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.     

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.     

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.     

Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli

(35)S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H(2)O(2), NH(2)OH, I(2), HNO(2), KMnO(4) and NaIO(4), under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH(2)OH, I(2) and NaIO(4) reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 2-thiocytidine (s(2)C) and 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A), to various extents. CNBr, HNO(2) and NaHSO(3) reacted with s(4)U, mnm(5)s(2)U and s(2)C, but not with ms(2)i(6)A. KMnO(4) and H(2)O(2) were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of (35)S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.     

Molecular dynamics simulations of human tRNA Lys,3 UUU: the role of modified bases in mRNA recognition

Accuracy in translation of the genetic code into proteins depends upon correct tRNA-mRNA recognition in the context of the ribosome. In human tRNA(Lys,3)UUU three modified bases are present in the anticodon stem-loop--2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms2t6A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm5s2U34) and pseudouridine (psi) at position 39--two of which, ms2t6A37 and mcm5s2U34, are required to achieve wild-type binding activity of wild-type human tRNA(Lys,3)UUU [C. Yarian, M. Marszalek, E. Sochacka, A. Malkiewicz, R. Guenther, A. Miskiewicz and P. F. Agris (2000) Biochemistry, 39, 13390-13395]. Molecular dynamics simulations of nine tRNA anticodon stem-loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stair-stepped conformation. The ms2t6 modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms2t6A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to psi39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps.     

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.     

The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium

Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations. In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene. This site may be suppressed by peptidyl-tRNAProcmo5UGG (cmo(5)U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNAGlnmnm5s2UUG (mnm(5)s(2)U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site. We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS. Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm(5)s(2)U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S. enterica were reduced in the iscS51 mutant. In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level. After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm(5)s(2)U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNAGlnmnm5s2UUG due to mnm(5)s(2)U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mnmA) lacking only mnm(5)s(2)U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm(5)s(2)U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant.     

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.     

Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities

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.     

Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.     

Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA

A selenium-containing nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is present in lysine- and glutamate-isoaccepting tRNA species of Escherichia coli. The synthesis of mnm5se2U is optimum (4 mol/100 mol tRNA) when selenium is present at about 1 microM concentration and is neither decreased by a high (8 mM) level of sulfur in the medium nor increased by excessive (10 or 100 microM) levels of selenium. Lysine- and glutamate-isoaccepting tRNA species that contain 5-methylaminomethyl-2-thiouridine (mnm5s2U) coexist with the seleno-tRNAs in E. coli cells and a reciprocal relationship between the mnm5se2U- and the mnm5s2U-containing species is maintained under a variety of growth conditions. The complete 5-methylaminomethyl side chain is not a prerequisite for introduction of selenium at the 2-position. In E. coli mutants deficient in the ability to synthesize the 5-methylaminomethyl substituent, both the 2-thiouridine and the corresponding 2-selenouridine derivatives of intermediate forms are accumulated. Broken cell preparations of E. coli synthesize mnm5se2U in tRNAs by an ATP-dependent process that appears to involve the replacement of sulfur in mnm5s2U with selenium.     

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.     

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.     

Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: the effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A

Previous nuclear magnetic resonance (NMR) studies of unmodified and pseudouridine39-modified tRNA(Lys) anticodon stem loops (ASLs) show that significant structural rearrangements must occur to attain a canonical anticodon loop conformation. The Escherichia coli tRNA(Lys) modifications mnm(5)s(2)U34 and t(6)A37 have indeed been shown to remodel the anticodon loop, although significant dynamic flexibility remains within the weakly stacked U35 and U36 anticodon residues. The present study examines the individual effects of mnm(5)s(2)U34, s(2)U34, t(6)A37, and Mg(2+) on tRNA(Lys) ASLs to decipher how the E. coli modifications accomplish the noncanonical to canonical structural transition. We also investigated the effects of the corresponding human tRNA(Lys,3) versions of the E. coli modifications, using NMR to analyze tRNA ASLs containing the nucleosides mcm(5)U34, mcm(5)s(2)U34, and ms(2)t(6)A37. The human wobble modification has a less dramatic loop remodeling effect, presumably because of the absence of a positive charge on the mcm(5) side chain. Nonspecific magnesium effects appear to play an important role in promoting anticodon stacking. Paradoxically, both t(6)A37 and ms(2)t(6)A37 actually decrease anticodon stacking compared to A37 by promoting U36 bulging. Rather than stack with U36, the t(6)A37 nucleotide in the free tRNAs is prepositioned to form a cross-strand stack with the first codon nucleotide as seen in the recent crystal structures of tRNA(Lys) ASLs bound to the 30S ribosomal subunit. Wobble modifications, t(6)A37, and magnesium each make unique contributions toward promoting canonical tRNA structure in the fundamentally dynamic tRNA(Lys)(UUU) anticodon.     

Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs.

The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases, suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t(6)A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) has a weaker positive effect depending on the context of other modifications. The s(2)U34 modification apparently has none and the pseudouridine (psi39) was inhibitory in most modification contexts. GC-rich but not IC-rich stems abolished the activity. Correlating the reported structural effects of the base modifications with their effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing interactions in the stem are destabilized. Moreover, the proposal that PrrC residue Asp(287) contacts mnm(5)s(2)U34 was reinforced by the observations that the mammalian tRNA(Lys-3) wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm(5)s(2)U) is inhibitory and that the D287H mutant favors tRNA(Lys-3) over Escherichia coli tRNA(Lys). The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNA(Lys-3) primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.     

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.     

Subcellular localization of cytokinins in transfer ribonucleic Acid

Evidence on the localization of cytokinins in chloroplast tRNA was obtained by comparison of Euglena gracilis var. bacillaris light-grown and dark-grown wild type cultures and chloroplast-bleached mutant strains. The several cytokinins characteristic of tRNA were separated by Sephadex LH-20 column chromatography of the hydrolysates and were quantitatively determined by tobacco bioassays of the eluates. The results indicate that 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine (i(6) A) is formed in both the cytoplasmic and chloroplast tRNA, whereas 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-d- ribofurano-sylpurine (c-io(6)A) is produced mainly in the cytoplasmic tRNA and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-d- ribofurano-sylpurine (ms(2)io(6)A) is localized exclusively in chloroplast tRNA. The restriction of the methiolation reaction to the chloroplast is supported by results of radioisotope experiments showing that (35)S-labeled MgSO(4) is incorporated into ms(2)io(6)A in the wild type cultures, but not in the chloroplast-bleached mutant strains.