mechanism for recombinant enzyme. Biochemistry 36:604–614. [PubMed] [CrossRef]
249. Motorin, Y., G. Bec, R. Tewari, and H. Grosjean. 1997. Transfer RNA recognition by the Escherichia coli Delta(2)-isopentenyl-pyrophosphate:tRNA Delta 2-isopentenyl transferase: dependence on the anticodon arm structure. RNA 3:721–733. [PubMed]
250. Motorin, Y., and H. Grosjean. 1998. Appendix 1: Chemical structures and classification of posttranscriptionally modified nucleosides in RNA, p. 543–549. In H. Grosjean and R. Benne (ed.), Modification and Editing of RNA. ASM Press, Washington, D.C.
251. Mueller, E. G. 2002. Chips off the old block. Nat. Struct. Biol. 9:320–322. [PubMed] [CrossRef]
252. Mueller, E. G., C. J. Buck, P. M. Palenchar, L. E. Barnhart, and J. L. Paulson. 1998. Identification of a gene involved in the generation of 4-thiouridine in tRNA. Nucleic Acids Res. 26:2606–2610. [PubMed] [CrossRef]
253. Mueller, E. G., P. M. Palenchar, and C. J. Buck. 2001. The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA. J. Biol. Chem. 276:33588–33595. [PubMed] [CrossRef]
254. Muramatsu, T., T. Miyazawa, and S. Yokoyama. 1992. Recognition of the nucleoside in the first position of the anticodon of isoleucine tRNA by isoleucyl-tRNA synthetase from Escherichia coli. Nucl. Nucl. 11:719–730.
255. Muramatsu, T., K. Nishikawa, F. Nemoto, Y. Kuchino, S. Nishimura, T. Miyazawa, and S. Yokoyama. 1988. Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification. Nature 336:179–181. [PubMed] [CrossRef]
256. Muramatsu, T., S. Yokoyama, N. Horie, A. Matsuda, T. Ueda, Z. Yamaizumi, Y. Kuchino, S. Nishimura, and T. Miyazawa. 1988. A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli. J. Biol. Chem. 263:9261–9267. [PubMed]
257. Murphy, F. V., V. Ramakrishnan, A. Malkiewicz, and P. F. Agris. 2004. The role of modifications in codon discrimination by tRNA(Lys)(UUU). Nat. Struct. Mol. Biol. 11:1186-1192. [CrossRef]
258. Nakanishi, S., T. Ueda, H. Hori, N. Yamazaki, N. Okada, and K. Watanabe. 1994. A UGU sequence in the anticodon loop is a minimum requirement for recognition by Escherichia coli tRNA-guanine transglycosylase. J. Biol. Chem. 269:32221–32225. [PubMed]
259. Nakayashiki, T., and H. Inokuchi. 1998. Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA(6)(Leu). J. Bacteriol. 180:2931–2935. [PubMed]
260. Näsvall, S. J., P. Chen, and G. R. Björk. 2004. The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAProcmo5UGG promotes reading of all four proline codons in vivo. RNA 10:1662-1673. [CrossRef]
261. Nègre, D., J. C. Cortay, P. Donini, and A. J. Cozzone. 1989. Relationship between guanosine tetraphosphate and accuracy of translation in Salmonella typhimurium. Biochemistry 28:1814–1819. [PubMed] [CrossRef]
262. Niimi, T., O. Nureki, T. Yokogawa, N. Hayashi, K. Nishikawa, K. Watanabe, and S. Yokoyama. 1994. Recognition of the anticodon loop of tRNAIle1 by isoleucyl-tRNA synthetase from Escherichia coli. Nucl. Nucl. 13:1231–1237.
263. Nilsson, K., H. K. Lundgren, T. G. Hagervall, and G. R. Björk. 2002. The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium. J. Bacteriol. 184:6830–6835. [PubMed] [CrossRef]
264. Nishimura, S. 1972. Minor components in transfer RNA: their characterization, location, and function. Prog. Nucl. Acid Res. Mol. Biol. 12:49–85. [PubMed] [CrossRef]
265. Nishimura, S. 1979. Modified nucleosides in tRNA, p. 59–79. In P. R. Schimmel, D. Söll, and J. N. Abelson (ed.), Transfer RNA: Structure, Properties, and Recognition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
266. Noguchi, S., Y. Nishimura, Y. Hirota, and S. Nishimura. 1982. Isolation and characterization of an Escherichia coli mutant lacking tRNA-guanine transglycosylase. Function and biosynthesis of queuosine in tRNA. J. Biol. Chem. 257:6544–6550. [PubMed]
267. Noguchi, S., Z. Yamaizumi, T. Ohgi, T. Goto, Y. Nishimura, Y. Hirota, and S. Nishimura. 1978. Isolation of Q nucleoside precursor present in tRNA of an E. coli mutant and its characterization as 7-(cyano)-7-deazaguanosine. Nucleic Acids Res. 5:4215–4223. [PubMed] [CrossRef]
268. Nonet, M. L., C. C. Marvel, and D. R. Tolan. 1987. The hisT-purF region of the Escherichia coli K-12 chromosome. Identification of additional genes of the hisT and purF operons. J. Biol. Chem. 262:12209–12217. [PubMed]
269. Nureki, O., T. Niimi, T. Muramatsu, H. Kanno, T. Kohno, C. Florentz, R. Giege, and S. Yokoyama. 1994. Molecular recognition of the identity-determinant set of isoleucine transfer rna from escherichia coli. J. Mol. Biol. 236:710–724. [PubMed] [CrossRef]
270. Nureki, O., K. Watanabe, S. Fukai, R. Ishii, Y. Endo, H. Hori, and S. Yokoyama. 2004. Deep knot structure for construction of active site and cofactor binding site of tRNA modification enzyme. Structure (Cambridge) 12:593–602. [PubMed] [CrossRef]
271. Ny, T., and G. R. Björk. 1977. Stringent regulation of the synthesis of a transfer ribonucleic acid biosynthetic enzyme: transfer ribonucleic acid(m5U)methyltransferase from Escherichia coli. J. Bacteriol. 130:635–641. [PubMed]
272. Ny, T., and G. R. Björk. 1980. Growth rate-dependent regulation of transfer ribonucleic acid (5-methyluridine) methyltransferase in Escherichia coli B/r. J. Bacteriol. 141:67–73. [PubMed]
273. Ny, T., and G. R. Björk. 1980. Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12. J. Bacteriol. 142:371–379.
274. Ny, T., H. R. Lindström, T. G. Hagervall, and G. R. Björk. 1988. Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA. Eur. J. Biochem. 177:467–475. [PubMed] [CrossRef]
275. Ny, T., J. Thomale, K. Hjalmarsson, G. Nass, and G. R. Björk. 1980. Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli. Biochim. Biophys. Acta 607:277–284. [PubMed]
276. O'Dwyer, K., J. M. Watts, S. Biswas, J. Ambrad, M. Barber, H. Brulé, C. Petit, D. J. Holmes, M. Zalacain, and W. M. Holmes. 2004. Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for growth. J. Bacteriol. 186:2346–2354. [PubMed] [CrossRef]
277. Oda, K., F. Kimura, F. Harada, and S. Nishimura. 1969. Restoration of valine acceptor activity by combining oligonucleotide fragments derived from a Bacillus subtilis ribonuclease digest of Escherichia coli valine transfer RNA. Biochim. Biophys. Acta 179:97–105. [PubMed]
278. Okada, N., S. Noguchi, H. Kasai, N. Shindo-Okada, T. Ohgi, T. Goto, and S. Nishimura. 1979. Novel mechanism of post-transcriptional modification of tRNA. Insertion of bases of Q precursors into tRNA by a specific tRNA transglycosylase reaction. J. Biol. Chem. 254:3067–3073. [PubMed]
279. Okada, N., T. Yasuda, and S. Nishimura. 1977. Detection of nucleoside Q precursor in methyl-deficient E.coli tRNA. Nucleic Acids Res. 4:4063–4075. [PubMed] [CrossRef]
280. Ollagnier-de-Choudens, S., T. Mattioli, Y. Takahashi, and M. Fontecave. 2001. Iron-sulfur cluster assembly: characterization of IscA and evidence for a specific and functional complex with ferredoxin. J. Biol. Chem. 276:22604–22607. [PubMed] [CrossRef]
281. Palenchar, P. M., C. J. Buck, H. Cheng, T. J. Larson, and E. G. Mueller. 2000. Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate. J. Biol. Chem. 275:8283–8286. [PubMed] [CrossRef]
282. Palmer, D. T., P. H. Blum, and S. W. Artz. 1983. Effects of the hisT mutation of Salmonella typhimurium on translation elongation rate. J. Bacteriol. 153:357–363. [PubMed]
283. Pan, H., S. Agarwalla, D. T. Moustakas, J. Finer-Moore, and R. M. Stroud. 2003. Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit. Proc. Natl. Acad. Sci. USA 100:12648–12653. [PubMed] [CrossRef]
284. Parker, J. 1982. Specific mistranslation in hisT mutants of Escherichia coli. Mol. Gen. Genet. 187:405–409. [PubMed] [CrossRef]
285. Perret, V., A. Garcia, H. Grosjean, J. P. Ebel, C. Florentz, and R. Giege. 1990. Relaxation of a transfer RNA specificity by removal of modified nucleotides. Nature 344:787–789. [PubMed] [CrossRef]
286. Persson, B. C., and G. R. Björk. 1993. Isolation of the gene (miaE) encoding the hydroxylase involved in the synthesis of 2-methylthio-cis-ribozeatin in tRNA of Salmonella typhimurium and characterization of mutants. J. Bacteriol. 175:7776–7785. [PubMed]
287. Persson, B. C., G. O. Bylund, D. E. Berg, and P. M. Wikström. 1995. Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics. J. Bacteriol. 177:5554–5560. [PubMed]
288. Persson, B. C., C. Gustafsson, D. E. Berg, and G. R. Björk. 1992. The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli. Proc. Natl. Acad. Sci. USA 89:3995–3998. [PubMed] [CrossRef]
289. Persson, B. C., G. Jäger, and C. Gustafsson. 1997. The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity. Nucleic Acids Res. 25:4093–4097. [CrossRef]
290. Petrullo, L. A., P. J. Gallagher, and D. Elseviers. 1983. The role of 2-methylthio-N6-isopentenyladenosine in readthrough and suppression of nonsense codons in Escherichia coli. Mol. Gen. Genet. 190:289–294. [PubMed] [CrossRef]
291. Pierrel, F., G. R. Björk, M. Fontecave, and M. Atta. 2002. Enzymatic modification of tRNAs: MiaB is an iron-sulfur protein. J. Biol. Chem. 277:13367–13370. [PubMed] [CrossRef]
292. Pierrel, F., T. Douki, M. Fontecave, and M. Atta. 2004. MiaB protein is a bifunctional "Radical SAM" enzyme involved in thiolation and methylation of tRNA. J. Biol. Chem. 279:47555-47563. [CrossRef]
293. Pierrel, F., H. L. Hernandez, M. K. Johnson, M. Fontecave, and M. Atta. 2003. MiaB protein from Thermotoga maritima. Characterization of an extremely thermophilic tRNA-methylthiotransferase. J. Biol. Chem. 278:29515–29524. [PubMed] [CrossRef]
294. Pope, W. T., and A. Brown, R. H. Reeves. 1978. The identification of the tRNA substrates for the supK tRNA methylase. Nucleic Acids Res. 5:1041–1057. [PubMed] [CrossRef]
295. Pope, W. T., and R. H. Reeves. 1978. Purification and characterization of a tRNA methylase from Salmonella typhimurium. J. Bacteriol. 136:191–200. [PubMed]
296. Precup, J., and J. Parker. 1987. Missense misreading of asparagine codons as a function of codon identity and context. J. Biol. Chem. 262:11351–11355. [PubMed]
297. Qian, Q., and G. R. Björk. 1997. Structural requirements for the formation of 1-methylguanosine in vivo in tRNAProGGG of Salmonella typhimurium. J. Mol. Biol. 266:283-296. [CrossRef]
298. Qian, Q., and G. R. Björk. 1997. Structural alterations far from the anticodon of the tRNAProGGG of Salmonella typhimurium induce +1 frameshifting at the peptidyl-site. J. Mol. Biol. 273:978-992. [CrossRef]
299. Qian, Q., J. N. Li, H. Zhao, T. G. Hagervall, P. J. Farabaugh, and G. R. Björk. 1998. A new model for phenotypic suppression of frameshift mutations by mutant tRNAs. Mol. Cell 1:471–482. [PubMed] [CrossRef]
300. Qian, Q. A., J. F. Curran, and G. R. Björk. 1998. The methyl group of the N-6-methyl-N-6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA. J. Bacteriol. 180:1808–1813. [PubMed]
301. Quay, S. C., R. P. Lawther, G. W. Hatfield, and D. L. Oxender. 1978. Branched-chain amino acid transport regulation in mutants blocked in tRNA maturation and transcriptional termination. J. Bacteriol. 134:683–686. [PubMed]
302. Quigley, G. J., A. H. Wang, N. C. Seeman, F. L. Suddath, A. Rich, J. L. Sussman, and S. H. Kim. 1975. Hydrogen bonding in yeast phenylalanine transfer RNA. Proc. Natl. Acad. Sci. USA 72:4866–4870. [PubMed] [CrossRef]
303. Ramabhadran, T. V., and J. Jagger. 1976. Mechanism of growth delay induced in Escherichia coli by near ultraviolet radiation. Proc. Natl. Acad. Sci. USA 73:59–63. [PubMed] [CrossRef]
304. Reader, J. S., D. Metzgar, P. Schimmel, and V. De Crecy-Lagard. 2004. Identification of four genes necessary for biosynthesis of the modified nucleoside queuosine. J. Biol. Chem. 279:6280–6285. [PubMed] [CrossRef]
305. Redlak, M., C. AndraosSelim, R. Giege, C. Florentz, and W. M. Holmes. 1997. Interaction of tRNA with tRNA (guanosine-1)methyltransferase: binding specificity determinants involve the dinucleotide G(36)pG(37) and tertiary structure. Biochemistry 36:8699–8709. [PubMed] [CrossRef]
306. Reeves, R. H., and J. R. Roth. 1975. Transfer ribonucleic acid methylase deficiency found in UGA supressor strains. J. Bacteriol. 124:332–340. [PubMed]
307. Reuter, K., R. Slany, F. Ullrich, and H. Kersten. 1991. Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes. J. Bacteriol. 173:2256–2264. [PubMed]
308. Romier, C., K. Reuter, D. Suck, and R. Ficner. 1996. Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile. Biochemistry 35:15734–15739. [PubMed] [CrossRef]
309. Romier, C., K. Reuter, D. Suck, and R. Ficner. 1996. Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange. EMBO J. 15:2850–2857. [PubMed]
310. Roovers, M., J. Wouters, J. M. Bujnicki, C. Tricot, V. Stalon, H. Grosjean, and L. Droogmans. 2004. A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase. Nucleic Acids Res. 32:465–476. [CrossRef]
311. Rosenberg, A. H., and M. L. Gefter. 1969. An iron-dependent modification of several transfer RNA species in Escherichia coli. J. Mol. Biol. 46:581–584. [PubMed] [CrossRef]
312. Rosenfeld, S. A., and J. E. Brenchley. 1980. Regulation of nitrogen utilization of hisT mutants of Salmonella typhimurium. J. Bacteriol. 143:801–808. [PubMed]
313. Ross, W., J. F. Thompson, J. T. Newlands, and R. L. Gourse. 1990. E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 9:3733–3742. [PubMed]
314. Rould, M. A., J. J. Perona, and T. A. Steitz. 1991. Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature 352:213–218. [CrossRef]
315. Ryals, J., R. Y. Hsu, M. N. Lipsett, and H. Bremer. 1982. Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant. J. Bacteriol. 151:899–904. [PubMed]
316. Sakamoto, K., G. Kawai, S. Watanabe, T. Niimi, N. Hayashi, Y. Muto, K. Watanabe, T. Satoh, M. Sekine, and S. Yokoyama. 1996. NMR studies of the effects of the 5'-phosphate group on conformational properties of 5-methylaminomethyluridine found in the first position of the anticodon of Escherichia coli tRNA(Arg)4. Biochemistry 35:6533–6538. [PubMed] [CrossRef]
317. Salazar, J. C., A. Ambrogelly, P. F. Crain, J. A. McCloskey, and D. Söll. 2004. A truncated aminoacyl-tRNA synthetase modifies RNA. Proc. Natl. Acad. Sci. USA 101:7536–7541. [PubMed] [CrossRef]
318. Sampson, J. R., and O. C. Uhlenbeck. 1988. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc. Natl. Acad. Sci. USA 85:1033–1037. [PubMed] [CrossRef]
319. Samuelsson, T., P. Elias, F. Lustig, T. Axberg, G. Fölsch, B. Åkesson, and U. Lagerkvist. 1980. Aberrations of the classic codon reading scheme during protein synthesis in vitro. J. Biol. Chem. 255:4583–4588. [PubMed]
320. Saneyoshi, M., and S. Nishimura. 1971. Selective inactivation of amino acid acceptor and ribosome-binding activities of Escherichia coli tRNA by modification with cyanogen bromide. Biochim. Biophys. Acta 246:123–131. [PubMed]
321. Santi, D. V., and L. W. Hardy. 1987. Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis. Biochemistry 26:8599–8606. [PubMed] [CrossRef]
322. Sarubbi, E., K. E. Rudd, H. Xiao, K. Ikehara, M. Kalman, and M. Cashel. 1989. Characterization of the spoT gene of Escherichia coli. J. Biol. Chem. 264:15074–15082. [PubMed]
323. Scannell, J. P., A. M. Crestfield, and F. W. Allen. 1959. Methylation studies on various uracil drivatives and on an isomer of uridine isolated from ribonucleic acid. Biochim. Biophys. Acta 32:406–412. [PubMed] [CrossRef]
324. Schimmel, P., and L. Ribas De Pouplana. 2000. Footprints of aminoacyl-tRNA synthetases are everywhere. Trends Biochem. Sci. 25:207–209. [PubMed] [CrossRef]
325. Schoenlein, P. V., B. B. Roa, and M. E. Winkler. 1989. Divergent transcription of pdxB and homology between the pdxB and serA gene products in Escherichia coli K-12. J. Bacteriol. 171:6084–6092. [PubMed]
326. Schön, A., A. Böck, G. Ott, M. Sprinzl, and D. Söll. 1989. The selenocysteine-inserting opal suppressor serine tRNA from E. coli is highly unusual in structure and modification. Nucleic Acids Res. 17:7159–7165. [PubMed] [CrossRef]
327. Schubert, H. L., R. M. Blumenthal, and X. Cheng. 2003. Many paths to methyltransfer: a chronicle of convergence. Trends Biochem. Sci. 28:329–335. [PubMed] [CrossRef]
328. Schulman, L. H., and H. Pelka. 1988. Anticodon switching changes the identity of methionine and valine transfer RNAs. Science 242:765–768. [PubMed] [CrossRef]
329. Schulman, L. H., and H. Pelka. 1990. An anticodon change switches the identity of E. coli tRNAMetm from methionine to threonine. Nucleic Acids Res. 18:285–289. [PubMed] [CrossRef]
330. Schulman, L. H., H. Pelka, and M. Susani. 1983. Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase. Nucleic Acids Res. 11:1439–1455. [PubMed] [CrossRef]
331. Schwartz, C. J., J. L. Giel, T. Patschkowski, C. Luther, F. J. Ruzicka, H. Beinert, and P. J. Kiley. 2001. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc. Natl. Acad. Sci. USA 98:14895–14900. [PubMed] [CrossRef]
332. Scrima, A., I. R. Vetter, M. E. Armengod, and A. Wittinghofer. 2005. The structure of the TrmE GTP-binding protein and its implications for tRNA modification. EMBO J. 24:23-33. [CrossRef]
333. Sekiya, T., K. Takeishi, and T. Ukita. 1969. Specificity of yeast glutamic acid transfer RNA for codon recognition. Biochim. Biophys. Acta 182:411–426. [PubMed]
334. Senger, B., S. Auxilien, U. Englisch, F. Cramer, and F. Fasiolo. 1997. The modified wobble base inosine in yeast tRNA(Ile) is a positive determinant for aminoacylation by isoleucyl tRNA synthetase. Biochemistry 36:8269–8275. [PubMed] [CrossRef]
335. Sengupta, R., S. Vainauskas, C. Yarian, E. Sochacka, A. Malkiewicz, R. H. Guenther, K. M. Koshlap, and P. F. Agris. 2000. Modified constructs of the tRNA T Psi C domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases. Nucleic Acids Res. 28:1374–1380. [PubMed] [CrossRef]
336. Seno, T., P. F. Agris, and D. Söll. 1974. Involvement of the anticodon region of Escherichia coli tRNAGln and tRNAGlu in the specific interaction with cognate aminoacyl-tRNA synthetase. Alteration of the 2-thiouridine derivatives located in the anticodon of the tRNAs by BrCN or sulfur deprivation. Biochim. Biophys. Acta 349:328–338. [PubMed]
337. Sha, J., E. V. Kozlova, A. A. Fadl, J. P. Olano, C. W. Houston, J. W. Peterson, and A. K. Chopra. 2004. Molecular characterization of a glucose-inhibited division gene, gidA, that regulates cytotoxic enterotoxin of Aeromonas hydrophila. Infect. Immun. 72:1084–1095. [PubMed] [CrossRef]
338. Singhal, R. P., R. A. Kopper, S. Nishimura, and N. Shindo-Okada. 1981. Modification of guanine to queuine in transfer RNAs during development and aging. Biochem. Biophys. Res. Commun. 99:120–126. [PubMed] [CrossRef]
339. Sivaraman, J., V. Sauvé, R. Larocque, E. A. Stura, J. D. Schrag, M. Cygler, and A. Matte. 2002. Structure of the 16S rRNA pseudouridine synthase RsuA bound to uracil and UMP. Nat. Struct. Biol. 9:353–358. [PubMed]
340. Slany, R. K., M. Bösl, P. F. Crain, and H. Kersten. 1993. A new function of S-adenosylmethionine: the ribosyl moiety of AdoMet is the precursor of the cyclopentenediol moiety of the tRNA wobble base queuine. Biochemistry 32:7811–7817. [PubMed] [CrossRef]
341. Slany, R. K., and H. Kersten. 1992. The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site. Nucleic Acids Res. 20:4193–4198. [PubMed] [CrossRef]
342. Smith, A. D., J. N. Agar, K. A. Johnson, J. Frazzon, I. J. Amster, D. R. Dean, and M. K. Johnson. 2001. Sulfur transfer from IscS to IscU: the first step in iron-sulfur cluster biosynthesis. J. Am. Chem. Soc. 123:11103–11104. [PubMed] [CrossRef]
343. Smith, J. D., and D. B. Dunn. 1959. The occurence of methylated guanines in ribonucleic acids from several sources. Biochem. J. 72:294–300. [PubMed]
344. Smith, W. S., H. Sierzputowska-Gracz, E. Sochacka, A. Malkiewicz, and P. F. Agris. 1992. Chemistry and structure of modified uridine dinucleosides are determined by thiolation. J. Am. Chem. Soc. 114:7989–7997. [CrossRef]
345. Soderberg, T., and C. D. Poulter. 2000. Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop. Biochemistry 39:6546–6553. [PubMed] [CrossRef]
346. Soderberg, T., and C. D. Poulter. 2001. Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues. Biochemistry 40:1734–1740. [PubMed] [CrossRef]
347. Sofia, H. J., G. Chen, B. G. Hetzler, J. F. ReyesSpindola, and N. E. Miller. 2001. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 29:1097–1106. [PubMed] [CrossRef]
348. Soma, A., Y. Ikeuchi, S. Kanemasa, K. Kobayashi, N. Ogasawara, T. Ote, J. Kato, K. Watanabe, Y. Sekine, and T. Suzuki. 2003. An RNA-modifying enzyme that governs both the codon and amino acid specificities of isoleucine tRNA. Mol. Cell 12:689–698. [PubMed] [CrossRef]
349. Spadaro, A., A. Spena, V. Santonastaso, and P. Conini. 1981. Stringency without ppGpp accumulation. Nature 291:256–258. [PubMed] [CrossRef]
350. Spanjaard, R. A., K. Chen, J. R. Walker, and J. van Duin. 1990. Frameshift suppression at tandem AGA and AGG codons by cloned tRNA genes: assigning a codon to argU tRNA and T4 tRNAArg. Nucleic Acids Res. 18:5031–5036. [PubMed] [CrossRef]
351. Sprinzl, M., and K. S. Vassilenko. 2003. tRNA sequences and tRNA genes database. [Online.] http://www.staff.uni-bayreuth.de/~btc914/search/
352. Sroga, G. E., F. Nemoto, Y. Kuchino, and G. R. Björk. 1992. Insertion (sufB) in the anticodon loop or base substitution (sufC) in the anticodon stem of tRNAProI2 from Salmonella typhimurium induces suppression of frameshift mutations. Nucleic Acids Res. 20:3463–3469. [PubMed] [CrossRef]
353. Stadtman, T. C. 1996. Selenocysteine. Annu. Rev. Biochem. 65:83–100. [PubMed] [CrossRef]
354. Stahl, G., G. P. McCarty, and P. J. Farabaugh. 2002. Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding. Trends Biochem. Sci. 27:178–183. [PubMed] [CrossRef]
355. Stern, L., and L. H. Schulman. 1977. Role of anticodon bases in aminoacylation of Escherichia coli methionine transfer RNAs. J. Biol. Chem. 252:6403–6408. [PubMed]
356. Stern, L., and L. H. Schulman. 1978. The role of the minor base N4-acetylcytidine in the function of the Escherichia coli noninitiator methionine transfer RNA. J. Biol. Chem. 253:6132–6139. [PubMed]
357. Stuart, J. W., K. M. Koshlap, R. Guenther, and P. F. Agris. 2003. Naturally-occurring Modification Restricts the Anticodon Domain Conformational Space of tRNA(Phe). J. Mol. Biol. 334:901–918. [PubMed] [CrossRef]
358. Sullivan, M. A., J. F. Cannon, F. H. Webb, and R. M. Bock. 1985. Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine. J. Bacteriol. 161:368–376. [PubMed]
359. Sundaram, M., P. C. Durant, and D. R. Davis. 2000. Hypermodified nucleosides in the anticodon of tRNA(Lys) stabilize a canonical U-turn structure. Biochemistry 39:12575–12584. [PubMed] [CrossRef]
360. Svensson, I., H. G. Boman, K. G. Eriksson, and K. Kjellin. 1963. Studies on microbial RNA. I. Transfer of methyl groups from methionine to soluble RNA from Escherichia coli. J. Mol. Biol. 7:254–271. [PubMed]
361. Sylvers, L. A., K. C. Rogers, M. Shimizu, E. Ohtsuka, and D. Söll. 1993. A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase. Biochemistry 32:3836–3841. [PubMed] [CrossRef]
362. Takai, K., N. Horie, Z. Yamaizumi, S. Nishimura, T. Miyazawa, and S. Yokoyama. 1994. Recognition of UUN codons by two leucine tRNA species from Escherichia coli. FEBS Lett. 344:31–34. [PubMed] [CrossRef]
363. Takai, K., S. Okumura, K. Hosono, S. Yokoyama, and H. Takaku. 1999. A single uridine modification at the wobble position of an artificial tRNA enhances wobbling in an Escherichia coli cell-free translation system. FEBS Lett. 447:1–4. [PubMed] [CrossRef]
364. Takai, K., and S. Yokoyama. 2003. Roles of 5-substituents of tRNA wobble uridines in the recognition of purine-ending codons. Nucleic Acids Res. 31:6383–6391. [PubMed] [CrossRef]
365. Tamura, K., H. Himeno, H. Asahara, T. Hasegawa, and M. Shimizu. 1992. In vitro study of E.coli tRNAArg and tRNALys identity elements. Nucleic Acids Res. 20:2335–2339. [PubMed] [CrossRef]
366. Thomale, J., and G. Nass. 1978. Alteration of the intracellular concentration of aminoacyl-tRNA synthetases and isoaccepting tRNAs during amino-acid limited growth in Escherichia coli. Eur. J. Biochem. 85:407–418. [PubMed] [CrossRef]
367. Thomas, G., and A. Favre. 1975. 4-Thiouridine as the target for near-ultraviolet light induced growth delay in Escherichia coli. Biochem. Biophys. Res. Commun. 66:1454–1461. [PubMed] [CrossRef]
368. Thomas, G., and A. Favre. 1980. 4-Thiouridine triggers both growth delay induced by near-ultraviolet light and photoprotection. Eur. J. Biochem. 113:67–74. [PubMed]
369. Thomas, G., K. Thiam, and A. Favre. 1981. tRNA thiolated pyrimidines as targets for near-ultraviolet-induced synthesis of guanosine tetraphosphate in Escherichia coli. Eur. J. Biochem. 119:381–387. [PubMed] [CrossRef]
370. Travers, A. 1980. Promoter sequence for stringent control of bacterial ribonucleic acid synthesis. J. Bacteriol. 141:973–976. [PubMed]
371. Tsang, T. H., M. Buck, and B. N. Ames. 1983. Sequence specificity of tRNA-modifying enzymes. An analysis of 258 tRNA sequences. Biochim. Biophys. Acta 741:180–196. [PubMed]
372. Tsui, H. C., G. Feng, and M. E. Winkler. 1996. Transcription of the mutL repair, miaA tRNA modification, hfq pleiotropic regulator, and hflA region protease genes of Escherichia coli K-12 from clustered Esigma32-specific promoters during heat shock. J. Bacteriol. 178:5719–5731. [PubMed]
373. Tsui, H.-C. T., P. J. Arps, D. M. Connolly, and M. E. Winkler. 1991. Absence of hisT-mediated tRNA pseudouridylation results in a uracil requirement that interferes with Escherichia coli K-12 cell division. J. Bacteriol. 173:7395–7400. [PubMed]
374. Tsui, H. C. T., G. Feng, and M. E. Winkler. 1997. Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12. J. Bacteriol. 179:7476–7487. [PubMed]
375. Tsui, H. C. T., H. C. E. Leung, and M. E. Winkler. 1994. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol. Microbiol. 13:35–49. [PubMed] [CrossRef]
376. Tsui, H. C. T., G. S. Zhao, G. Feng, H. C. E. Leung, and M. E. Winkler. 1994. The mutL repair gene of Escherichia coli K-12 forms a superoperon with a gene encoding a new cell-wall amidase. Mol. Microbiol. 11:189–202. [PubMed] [CrossRef]
377. Turnbough, C. L., Jr., R. J. Neill, R. Landsberg, and B. N. Ames. 1979. Pseudouridylation of tRNAs and its role in regulation in Salmonella typhimurium. J. Biol. Chem. 254:5111–5119. [PubMed]
378. Turner, D. H., and P. C. Bevilacqua. 1993. Thermodynamic considerations for evolution by RNA, p. 447–464. In R. F. Gesterland and J. F. Atkins (ed.), The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
379. Urbina, H. D., J. J. Silberg, K. G. Hoff, and L. E. Vickery. 2001. Transfer of sulfur from IscS to IscU during Fe/S cluster assembly. J. Biol. Chem. 276:44521–44526. [PubMed] [CrossRef]
380. Urbonavicius, J., J. M. Durand, and G. R. Björk. 2002. Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri. J. Bacteriol. 184:5348–5357. [PubMed] [CrossRef]
381. Urbonavicius, J., Q. Qian, J. M. Durand, T. G. Hagervall, and G. R. Björk. 2001. Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J. 20:4863–4873. [PubMed] [CrossRef]
382. Urbonavicius, J., G. Stahl, J. M. Durand, S. N. Ben Salem, Q. Qian, P. J. Farabaugh, and G. R. Björk. 2003. Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting. RNA 9:760–768. [PubMed] [CrossRef]
383. Vacher, J., H. Grosjean, C. Houssier, and R. H. Buckingham. 1984. The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression. J. Mol. Biol. 177:329–342. [PubMed] [CrossRef]
384. Van Lanen, S. G., S. Daoud Kinzie, S. Matthieu, T. Link, J. Culp, and D. Iwata-Reuyl. 2003. tRNA modification by S-adenosylmethionine: tRNA ribosyltransferase-isomerase (QueA): assay development and characterization of the recombinant enzyme. J. Biol. Chem. 278:10491–10499. [PubMed] [CrossRef]
385. Verbeek, H., L. Nilsson, G. Baliko, and L. Bosch. 1990. Potential binding sites of the trans-activator FIS are present upstream of all rRNA operons and of many but not all tRNA operons. Biochim. Biophys. Acta 1050:302–306. [PubMed]
386. Veres, Z., I. Y. Kim, T. D. Scholz, and T. C. Stadtman. 1994. Selenophosphate synthetase—enzyme properties and catalytic reaction. J. Biol. Chem. 269:10597–10603. [PubMed]
387. Veres, Z., and T. C. Stadtman. 1994. A purified selenophosphate-dependent enzyme from Salmonella typhimurium catalyzes the replacement of sulfur in 2-thiouridine residues in tRNAs with selenium. Proc. Natl. Acad. Sci. USA 91:8092–8096. [PubMed] [CrossRef]
388. Weiss, R. B., D. M. Dunn, J. F. Atkins, and R. F. Gesteland. 1990. Ribosomal frameshifting from -2 to +50 nucleotides. Prog. Nucleic Acid Res. Mol. Biol. 39:159–183. [PubMed] [CrossRef]
389. Weissenbach, J., and H. Grosjean. 1981. Effect of threonylcarbamoyl modification (t6A) in yeast tRNAArgIII on codon-anticodon and anticodon-anticodon interactions. A thermodynamic and kinetic evaluation. Eur. J. Biochem. 116:207–213. [PubMed] [CrossRef]
390. Wettstein, F. O., and G. S. Stent. 1968. Physiologically induced changes in the property of phenylalanine tRNA in Escherichia coli. J. Mol. Biol. 38:25–40. [PubMed] [CrossRef]
391. White, D. J., R. Merod, B. Thomasson, and P. L. Hartzell. 2001. GidA is an FAD-binding protein involved in development of Myxococcus xanthus. Mol. Microbiol. 42:503–517. [PubMed] [CrossRef]
392. Wikström, P. M., and G. R. Björk. 1988. Noncoordinate translation-level regulation of ribosomal and nonribosomal protein genes in the Escherichia coli trmD operon. J. Bacteriol. 170:3025–3031. [PubMed]
393. Wikström, P. M., A. S. Byström, and G. R. Björk. 1988. Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli. J. Mol. Biol. 203:141–152. [PubMed] [CrossRef]
394. Wikström, P. M., L. K. Lind, D. E. Berg, and G. R. Björk. 1992. Importance of mRNA folding and start codon accessibility in the expression of genes in a ribosomal protein operon of Escherichia coli. J. Mol. Biol. 224:949–966. [PubMed] [CrossRef]
395. Willick, G. E., and C. M. Kay. 1976. Circular dichroism study of the interaction of glutamyl-tRNA synthetase with tRNAGlu2. Biochemistry 15:4347–4352. [PubMed] [CrossRef]
396. Wilson, R. K., and B. A. Roe. 1989. Presence of the hypermodified nucleotide N6-(delta2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA. Proc. Natl. Acad. Sci. USA 86:409–413. [PubMed] [CrossRef]
397. Winkler, M. E. 1998. Genetics and regulation of base modification in tRNA and rRNA of prokaryotes and eukaryotes. In H. Grosjean and R. Benne (ed.), Modification and Editing of RNA. ASM Press, Washington, D.C.
398. Wittwer, A. J. 1983. Specific incorporation of selenium into lysine- and glutamate-accepting tRNAs from Escherichia coli. J. Biol. Chem. 258:8637–8641. [PubMed]
399. Wittwer, A. J., and W. M. Ching. 1989. Selenium-containing tRNAGlu and tRNALys from Escherichia coli: purification, codon specificity and translational activity. Biofactors 2:27–34. [PubMed]
400. Wittwer, A. J., and T. C. Stadtman. 1986. Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA. Arch. Biochem. Biophys. 248:540–550. [PubMed] [CrossRef]
401. Wittwer, A. J., L. Tsai, W. M. Ching, and T. C. Stadtman. 1984. Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-:(methylamino)methyl:-2-selenouridine. Biochemistry 23:4650–4655. [PubMed] [CrossRef]
402. Woese, C. R., O. Kandler, and M. L. Wheelis. 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87:4576–4579. [PubMed] [CrossRef]
403. Wolf, J., A. P. Gerber, and W. Keller. 2002. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. EMBO J. 21:3841–3851. [PubMed] [CrossRef]
404. Wolfe, M. D., F. Ahmed, G. M. Lacourciere, C. T. Lauhon, T. C. Stadtman, and T. J. Larson. 2004. Functional diversity of the Rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase. J. Biol. Chem. 279:1801–1809. [PubMed] [CrossRef]
405. Wright, C. M., P. M. Palenchar, and E. G. Mueller. 2002. A paradigm for biological sulfur transfers via persulfide groups: a persulfide-disulfide-thiol cycle in 4-thiouridine biosynthesis. Chem. Commun. 22:2708–2709. [PubMed] [CrossRef]
406. Wrzesinski, J., K. Nurse, A. Bakin, B. G. Lane, and J. Ofengand. 1995. A dual-specificity pseudouridine synthase–an Escherichia coli synthase purified and cloned on the basis of its specificity for psi-746 in 23S RNA is also specific for psi-32 in tRNA(Phe). RNA 1:437–448. [PubMed]
407. Xie, W., X. Liu, and R. H. Huang. 2003. Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate. Nat. Struct. Biol. 10:781–788. [PubMed] [CrossRef]
408. Yamamoto, Y., S. Yokoyama, T. Miyazawa, K. Watanabe, and S. Higuchi. 1983. NMR analyses on the molecular mechanism of the conformational rigidity of 2-thioribothymidine, a modified nucleoside in extreme thermophile tRNAs. FEBS Lett. 157:95–99. [PubMed] [CrossRef]
409. Yao, L. J., T. L. James, J. T. Kealey, D. V. Santi, and U. Schmitz. 1997. The dynamic NMR structure of the T psi C-loop: implications for the specificity of tRNA methylation. J. Biomol. NMR 9:229–244. [PubMed] [CrossRef]
410. Yarian, C., H. Townsend, W. Czestkowski, E. Sochacka, A. J. Malkiewicz, R. Guenther, A. Miskiewicz, and P. F. Agris. 2002. Accurate translation of the genetic code depends on tRNA modified nucleosides. J. Biol. Chem. 277:16391–16395. [PubMed] [CrossRef]
411. Yarus, M. 1982. Translational efficiency of transfer RNAs: uses of an extended anticodon. Science 218:646–652. [PubMed] [CrossRef]
412. Yim, L., M. Martínez-Vicente, M. Villarroya, C. Aguado, E. Knecht, and M. E. Armengod. 2003. The GTPase activity and C-terminal cysteine of the Escherichia coli MnmE protein are essential for its tRNA modifying function. J. Biol. Chem. 278:28378–28387. [PubMed] [CrossRef]
413. Yokoyama, S., and S. Nishimura. 1995. Modified nucleosides and codon recognition, p. 207–223. In D. Söll and U. L. Rajbhandary (ed.), tRNA: Structure, Biosynthesis, and Function. ASM Press, Washington, D.C.
414. Yokoyama, S., T. Watanabe, K. Murao, H. Ishikura, Z. Yamaizumi, S. Nishimura, and T. Miyazawa. 1985. Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon. Proc. Natl. Acad. Sci. USA 82:4905–4909. [PubMed] [CrossRef]
415. Yu, C.-T., and F. W. Allen. 1959. Studies on an isomer of uridine isolated from ribonucleic acid. Biochim. Biophys. Acta 32:393–406. [PubMed] [CrossRef]
416. Zhang, A., K. M. Wassarman, C. Rosenow, B. C. Tjaden, G. Storz, and S. Gottesman. 2003. Global analysis of small RNA and mRNA targets of Hfq. Mol. Microbiol. 50:1111–1124.[PubMed] [CrossRef]
Module4.6.2
