The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA.

A Salmonella typhimurium LT2 mutant which harbors a mutation (miaB2508::Tn10dCm) that results in a reduction in the activities of the amber suppressors supF30 (tRNA(CUATyr)), supD10 (tRNA(CUASer)), and supJ60 (tRNA(CUALeu)) was isolated. The mutant was deficient in the methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), a modified nucleoside that is normally present next to the anticodon (position 37) in tRNAs that read codons that start with uridine. Consequently, the mutant had i6A37 instead of ms2io6A37 in its tRNA. Only small amounts of io6A37 was found. We suggest that the synthesis of ms2io6A occurs in the following order: A-37-->i6A37-->ms2i6A37-->ms2io6A37. The mutation miaB2508::Tn10dCm was 60% linked to the nag gene (min 15) and 40% linked to the fur gene and is located counterclockwise from both of these genes. The growth rates of the mutant in four growth media did not significantly deviate from those of a wild-type strain. The polypeptide chain elongation rate was also unaffected in the mutant. However, the miaB2508::Tn10dCm mutation rendered the cell more resistant or sensitive, compared with a wild-type cell, to several amino acid analogs, suggesting that this mutation influences the regulation of several amino acid biosynthetic operons. The efficiencies of the aforementioned amber suppressors were decreased to as low as 16%, depending on the suppressor and the codon context monitored, demonstrating that the ms2 group of ms2io6A contributes to the decoding efficiency of tRNA. However, the major impact of the ms2io6 modification in the decoding process comes from the io6 group alone or from the combination of the ms2 and io6 groups, not from the ms2 group alone.     

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.     

tRNA anticodons with the modified nucleoside 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine distinguish between bases 3' of the codon

The modified nucleoside 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) is present immediately to the 3' side of the anticodon (position 37) in tRNAs that read codons starting with uridine and hence include amber (UAG) suppressor tRNAs. We have used strains of Salmonella typhimurium that differ only in their ability to synthesize ms2io6A in order to determine specifically how this modified nucleoside influences the efficiency of amber suppression in two codon contexts differing by only which base is 3' of the codon. The results show that the presence of the modified nucleoside ms2io6A not only improves the efficiency of the suppressor tRNAs but also allows them to distinguish between at least two bases 3' of the codon. Thus, the presence of ms2io6A reduces the intrinsic codon context sensitivity of the tRNA and specifically counteracts an unfavourable nucleotide on the 3' side of the codon. The possible codon-anticodon interactions responsible for this effect are discussed.     

Role of tRNA modification in translational fidelity

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.     

Pleiotropic effects induced by modification deficiency next to the anticodon of tRNA from Salmonella typhimurium LT2.

A strain of Salmonella typhimurium LT2 was isolated, which harbors a mutation acting as an antisuppressor toward an amber suppressor derivative, supF30, of tRNATyr1. The mutant is deficient in cis-2-methylthioribosylzeatin[N6-(4-hydroxyisopentenyl)-2-me thylthioadenosine, ms2io6A], which is a modification normally present next to the anticodon (position 37) in tRNA reading codons starting with uridine. The gene miaA, defective in the mutant, is located close to and counterclockwise of the purA gene at 96 min on the chromosomal map of S. typhimurium with the gene order mutL miaA purA. Growth rate of the mutant was reduced 20 to 50%, and the effect was more pronounced in media supporting fast growth. Translational chain elongation rate at 37 degrees C was reduced from 16 amino acids per s in the wild-type cell to 11 amino acids per s in the miaA1 mutant in the four different growth media tested. The cellular yield in limiting glucose, glycerol, or succinate medium was reduced for the miaAI mutant compared with wild-type cells, with 49, 41, and 57% reductions, respectively. The miaAI mutation renders the cell more sensitive or resistant toward several amino acid analogs, suggesting that the deficiency in ms2io6A influences the regulation of several amino acid biosynthetic operons. We suggest that tRNAPhe, lacking ms2io6A, translates a UUU codon in the early histidine leader sequence with lowered efficiency, leading to repression of the his operon.     

Structural alterations of the cysteine desulfurase IscS of Salmonella enterica serovar Typhimurium reveal substrate specificity of IscS in tRNA thiolation

The cysteine desulfurase IscS in Salmonella enterica serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s(4)U) and 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s(2)C) and N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm(5)s(2)U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms(2)io(6)A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms(2)io(6)A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.     

Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting

Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts errors at various heptameric sequences, at which at least one codon is decoded by tRNAs having these modified nucleosides. The frequency of -1 frameshifting was the same in congenic strains only differing in the allelic state of the various tRNA modification genes. In fact, in one case (deficiency of mnm(5)s(2)U34), we observed a reduced ability of the undermodified tRNA to make a -1 frameshift error. These results are in sharp contrast to earlier observations that tRNA modification prevents +1 frameshifting suggesting that the mechanisms by which -1 and +1 frameshift errors occur are different. Possible mechanisms explaining these results are discussed.     

Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways

tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s(2)C), 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms(2)io(6)A (10-fold) and s(2)C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms(2)io(6)A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s(4)U and the s(2)U moiety of (c)mnm(5)s(2)U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s(2)C and MiaB in the synthesis of ms(2)io(6)A) in the transfer of sulfur to the tRNA.     

MiaB protein is a bifunctional radical-S-adenosylmethionine enzyme involved in thiolation and methylation of tRNA

The last biosynthetic step for 2-methylthio-N(6)-isopentenyl-adenosine (ms(2)i(6)A), present at position 37 in some tRNAs, consists of the methylthiolation of the isopentenyl-adenosine (i(6)A) precursor. In this work we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. We show that a synthetic i(6)A-containing RNA corresponding to the anticodon stem loop of tRNA(Phe) is also a substrate. This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i(6)A. In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. The origin of the sulfur atom is discussed.     

The transfer RNA of certain Enterobacteriacae contain 2-methylthiozeatin riboside (ms2io6A) an isopentenyl adenosine derivative

Isopentenyl adenosine derivatives are always located adjacent to the 3' end of the anticodon in transfer RNA and have been implicated in certain biological functions. In the enteric bacterium, E. coli, the derivative is ms2i6A whereas in some plant associated bacteria the derivative is the hydroxylated form, ms2io6A. Anti-i6A immunoadsorbent chromatography has been employed to detect isopentenyl adenosine compounds. In the present study we show that the transfer RNA of three species of enteric bacteria, S. typhimurium, K. pneumoniae, and S. marcescens contains both ms2io6A and ms2i6A. Under the growth conditions utilized the ms2io6A is predominant. The presence of ms2io6A in Enterobacteriacae is particularly noteworthy since in previous work it has been found only in plant-associated species of bacteria.     

cis 2-Methylthio-ribosylzeatin (ms2io6A) is present in the transfer RNA of Salmonella typhimurium, but not Escherichia coli

We have identified the cis isomer of N6-(4-hydroxy-isopentenyl)-2-methylthioadenosine (ms2io6A) as a component of the tRNA of Salmonella typhimurium. This is the first report of this compound in the tRNA of any member of the enterobacteriaceae: the nucleoside was previously thought to be found exclusively in plants or plant associated bacteria. Interestingly, all E. coli strains examined were found to lack ms2io6A. Evidence is presented which suggests S. typhimurium tRNA also contains low levels of 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U) in addition to 5-methylaminomethyl-2-thiouridine (mnm5s2U).     

Human Cells Have a Limited Set of tRNA Anticodon Loop Substrates of the tRNA Isopentenyltransferase TRIT1 Tumor Suppressor

Human TRIT1 is a tRNA isopentenyltransferase (IPTase) homologue of Escherichia coli MiaA, Saccharomyces cerevisiae Mod5, Schizosaccharomyces pombe Tit1, and Caenorhabditis elegans GRO-1 that adds isopentenyl groups to adenosine 37 (i6A37) of substrate tRNAs. Prior studies indicate that i6A37 increases translation fidelity and efficiency in codon-specific ways. TRIT1 is a tumor suppressor whose mutant alleles are associated with cancer progression. We report the systematic identification of i6A37-containing tRNAs in a higher eukaryote, performed using small interfering RNA knockdown and other methods to examine TRIT1 activity in HeLa cells. Although several potential substrates contained the IPTase recognition sequence A36A37A38 in the anticodon loop, only tRNA^SerAGA, tRNA^SerCGA, tRNA^SerUGA, and selenocysteine tRNA with UCA (tRNA^[Ser]SecUCA) contained i6A37. This subset is a significantly more restricted than that for two distant yeasts (S. cerevisiae and S. pombe), the only other organisms comprehensively examined. Unlike the fully i6A37-modified tRNAs for Ser, tRNA^[Ser]SecUCA is partially (∼40%) modified. Exogenous selenium and other treatments that decreased the i6A37 content of tRNA^[Ser]SecUCA led to increased levels of the tRNA^[Ser]SecUCA. Of the human mitochondrion (mt)-encoded tRNAs with A36A37A38, only mt tRNAs tRNA^SerUGA and tRNA^TrpUCA contained detectable i6A37. Moreover, while tRNA^Ser levels were unaffected by TRIT1 knockdown, the tRNA^[Ser]SecUCA level was increased and the mt tRNA^SerUGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.     

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.     

Modified nucleotides in Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli.

In the present study, modified nucleotides in the B. subtilis tRNA(Trp) cloned and hyperexpressed in E. coli have been identified by TLC and HPLC analyses. The modification patterns of the two isoacceptors of cloned B. subtilis tRNA(Trp) have been compared with those of native tRNA(Trp) from B. subtilis and from E. coli. The modifications of the A73 mutant of B. subtilis tRNA(Trp), which is inactive toward its cognate TrpRS, were also investigated. The results indicate the formation of the modified nucleotides S4U8, Gm18, D20, Cm32, i6A/ms2i6A37, T54 and psi 55 on cloned B. subtilis tRNA(Trp). This modification pattern resembles the pattern of E. coli tRNA(Trp), except that m7G is missing from the cloned tRNA(Trp), probably on account of its short extra loop. In contrast, the pattern departs substantially from that of native B. subtilis tRNA(Trp). Therefore, the cloned B. subtilis tRNA(Trp) has taken on largely the modification pattern of E. coli tRNA(Trp) despite the 26% sequence difference between the two species of tRNA, gaining in particular the Cm32 and Gm18 modifications from the E. coli host. A notable difference between the isoacceptors of the cloned tRNA(Trp) was seen in the extent of modification of A37, which occurred as either the hypomodified i6A or the hypermodified ms2i6A form. Surprisingly, base substitution of guanosine by adenosine at position 73 of the cloned tRNA(Trp) has led to the abolition of the 2'-O-methylation modification of the remote G18 residue.     

1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium.

1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression.     

The role of modifications in codon discrimination by tRNA(Lys)UUU

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.     

Hypermodified nucleoside carboxyl group as a target site for specific tRNA modification

The free carboxyl group of hypermodified nucleosides N6-methyl-N6-(threoninocarbonyl)adenosine (mt6A37) and 3-(3-amino-3-carboxypropyl)uridine (acp3U20:1) in tRNAmMet (yellow lupine), and N6-(threoninocarbonyl)adenosine (t6A37) in tRNAiMet (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water-soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD-DEAE-cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono-modified (at mt6A37) and bis-modified (at mt6A37 and acp3U20:1) tRNAmMet, and for mono-modified (at t6A37) tRNAiMet by sequence analysis using 5'end 32P-labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.