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PubMed1986

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.

Anticodon↗

PubMed1995

An anticodon sequence mutant of Escherichia coli initiator tRNA: possible importance of a newly acquired base modification next to the anticodon on its activity in initiation.

Initiator tRNAs from eubacteria and chloroplasts lack a base modification next to the anticodon. This is in contrast to virtually all other tRNAs from these sources. We show that a mutant Escherichia coli initiator tRNA which has an anticodon sequence change from CAU to CUA now has a 2-methylthio-N6-(delta 2-isopentenyl)adenosine (ms2i6A) modification, produced by posttranscriptional modification of A, next to the anticodon. This newly acquired base modification may be important for the function of the mutant tRNA in initiation. In a miaA mutant strain of E. coli defective in biosynthesis of ms2i6A, the mutant initiator tRNA is 10- to 12-fold less active in initiation. The mutant tRNA is aminoacylated and formylated normally in the miaA strain. Thus, the absence of the base modification affects the activity of the mutant tRNA at a step subsequent to its formylation.

Anticodon↗

PubMed1993

UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin.

Mutant UGA codon contexts which previously have been identified at different positions in the lacI part of a fused lacIlacZ gene were characterized with respect to translational readthrough in another genetic surrounding at a constant location. Although readthrough levels are systematically higher in this new location the "tight/leaky" characteristics of these codon contexts are essentially fully determined by the two codons flanking the nonsense codon itself. Analysis of some UGA hybrid contexts shows that the contribution to the codon context character by the codon either at the 5'-side (CCA or AGC) or at the 3'-side (NGU) is independent of the nature of the codon at the other side of UGA if this codon is decoded by trpT(Su9) suppressor tRNA. In a trpT(Su9), miaA double mutant strain, which lacks the ms2i6A37 modification in this tRNA, suppression is decreased at all UGA contexts investigated. However, in one case the contribution to the codon context character by the determinant flanking at one side is negatively affected by the nature of the codon at the other side of UGA. Thus, the character of a nonsense codon context in this case results from both flanking codons acting in a co-operative manner with the tRNA reading the middle UGA codon. This negative context effect is counteracted by a rpsD12 (ribosomal protein S4) mutation or by a sublethal concentration of streptomycin in the growth medium. It is suggested that the ms2i6A37 base in trpT(Su9) suppressor tRNA increases the efficiency of this tRNA by protecting it from ribosomal proofreading which is induced by codon context.

Bacterial Proteins↗

PubMed2002

Prevention of mis-aminoacylation of a dual-specificity aminoacyl-tRNA synthetase.

Accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetase is essential for the fidelity of protein synthesis. For Methanococcus jannaschii tRNA(Pro), accuracy is difficult because the cognate prolyl-tRNA synthetase also recognizes and aminoacylates tRNA(Cys) with cysteine. We show here that the unmodified transcript of M. jannaschii tRNA(Pro) is indeed mis-acylated with cysteine. However, the origin of mis-charging is not at the anticodon or acceptor stem, the two hotspots for tRNA(Pro) and tRNA(Cys) identity determinants. Instead, replacement of the D loop in the tRNA core with that of tRNA(Cys) suppresses mis-charging with cysteine without compromising the activity of aminoacylation with proline. The reduced level of cysteine activity of the chimera is not due an editing response of the synthetase and is consistent with a relaxed sensitivity of the tRNA to the analog thiaproline in aminoacylation with cysteine. We suggest that mis-acylation is not due to the presence of cysteine determinants, but to a mis-placed 3' end into the cysteine catalytic site that activates and transfers cysteine to the tRNA. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase.

Acylation↗

PubMed2004

Nuclear control of cloverleaf structure of human mitochondrial tRNA(Lys).

The evolutionary loss in eukaryotic cells of mitochondrial (mt) tRNA genes and of tRNA structural information in the surviving genes has led to the appearance of mt-tRNAs with highly unusual structural features. One such mt-tRNA is the human mt-tRNALys, which relies on post-transcriptional base modification to achieve correct three-dimensional structure. It has been shown that the in vitro transcript of human mt-tRNALys adopts a particular, non-cloverleaf structure when devoid of modified bases, while the native, fully modified tRNA shows the expected cloverleaf structure. Furthermore, a methyl group at position A9-N1, introduced chemically in an otherwise unmodified mt-tRNALys transcript, was found to induce a stable cloverleaf conformation, raising the question of how the specific methyltransferase recognizes the unmodified transcript. In order to shed light on this unusual case of tRNA maturation, the tRNA modification enzymes contained in protein extracts from either highly purified HeLa cell mitochondria or HeLa cell cytosol were first identified and compared, and then used to analyze the mt-tRNALys. An initial screening for modification activities, using as substrates unmodified in vitro transcripts of tRNA genes with well characterized structures, namely yeast cytosolic tRNAPhe, human cytosolic tRNA3Lys, and human mt-tRNAIle, revealed the presence of nine and 11 modification activities in the mitochondrial and cytosolic protein extracts, respectively, the mitochondrial extract including a tRNA (adenine-9,N1)-methyltransferase activity. The comparison of the level and kinetics of A9-N1 methylation and other secondary modifications in the unmodified, misfolded mt-tRNALys and in a cloverleaf-shaped structural mutant, engineered to adopt the tRNALys cloverleaf structure without post-transcriptional modifications, suggested strongly that the methylation of A9-N1 in tRNALys proceeds via a cloverleaf-shaped intermediate. Therefore, it is proposed that this intermediate is present in the in vitro transcript as part of a dynamic equilibrium, and that the mitochondrial protein extract contains an activity that stabilizes, by secondary modification, such a transient cloverleaf-shaped intermediate. Thus, countering the evolutionary loss of structural information in mt-tRNA genes, the mt-tRNA structure is maintained by a modification enzyme encoded in nuclear DNA.

Cytosol↗

PubMed1998

The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA.

Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression of GCN4 mRNA. The phenotypes of gcd10 mutants were suppressed by high-copy-number IMT genes, encoding initiator methionyl tRNA (tRNAiMet), or LHP1, encoding the yeast homolog of the human La autoantigen. The gcd10-504 mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet. Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNAiMet is the essential function of Gcd10p. A gcd14-2 mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMet processing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMet maturation. The chromatographic behavior of elongator and initiator tRNAMet on a RPC-5 column indicated that both species are altered structurally in gcd10Delta cells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable in gcd10Delta tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

Adenosine↗

PubMed1999

The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2'-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs.

The protein sequences of three known RNA 2'-O-ribose methylases were used as probes for detecting putative homologs through iterative searches of genomic databases. We have identified 45 new positive Open Reading Frames (ORFs), mostly in prokaryotic genomes. Five complete eukaryotic ORFs were also detected, among which was a single ORF (YDL112w) in the yeast Saccharomyces cerevisiae genome. After genetic depletion of YDL112w, we observed a specific defect in tRNA ribose methylation, with the complete disappearance of Gm18 in all tRNAs that naturally contain this modification, whereas other tRNA ribose methylations and the complex pattern of rRNA ribose methylations were not affected. The tRNA G18 methylation defect was suppressed by transformation of the disrupted strain with a plasmid allowing expression of YDL112wp. The formation of Gm18 on an in vitro transcript of a yeast tRNASer naturally containing this methylation, which was efficiently catalyzed by cell-free extracts from the wild-type yeast strain, did not occur with extracts from the disrupted strain. The protein encoded by the YDL112w ORF, termed Trm3 (tRNA methylation), is therefore likely to be the tRNA (Gm18) ribose methylase. In in vitro assays, its activity is strongly dependent on tRNA architecture. Trm3p, the first putative tRNA ribose methylase identified in an eukaryotic organism, is considerably larger than its Escherichia coli functional homolog spoU (1,436 amino acids vs. 229 amino acids), or any known or putative prokaryotic RNA ribose methyltransferase. Homologs found in human (TRP-185 protein), Caenorhabditis elegans and Arabidopsis thaliana also exhibit a very long N-terminal extension not related to any protein sequence in databases.

Amino Acid Sequence↗

PubMed1984

tRNA binding modifies the properties of the small ribosomal subunit of rat liver.

The binding of a specific tRNA (acylated or not) to the 40S subunits in the presence of the proper codon was shown to produce two striking effects on the subunits. First, the subunits were no longer able to dimerize at low ionic strength. Second, they became fully resistant to 1.25 M LiCl treatment: bound tRNA prevented subunit inactivation as measured by polyphenylalanine synthesis; it also prevented large sedimentation changes of subunits and ribosomal protein release induced by LiCl. The number of protected proteins far exceeded that of the proteins crosslinked with tRNA after irradiation at 254 nm (A.M. REBOUD, S. DUBOST and J.P. REBOUD (1983) FEBS Lett. 158, 285-288). These results strongly suggest that tRNA binding induces modifications of rRNA-protein interactions in large domains of the subunits. A weak interaction of tRNA with the 40S subunit was demonstrated in the absence of the codon.

Animals↗

PubMed1982

Implication of arginyl residues in aminoacyl-tRNA binding to ribosomes.

Modification of the 50-S subunits of Escherichia coli ribosomes with the arginine reagent phenylglyoxal produces inactivation of peptidyl transferase and inhibition of the binding of C(U)-A-C-C-A-LeuAc, phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to the ribosome. Hybridization experiments, using 1.25 M LiCl core particles and the corresponding split proteins from untreated and phenylglyoxal-treated 50-S subunits, indicate that inactivation and inhibition of binding are the effects of modification of a protein fraction, the functionality of the RNA moiety being unaffected by the reagent. The split proteins from phenylglyoxal-modified 50-S subunits are incorporated to 1.25 M LiCl core particles as well as those obtained from unmodified subunits, thus excluding the failure to bind as the cause of inactivation. In agreement with the general role played by the arginyl residues as positive binding sites for anionic ligands, the present results indicate that the arginyl residues of a protein fraction from 50-S subunits might be important in the binding of aminoacyl-tRNA and peptidyl-tRNA to ribosomes.

Arginine↗

PubMed1999

Coordination of tRNA nuclear export with processing of tRNA.

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding.

Animals↗

PubMedDate not supplied

[Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center].

Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.

Affinity Labels↗

PubMed1993

Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure.

Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity. A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs). The polymerase chain reaction was used for the production of cptRNA templates. After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA. Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension. These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site. The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

Affinity Labels↗

PubMed1997

The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity.

We have evidence that the open reading frame previously denoted spoU is necessary for tRNA (Gm18) 2'-O-methyltransferase activity. The spoU gene is located in the gmk-rpoZ-spoT-spoU-recG operon at 82 minutes on the Escherichia coli chromosome. The deduced amino acid sequence of spoU shows strong similarities to previously characterized 2'-O-methyltransferases. Comparison of the nucleoside modification pattern of hydrolyzed tRNA, 16S rRNA and 23S rRNA from wild-type and spoU null mutants showed that the modified nucleoside 2'-O-methylguanosine (Gm), present in a subset of E. coli tRNAs at residue 18, is completely absent in the spoU mutant, suggesting that spoU encodes tRNA (Gm18) 2'-O-methyltransferase. Nucleoside modification of 16S and 23S rRNA was unaffected in the spoU mutant. Insertions in the downstream recG gene did not affect RNA modification. Absence of Gm18 in tRNA does not influence growth rate under the tested conditions and does not interfere with activity of the SupF amber suppressor, a suppressor tRNA that normally has the Gm18 modification. We suggest that the spoU gene be renamed trmH (tRNA methylation).

Amino Acid Sequence↗

PubMed1981

Reversible inactivation of Escherichia coli methionyl-tRNA synthetase by covalent attachment of formylmethionine tRNA to the tRNA binding site with a cleavable cross-linker.

Protein affinity labeling groups have been attached to single-stranded cytidine residues in four structural regions of tRNAfMet. Modification of the tRNA with an average of one cross-linking group per molecule is achieved with retention of 75% of the original methionine acceptor activity. Incubation of the modified tRNA with methionyl-tRNA synthetase (MetRS) results in covalent coupling of the protein and nucleic acid by reaction of N-hydroxysuccinimide ester groups attached to the tRNA with lysine residues in the enzyme. In the presence of excess MetRS, approximately 30% of the input tRNA can be covalently bound to protein, indicating that lysine residues are appropriately oriented for reaction with cross-linking groups attached to certain sites in the tRNA but not to others. The cross-linking reaction results in loss of aminoacylation activity of MetRS equal to the amount of covalently bound tRNA. Enzyme activity is restored by release of bound tRNA following cleavage of the disulfide bond of the cross-linker with a sulfhydryl reagent. The data indicate that cross-linking occurs at the tRNA binding site of the enzyme. In the presence of excess modified tRNAfMet, a maximum of 1 mol of tRNA is cross-linked per mol of MetRS, in keeping with the known anticooperative tRNA binding properties of the native dimeric synthetase. In addition, the coupling reaction is effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAs.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Purification of hyperexpressed Bacillus subtilis tRNA(Trp) cloned in Escherichia coli.

To study the effects of evolutionary sequence changes on the molecular interactions of tRNA inside the cell, the Bacillus subtilis tRNA(Trp) gene has been cloned into Escherichia coli JM109 under the control of the lac promotor. Hyperexpression of the gene in minimal medium upon induction yielded 28% of total tRNA in the form of B. subtilis tRNA(Trp). The tRNA(Trp) gene product was purified by the use of a single Vydac C4 high-performance liquid chromatography (HPLC) matrix. This experimental system provided a valuable system for the hyperexpression and purification of a heterologous tRNA for studies in vitro. Moreover, because HPLC fractionation of the heterologous tRNA(Trp) gene product yielded multiple peaks, the system made possible an analysis of the molecular mechanisms for the transcriptional modifications of the tRNA(Trp) gene product in vivo.

Bacillus subtilis↗

PubMed2000

Role of modified nucleosides of yeast tRNA(Phe) in ribosomal binding.

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.

Nucleosides↗

PubMed1983

Alterations in post-transcriptional modification of the Y base in phenylalanine tRNA from tumor cells.

Various tumor cells contain chromatographically distinct isoacceptor tRNA species. To decide whether the tumor-specific species represent an expression of a separate tRNA gene or only an undermodified form of normal tRNAPhe, nucleotide sequences of tRNAPhe isolated from neuroblastoma and normal mouse liver were determined by postlabeling techniques. The results showed identical sequences except for the changes of post-transcriptional modifications in the anticodon loop. Normal mouse liver tRNAPhe contained Cm32, Gm34, and the hypermodified YOH next to the 3' end of the anticodon. On the contrary, tRNAPhe from neuroblastoma contained C32, G34, and, instead of YOH base m1G. A small proportion of tRNAPhe species contained an undermodified YOH base. For the examination of the conditions leading to the undermodified tRNAPhe, Vero cells derived from the kidney of African green monkey in culture were used. In these cells, deprivation of methionine or lysine resulted in changes in tRNAPhe modification similar to those in tumor cells. Ehrlich ascites tumor cells were examined to determine whether the presence of altered tRNAPhe species in various tumors is also the result of starvation of some nutritional factors. Results obtained with these cells showed that tRNAPhe species lacking the Y base disappeared in tumor-bearing mice after intraperitoneal injection with a mixture of amino acids and vitamins. Thus it is concluded that tumor-specific tRNAPhe species are the products of aberrant post-transcriptional modification, not the transcripts of different, normally repressed genes.

Animals↗

PubMed1991

Evolution of tRNAs and tRNA genes in Acholeplasma laidlawii.

The genes for 22 tRNA species from Acholeplasma laidawii, belonging to the class Mollicutes (Mycoplasmas), have been cloned and sequenced. Sixteen genes are organized in 3 clusters consisting of eleven, three and two tRNA genes, respectively, and the other 6 genes exist as a single gene. The arrangement of tRNA genes in the 11-gene, the 3-gene and the 2-gene clusters reveals extensive similarity to several parts of the 21-tRNA or 16-tRNA gene cluster in Bacillus subtilis. The 11-gene cluster is also similar to the tRNA gene clusters found in other mycoplasma species, the 9-tRNA gene cluster in M.capricolum and in M.mycoides, and the 10-tRNA gene cluster in Spiroplasma meliferm. The results suggest that the tRNA genes in mycoplasmas have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to mycoplasmas and B.subtilis. The anticodon sequences including base modifications of 15 tRNA species from A.laidlawii were determined. The anticodon composition and codon-recognition patterns of A.laidlawii resemble those of Bacillus subtilis rather than those of other mycoplasma species.

Acholeplasma laidlawii↗