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Transfer RNAs from all organisms contain many modified nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their influence on different reactions in which tRNA participates suggest that each modified nucleoside may have its own specific function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modification was higher compared with the corresponding wild-type controls, these modifications have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl-tRNA slippage, which is influenced by the hypomodified tRNA in two ways: (i) a hypomodified tRNA in the ternary complex may decrease the rate by which the complex is recruited to the A-site and thereby increasing peptidyl-tRNA slippage; or (ii) a hypomodified peptidyl-tRNA may be more prone to slip than its fully modified counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modified nucleosides.

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Transfer RNA modification improves the rate of aa-tRNA selection at the A-site and the fitness in the P-site and thereby prevents frameshifting according to a new model how frameshifting occurs [Qian et al. (1998) Mol. Cell 1, 471-482]. Evidence that the presence of various modified nucleosides in tRNA also influences central metabolism, thiamine metabolism, and bacterial virulence is reviewed.

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It has been suggested that modified nucleosides of the xm5(s2)U(m)34-type restrict the wobble capacity of the base, and that their function is to prevent misreading in the third position of the codon in mixed codon family boxes that encode two different amino acids. In this study in Escherichia coli, the misreading in vivo of asparagine codons in bacteriophage MS2 mRNA by different hypomodified derivatives of tRNALys, normally containing 5-methylaminomethyl-2-thiouridine (mnm5s2U34) in the wobble position, has been analysed. Contrary to what would be predicted from the general hypothesis for the function of mnm5s2U, it was found that the misreading of asparagine codons by tRNALys was greatly reduced in the mnmA (formerly asuE or trmU) and mnmE (formerly trmE) mutants which contain the hypomodified mnm5U34 and s2U34, respectively, instead of the fully modified mnm5s2U34. In addition, it was found that these hypomodified tRNAs were efficiently charged with lysine in vivo, under the growth conditions employed. The latter result is at variance with results obtained in vitro. The results are discussed in relation to the postulated function for modified nucleosides of the xm5s2U type.

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In Escherichia coli, uridine in the wobble position of tRNAGlu and tRNALys is modified to mnm5s2U34. This modification is believed to restrict the base-pairing capability, i.e. to prevent misreading of near-cognate codons and reduce the efficiency of cognate codon reading, especially of codons ending in G. We have determined the influence of the 5-methylaminomethyl and the 2-thio modifications of mnm5s2U34 in tRNAGlu on the translation rate of the glutamate codons GAA and GAG in vivo. In wild-type cells, GAG is translated slower (7. 7 codons/second) and GAA faster (18 codons/second) than the average codon (13 codons/second). Surprisingly, tRNAGlu lacking the 5-methylaminomethyl group, thus containing s2U34, translated GAA twofold faster (47 codons/second) and GAG fourfold slower (1.9 codons/second) than fully modified tRNAGlu. In contrast, tRNAGlu that contains mnm5U34 instead of mnm5s2U34 translated GAA fourfold slower (4.5 codons/second) and GAG only 20% slower (6.2 codons/second). Clearly, the 5-methylaminomethyl group of mnm5s2U34 facilitates base-pairing with G while decreasing base-pairing with A, resulting in rates of translation of GAG and GAA that approach that of the average codon. The 2-thio group increases the recognition of GAA and has only a minor effect on the decoding of GAG. Furthermore, the 2-thio group is important for aminoacylation (see the accompanying paper). These data imply that the function of mnm5s2U34 may be different from what has been suggested previously.

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According to the prevailing model, frameshift-suppressing tRNAs with an extra nucleotide in the anticodon loop suppress +1 frameshift mutations by recognizing a four-base codon and promoting quadruplet translocation. We present three sets of experiments that suggest a general alternative to this model. First, base modification should actually block such a four-base interaction by two classical frameshift suppressors. Second, for one Salmonella suppressor tRNA, it is not mutant tRNA but a structurally normal near cognate that causes the +1 shift in-frame. Finally, frameshifting occurs in competition with normal decoding of the next in-frame codon, consistent with an event that occurs in the ribosomal P site after the translocation step. These results suggest an alternative model involving peptidyl-tRNA slippage at the classical CCC-N and GGG-N frameshift suppression sites.

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Eukaryotic ribosomal frameshift signals generally contain two elements, a heptanucleotide slippery sequence (XXXYYYN) and an RNA secondary structure, often an RNA pseudoknot, located downstream. Frameshifting takes place at the slippery sequence by simultaneous slippage of two ribosome-bound tRNAs. All of the tRNAs that are predicted to decode frameshift sites in the ribosomal A-site (XXXYYYN) possess a hypermodified base in the anticodon-loop and it is conceivable that these modifications play a role in the frameshift process. To test this, we expressed slippery sequence variants of the coronavirus IBV frameshift signal in strains of Escherichia coli unable to modify fully either tRNA(Lys) or tRNA(Asn). At the slippery sequences UUUAAAC and UUUAAAU (underlined codon decoded by tRNA(Asn), anticodon 5' QUU 3'), frameshifting was very inefficient (2 to 3%) and in strains deficient in the biosynthesis of Q base, was increased (AAU) or decreased (AAC) only two-fold. In E. coli, therefore, hypomodification of tRNA(Asn) had little effect on frameshifting. The situation with the efficient slippery sequences UUUAAAA (15%) and UUUAAAG (40%) (underlined codon decoded by tRNA(Lys), anticodon 5' mnm5s2UUU 3') was more complex, since the wobble base of tRNA(Lys) is modified at two positions. Of four available mutants, only trmE (s2UUU) had a marked influence on frameshifting, increasing the efficiency of the process at the slippery sequence UUUAAAA. No effect on frameshifting was seen in trmC1 (cmnm5s2UUU) or trmC2 (nm5s2UUU) strains and only a very small reduction (at UUUAAAG) was observed in an asuE (mnm5UUU) strain. The slipperiness of tRNA(Lys), therefore, cannot be ascribed to a single modification site on the base. However, the data support a role for the amino group of the mnm5 substitution in shaping the anticodon structure. Whether these conclusions can be extended to eukaryotic translation systems is uncertain. Although E. coli ribosomes changed frame at the IBV signal (UUUAAAG) with an efficiency similar to that measured in reticulocyte lysates (40%), there were important qualitative differences. Frameshifting of prokaryotic ribosomes was pseudoknot-independent (although secondary structure dependent) and appeared to require slippage of only a single tRNA.

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The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons. A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity. In this study, the mechanism of this trmD3-induced frameshift involving mutant tRNA(Pro) and tRNA(Leu) species has been investigated. Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain. Hybrid beta-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing. The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNA(Pro) deficient in m1G is the frameshifting agent. Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3-dependent +1 frameshifting. Of the four potential sites for tRNA(Leu) (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant. Thus, species of tRNA(Leu) may also act as +1 frameshift suppressors. No -1 frameshifting activity was found with the trmD3 mutant.

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The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and upstream from the -10 region is a sequence, TCCC, in the trmA promoter that is present in all of the seven rRNA P1 promoters and in some tRNA promoters but not in any other sigma 70 promoter. However, a similar motif is also found in promoters transcribed by the heat shock sigma factor sigma 32. The trmA gene is transcribed as a monocistronic operon, and the 3' end of the transcript is shown to be located downstream from a dyad symmetry region not followed by a poly(U) stretch. Using a trmA-cat operon fusion, we show that the growth rate-dependent regulation of trmA resembles that of rRNA and operates at the level of transcription.

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In transfer RNA many different modified nucleosides are found, especially in the anticodon region. In this region, pseudouridine (psi) is found in positions 38, 39 or 40 in a subset of tRNA species, 2-methylthio-6-hydroxyisopentenyladenosine (ms2io6A) is found in position 37 in tRNAs that read codons starting with U and 1-methylguanosine (m1G) is found in position 37 in tRNAs reading codons of the UCCNG type. We have used the mutants hisT, miaA and miaB and trmD, which are deficient in the biosynthesis of psi, ms2io6A, and m1G, respectively, to study the functional aspects of the respective modified nucleosides. We have shown: (1) Presence of psi improved the cellular growth rate, the polypeptide step-time, and the efficiency of an amber suppressor, but did not appreciably sense the codon context. (2) Presence of ms2io6A improved the cellular growth rate, the polypeptide step-time and the efficiency of several amber suppressor tRNAs. It also had a profound effect on the codon context sensitivity of the tRNA. (3) Presence of m1G improved the cellular growth rate and the polypeptide steptime and also prevented the tRNA from shifting the reading frame. Thus, these three modified nucleosides present in the anticodon region have apparently different functions.

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Chorismic acid is the common precursor for the biosynthesis of the three aromatic amino acids as well as for four vitamins. Mutants of Escherichia coli defective in any of the genes involved in the synthesis of chorismic acid are also unable to synthesize uridine 5-oxyacetic acid (cmo5U) and its methyl ester (mcmo5U). Both modified nucleosides are normally present in the wobble position of some tRNA species. Mutants defective in any of the specific pathways leading to phenylalanine, tyrosine, tryptophan, folate, enterochelin, ubiquinone, and menaquinone have normal levels of cmo5U and mcmo5U in their tRNA. The presence of shikimic acid in the growth medium restores the ability of an aroD mutant to synthesize cmo5U, while O-succinylbenzoate, which is an early intermediate in the synthesis of menaquinone, does not. Thus, chorismic acid is a key metabolite in the synthesis of these two modified nucleosides in tRNA. The absence of chorismic acid blocks the formation of cmo5U and mcmo5U at the first step, which might be the formation of 5-hydroxyuridine. This results in an unmodified U in the wobble position of tRNA(1Val) and in most of the tRNAs normally containing cmo5U and mcmo5U. Since cmo5U and mcmo5U are synthesized under anaerobic conditions, the formation of these nucleosides does not require molecular oxygen. One of the carbon atoms of the side chain, --O--CH2--COOH, originates from the methyl group of methionine. The other carbon atom does not originate directly from the C-1 pool, from the carboxyl group methionine, or from bicarbonate. This metabolic link between intermediary metabolism and translation also exists for another member of the family Enterobacteriaceae, Salmonella typhimurium, as well as for the distantly related gram-positive organism Bacillus subtilis.

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tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex. All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively.

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The tRNA(5-methylaminomethyl-2-thiouridine)-methyltransferase, which is involved in the biosynthesis of the modified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) present in the wobble position of some tRNAs, was purified close to homogeneity (95% purity). The molecular mass of the enzyme is 79,000 daltons. The enzyme activity has a pH optimum of 8.0-8.5, is inhibited by magnesium ions, and stimulated by ammonium ions. Two different intermediates in the biosynthesis of mnm5s2U34 are present in tRNA from the mutants trmC1 and trmC2. Unexpectedly, the product present in tRNA from trmC1 cells was identified by mass spectrometric and chromatographic analyses as 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), i.e. a more complex derivative than the final product mnm5s2U. The product present in tRNA from trmC2 cells was identified as 5-aminomethyl-2-thiouridine (nm5s2U). In the presence of S-adenosylmethionine the most purified enzyme fraction converts both cmnm5s2U34 and nm5s2U34 into mnm5s2U34. In the absence of S-adenosylmethionine, however, cmnm5s2U34 is converted into nm5s2U by this enzyme fraction. We conclude that the purified polypeptide has two enzymatic activities; one actually demodifies cmnm5s2U to nm5s2U and the other catalyzes the transfer of a methyl group from S-adenosylmethionine to nm5s2U, thus forming mnm5s2U. The sequential order of the biosynthesis of mnm5s2U34 is suggested to be: (Formula: see text). The molecular activity of the methyltransferase activity (nm5s2U34----mnm5s2U34) is 74 min-1, and the steady state concentration of the enzyme is only 78 molecules/genome equivalent in cells growing at a specific growth rate of 1.0/h.

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Two mutants of Escherichia coli, trmC1 and trmC2, which are both defective in the synthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U) were utilized to study the function of this complex modified nucleoside. Transfer RNAs specific for glutamine, glutamic acid and lysine as well as a specific ochre suppressor derived from lysine tRNA (tRNAUAAlys encoded by the supG allele), contain this modified nucleoside at position 34 (the wobble position). It was found that two different undermodified derivatives of mnm5s2U were present in the two trmC mutants, which suggests that the two mutations affect two different enzymatic activities. Using the lacI-Z fusion system (Miller and Albertini 1983), we found that the efficiency of supG-mediated suppression was reduced to 30%-90% of the wild-type value in the trmC mutants. The modification-deficient supG-tRNA in the mutants showed a higher sensitivity to codon context than the normal tRNAUAAlys.

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The trmC gene, responsible for the formation of 5-methylaminomethyl-2-thiouridine (mnm5s2U) from 2-thiouridine, present in the first position in the anticodon of some tRNAs, has been located at 50.5 min on the Escherichia coli K12 chromosome. Results from transductional mapping suggest that the trmC gene is located counter-clockwise of aroC. A ColE1 hybrid plasmid carrying the aroC+, trmC+ and hisT+ genes was isolated, and the gene order was established, by subcloning, to be hisT-trmC-aroC. The trmC gene is located 1.9 kb from the aroC gene. Two mutations (trmC1 and trmC2) were shown to be recessive, suggesting that the trmC gene is the structural gene for the tRNA-(mnm5s2U)methyltransferase.

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