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PubMed1994

Recognition of UUN codons by two leucine tRNA species from Escherichia coli.

Codon recognition by Escherichia coli tRNA(Leu)4 and tRNA(Leu)5 was investigated by analysis of the competition between two aminoacyl-tRNA species in an in vitro protein synthesis. Both tRNA species strictly obey the wobble rule when they are in competition with other tRNA species. This is probably due to the post-transcriptional modifications at the first position of the anticodon of these tRNA(Leu) species, supporting the proposal that the conformational rigidity of post-transcriptionally modified pyrimidine nucleotides guarantees the correct codon recognition.

Amino Acid Sequence↗
PubMed2006

Identification of two tRNA thiolation genes required for cell growth at extremely high temperatures.

Thermostability of tRNA in thermophilic bacteria is effected by post-transcriptional modifications, such as 2-thioribothymidine (s2T) at position 54. Using a proteomics approach, we identified two genes (ttuA and ttuB; tRNA-two-thiouridine) that are essential for the synthesis of s2T in Thermus thermophilus. Mutation of either gene completely abolishes thio-modification of s2T, and these mutants exhibit a temperature-sensitive phenotype. These results suggest that bacterial growth at higher temperatures is achieved through the thermal stabilization of tRNA by a 2-thiolation modification. TtuA (TTC0106) is possibly an ATPase possessing a P-loop motif. TtuB (TTC0105) is a putative thio-carrier protein that exhibits significant sequence homology with ThiS of the thiamine synthesis pathway. Both TtuA and TtuB are required for in vitro s2T formation in the presence of cysteine and ATP. The addition of cysteine desulfurases such as IscS (TTC0087) or SufS (TTC1373) enhances the sulfur transfer reaction in vitro.

Amino Acid Motifs↗
PubMed1997

A conditional lethal yeast phosphotransferase (tpt1) mutant accumulates tRNAs with a 2'-phosphate and an undermodified base at the splice junction.

tRNA splicing is essential in yeast and humans and presumably all eukaryotes. The first two steps of yeast tRNA splicing, excision of the intron by endonuclease and joining of the exons by tRNA ligase, leave a splice junction bearing a 2'-phosphate. Biochemical analysis suggests that removal of this phosphate in yeast is catalyzed by a highly specific 2'-phosphotransferase that transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate. 2'-Phosphotransferase catalytic activity is encoded by a single essential gene, TPT1, in the yeast Saccharomyces cerevisiae. We show here that Tpt1 protein is responsible for the dephosphorylation step of tRNA splicing in vivo because, during nonpermissive growth, conditional lethal tpt1 mutants accumulate 2'-phosphorylated tRNAs from eight different tRNA species that are known to be spliced. We show also that several of these tRNAs are undermodified at the splice junction residue, which is always located at the hypermodified position one base 3' of the anticodon. This result is consistent with previous results indicating that modification of the hypermodified position occurs after intron excision in the tRNA processing pathway, and implies that modification normally follows the dephosphorylation step of tRNA splicing in vivo.

Genes, Fungal↗
PubMed1993

tRNA-guanine transglycosylase from Escherichia coli: gross tRNA structural requirements for recognition.

tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the post-transcriptional modification of specific tRNAs (those for Asn, Asp, His, and Tyr) with the hypermodified base, queuine. In Escherichia coli this enzyme catalyzes the exchange of guanine-34 in the anticodon with preQ1, which is subsequently further modified to queuine. There is evidence that such hypermodified tRNA molecules may play a role in the control of cell proliferation and differentiation. In order to perform detailed, in vitro mechanistic studies and to probe the tRNA-enzyme interaction, we have generated unmodified E. coli tRNA(Tyr) and truncated analogues using an in vitro RNA synthesis system suggested by Milligan and Uhlenbeck [Milligan, J. F., & Uhlenbeck, O. C. (1989) Methods Enzymol. 180, 51-62]. From this system we have generated three tRNA analogues totally devoid of any post-transcriptional modifications. In order to compare the unmodified tRNA with the true physiological substrate for TGT, that is, tRNA that contains all modified bases except queuine, we have isolated E. coli tRNA(Tyr) from an overexpressing clone in a TGT-deficient strain of E. coli. We report here that unmodified, full-length tRNA(Tyr) serves as a substrate for TGT with kinetic parameters that are, within experimental error, the same as those for in vivo isolated tRNA(Tyr). This indicates that other post-transcriptional modifications have negligible effects upon TGT recognition of tRNA. A 17-base oligoribonucleotide, corresponding to the anticodon loop and stem, is also a substrate for TGT with only a 20-fold loss in Vmax/KM, versus the full-length tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1976

Location of accessible bases in Escherichia coli formylmethionine transfer RNA as determined by chemical modification.

Chemical modification of Escherichia coli tRNAfMet with 1 M chloroacetaldehyde, pH 5.5-6.0 at 25 degrees C, has been found to result in alteration of six cytidine and five adenosine residues in the molecule. The modified cytidine residues are the same as those previously found to be reactive with sodium bisulfite at pH 6.0. The accessible adenosine residues are A36 in the anticodon, A58 in the T psi C loop, and A73, A74, and A77 in the 3; terminal sequence. No modification of adenosine residues in the dihydrouridine or variable loops or of adenosine residues on the 3' side of the anticodon loop could be detected. Treatment of fMet-tRNAfMet with chloracetaldehyde gave the same pattern of midofication as was observed with deacylated tRNAfMet. Chemical modification of E. coli tRNAfMet with 2 sodium bisulfite, pH 7.0 at 25 degrees C, resulted in selective modification of exposed uridine residues in the tRNA. Only three sites were found to be reactive: U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. The overall pattern of chemical modification of tRNAfMet is very similar to that found by others for yeast tRNAPhe, supporting the idea that many of the tertiary interactions in the two tRNAs are the same. The adenosine residue at position 58 in the center of the T psi C loop of the initiator tRNA shows unusual reactivity, however, being modified by chloroacetaldehyde at the same rate as the 3' terminal adenosine residue. This result is in sharp contrast to the uniform resistance of nucleotides in the T psi C loop of yeast tRNAPhe to chemical modification.

Acetaldehyde↗
PubMed1987

The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs.

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.

Animals↗
PubMed1995

Native bovine selenocysteine tRNA(Sec) secondary structure as probed by two plant single-strand-specific nucleases.

Two single-strand-specific nucleases, discovered in plants, have been used to investigate the secondary and tertiary structures of the native bovine liver selenocysteine tRNA(Sec). To check the possible influence of nucleotide modifications on these structures, we compared the results obtained with the fully modified tRNA to the unmodified transcript prepared by in vitro T7 transcription of the Xenopus laevis tRNA(Sec) gene. We found that the structures in solution of the native tRNA(Sec) and the transcript are very similar despite some differences in accessibility to the enzymatic probes. Indeed, the modified anticodon-loop of native bovine tRNA(Sec), containing 5-methylcarboxymethyluridine (mcm5U34) and N6-isopentenyladenosine (i6A37), is less accessible to Rn nuclease than that of the transcript: the intensity of bands representing cuts at A36 and A38 is much lower as compared to those of the transcript, whereas no cuts were found at the level of i6A37 in the anticodon loop of the native molecule. Surprisingly, the variable arm of the native molecule has been found to be more susceptible to single-strand-specific nuclease action, suggesting a looser structure of the variable arm in native bovine tRNA(Sec) than in the transcript.

Animals↗
PubMed1998

Recognition of a pre-tRNA substrate by the Bacillus subtilis RNase P holoenzyme.

The holoenzyme of the bacterial RNase P has broader selectivity for biological substrates compared to the RNA alone (denoted P RNA) reaction. The structural basis of the substrate selectivity is investigated using a pre-tRNA substrate containing single-atom modifications by single turnover kinetics. Hydroxyl radical protection of the holoenzyme in the absence of the substrate shows that the RNase P protein binds to several regions in P RNA. The holoenzyme interacts with a subset of functional groups in the T stem-loop region of a pre-tRNA substrate previously identified to directly contact P RNA. The subtle change in structural recognition allows the holoenzyme to recognize RNA structures with only a small perturbation in an A-form helix at the corresponding position of the T stem-loop. This altered profile may permit the holoenzyme to bind non-tRNA substrates with little change in catalytic efficiency. The holoenzyme recognizes the same set of functional groups as the P RNA reaction in the region around the cleavage site and shows similar cleavage site selection compared to the P RNA reaction. These results suggest that the holoenzyme does not alter the fundamental mechanism of this enzymatic reaction. Rather, the holoenzyme significantly affects the binding affinity of an RNA substrate through additional interactions with the 5' leader [Kurz, C. A., Niranjanakumari, S., and Fierke, C. A. (1998) Biochemistry 37, 2393] and through altered recognition of the substrate structure.

Bacillus subtilis↗
PubMed1992

Formation and 32P-postlabeling of DNA and tRNA adducts derived from peroxidative activation of carcinogenic azo dye N,N-dimethyl-4-aminoazobenzene.

Peroxidase in the presence of hydrogen peroxide catalyzes in vitro the activation of carcinogenic N,N-dimethyl-4-aminoazobenzene (DAB) to DNA-, tRNA- and homopolydeoxyribonucleotide-bound products. tRNA is the most susceptible to modification by the activated DAB. Binding of DAB products to macromolecules is inhibited by methyl viologen, nitrosobenzene, ascorbate, glutathione, NADH and MgCl2. The mechanism of these inhibitions was studied. The nuclease P1 version of the 32P-postlabeling assay was employed for detection and quantitation of some major DNA or tRNA adducts formed with DAB activated by a peroxidase system. tRNA modified by activated DAB shows a significantly increased acceptance for L-methionine.

Animals↗
PubMed1976

Subcellular localization of S-adenosyl-L-methionine:tRNA methyltransferases with aminoacyl-tRNA synthetases in human and mouse: normal and leukemic leukocytes.

The subcellular distributions of S-adenosyl-L-methionine:tRNA methyltransferases and aminoacyl-tRNA synthetases were investigated with the use of human and mouse normal and leukemic leukocyte cell lines. Differential centrifugation of homogenized cell suspensions produced three pelleted subcellular fractions (nuclear and membrane, microsomal, and postribosomal) and a supernatant fraction. Each fraction was assayed for both methyltransferase activity and synthetase activity. The largest amounts, 40-50%, of total methyltransferase and synthetase activities were localized in either the microsomal or the postribosomal fractions, depending on cell type. In addition, the highest specific activities of these two enzyme systems were found to be present in the microsomal and postribosomal fractions. The psotribosomal fraction from leukemic leukocytes had a methyltransferase specific activity higher than that of the microsomal fraction, while the same two fractions of normal leukocytes had approximately equal activities. Specific activities of aminoacyl-tRNA synthetases were found to be approximately equal for these two fractions, whether they were from normal or leukemic leukocytes. The activity of tRNA methyltransferases and synthetases within the postribosomal fraction of the cytoplasm suggests the existence of high-molecular-weight enzyme complexes for the modification as well as the aminoacylation of tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Binding of tRNA alters the chemical accessibility of nucleotides within the large ribosomal RNAs of E. coli ribosomes.

Functionally active 70S ribosomes were chemically modified with dimethylsulfate (DMS) in the presence and absence of bound tRNA. The ribosomal 16S RNA and 23S RNA were extracted, separated and labeled radioactively at their 3'-ends. DMS modification sites within the last 200 nucleotides from the 3'-ends were investigated on sequencing gels, after borohydride reduction and aniline catalyzed strand scission of the isolated RNA's. tRNA binding caused enhanced reactivity at 9 nucleotide positions while three sites showed decreased reactivity in the 16S RNA. The effects of bound tRNA on the modification of 23S RNA were limited. Only one enhancement was observed in the presence of bound tRNA. mRNA binding alone showed two more sites with enhanced reactivity, however. The results are consistent with the view that the sequence 1400-1500 of the 16S RNA plays an important functional role in the translating ribosome and possibly constitutes part of the tRNA binding site.

Base Sequence↗
PubMed1988

Import of several tRNAs from the cytoplasm into the mitochondria in bean Phaseolus vulgaris.

Four P. Vulgaris mitochondrial tRNA(Leu) species have been shown to be nuclear encoded. These mt tRNAs(Leu) can be used for in vitro protein synthesis. We found that the sequences of P. vulgaris mitochondrial and cytoplasmic tRNAs(Leu)(NAG) are identical except for a post-transcriptional modification occurring at position 18 (Gm in mt tRNA(Leu) instead of G in cyt tRNA(Leu], as in the case of mt and cyt tRNAs(Leu)(NAA) already sequenced. This post-transcriptional modification has also been found in two other bean mt tRNA(Leu) species, but not in P. vulgaris cytoplasmic tRNA(Leu) species that we have purified so far. Furthermore, comparison of the 2-D polyacrylamide gel electrophoretic patterns of tRNAs eluted from bean mt tRNA-mtDNA and mt tRNA-nDNA hybrids revealed at least 8 mt tRNAs coded for by the nuclear genome.

Base Sequence↗
PubMed1983

tRNA alterations in cancer.

1. 3H-, 125I-, and 32P-labeling methods were developed for base composition and sequence analysis of minute amounts of nonradioactive nucleic acids containing modified constituents. 2. Base composition analysis showed tRNA from two "liver-like" minimal deviation hepatomas, Morris hepatomas 5123D and 7777, to exhibit typical alterations when compared with liver tRNA. Our observations, which were made for different transplant generations of the tumors, indicated a trend toward undermethylation and undermodification of tRNA. 3. Sequence analysis of several cytoplasmic and mitochondrial tRNAs from hepatoma 5123D showed partial lack of m2G and complete lack of Gm and Q. 4. Sequence analysis of mitochondrial tRNAs from hepatoma 5123D indicated several instances of alterations of primary structure, a phenomenon not previously observed for cytoplasmic tRNAs from neoplasms. 5. Biochemical mechanisms underlying these alterations, as well as their functional implications, have yet to be investigated. 6. Modification patterns, but not primary structures, of mitochondrial tRNAs have been highly conserved when compared to prokaryotic and eukaryotic cytoplasmic tRNAs. This implies that (a) post-transcriptional modifications must play a crucial role in tRNA function, and (b) alterations of post-transcriptional modifications in tumor tRNAs have to be regarded as highly significant deviations from the norm.

Animals↗
PubMed2006

Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

Archaea↗
PubMed1983

Structural investigation of Phe-tRNAPhe from E.coli bound to the ribosomal A-site.

Kethoxal modification of guanosines within Phe-tRNAPhe from E. coli was studied for tRNA in the free state and specifically bound to the ribosomal A-site. Complex formation with the ribosome results in a protection from chemical modification of two distant sites in the tRNA molecule. The guanosines affected are G-18 and G-19, located in the D-loop, and G-34 in the anticodon loop. Modification of Phe-tRNAPhe in the absence of ribosomes leads to a destabilisation of the tRNA structure. Our data are consistent with the conclusion that modification of G-34 at the anticodon loop triggers a conformational instability in distant parts of the tRNA molecule.

Aldehydes↗
PubMed1998

tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases.

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.

Acylation↗
PubMed1978

Specific transcription of eukaryotic tRNA genes in Xenopus germinal vesicle extracts.

Cloned tRNA genes from Drosophila and from yeast have been transcribed faithfully in extracts prepared from Xenopus germinal vesicles. The newly formed RNA is composed of precursor tRNAs (of 5S RNA size) and of tRNAs. The plasmid pCIT12 carries genes for Drosophila tRNALys, tRNAArg, and tRNAAsn, Nucleotide analysis of one RNA species transcribed from pCIT12 DNA showed it to be identical to Drosophila tRNALys; it even contained some of the modified nucleotides expected for this tRNA. This RNA species is formed in the germinal vesicle extract via a larger precursor tRNA molecule that does not contain nucleotide modifications. This simple transcription system should aid studies aimed at defining the regulatory DNA regions responsible for eukaryotic gene transcription. In addition, it may provide tRNA precursors that are needed for detailed investigations of eukaryotic tRNA biosynthesis.

Animals↗
PubMed1976

Effects of 5-fluorouridine on modified nucleosides in mouse liver transfer RNA.

Administration of the pyrimidine antimetabolite, 5-fluorouridine, to mice was found to cause a marked specific reduction of the amounts of 5-methyluridine, pseudouridine, and dihydrouridine but not of 3-(3-amino-3-carboxypropyl)uridine in tRNA from the livers of the treated animals. The data presented indicate that this effect is not simply due to the incorporation of 5-fluorouridine into tRNA; the drug appears to interfere directly with the enzymic reactions involved in the modification of the 5-position of uridine. 5-Fluorouridine was found to have no effect on the modification of adenosine, guanosine, and cytidine in mouse liver tRNA.

Animals↗