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At least 649 records · Page 36Linked to original sources
PubMed2004

Essential role of histidine 20 in the catalytic mechanism of Escherichia coli peptidyl-tRNA hydrolase.

The peptidyl-tRNA hydrolase (Pth) enzyme plays an essential role in recycling tRNA from peptidyl-tRNA that has prematurely dissociated from the ribosome. In this study of Escherichia coli Pth, the critical role of histidine 20 was investigated by site-directed mutagenesis, stopped-flow kinetic measurements, and chemical modification. The histidine residue at position 20 is known to play an important role in the hydrolysis reaction, but stopped-flow fluorescence measurements showed that, although the His20Asn Pth mutant enzyme was unable to hydrolyze the substrate, the enzyme retained the ability to bind peptidyl-tRNA. Chemical modification of Pth with diethyl pyrocarbonate (DEPC) showed that a residue, with a pK(a) value of 6.3, was essential for substrate hydrolysis and that the stoichiometry of inhibition was 0.70 +/- 0.06 mol of DEPC/mol of enzyme, indicating that modification of only a single residue by DEPC was responsible for the loss of activity. Parallel chemical modification studies with the His20Asn and Asp93Asn mutant enzymes showed that this essential residue was His20. These studies indicate that histidine 20 acts as the catalytic base in the hydrolysis of peptidyl-tRNA by Pth.

Asparagine↗
PubMed2024

Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.

tRNA-derived RNAs (tDRs), resulting from enzyme-mediated hydrolysis of tRNAs, have been implicated as active small RNAs in various molecular processes. While the molecular modes of action for these small RNAs remain unclear, attempts to decipher the mechanistic details of tDR functionality have mostly used synthetic tDR sequences. Since parental tRNAs are extensively post-transcriptionally modified, tDR functionality is likely affected by chemical modifications. To help approach the biological function of endogenously modified tDRs, this contribution details a protocol that allows purifying specific tDRs carrying post-transcriptional modifications from both in vivo and in vitro sources. Purified tDRs can be used for various downstream applications including differential affinity capture of tDR-binding proteins, the details of which are also described in this contribution.

RNA, Transfer↗
PubMed1982

Comparison of the hydrolysis patterns of several tRNAs by cobra venom ribonuclease in different steps of the aminoacylation reaction.

The hydrolysis of several tRNAs by an endonuclease extracted from the venom of Naja oxiana and specific for double-stranded, or at least highly ordered, regions has been studied under various experimental conditions. It is shown that the hydrolysis patterns of yeast tRNAPhe, tRNAVal and tRNAAsp in the isolated state are similar, most of the cuts occurring in the anticodon and acceptor stems. Ionic conditions are able to modify the hydrolysis pattern. The origin of these modifications is discussed. The protection against ribonuclease action, afforded to tRNAPhe, tRNAVal and tRNAAsp by the cognate aminoacyl-tRNA synthetase, is analyzed. It is shown that in all cases the anticodon stem is protected. The 3'-terminal region does not seem to be tightly engaged in the complex with the aminoacyl-tRNA synthetase. These results are discussed in the light of information on contact areas previously obtained by ultraviolet cross-linking techniques. The effects of the small ligands (ATP and amino acid) on the protection afforded to the tRNA by the cognate synthetase, have been studied. In the valine and aspartic acid systems, ATP induced a modification of the tRNA-enzyme complex leading to differences in the hydrolysis pattern of the 3'-accepting region. The effects of aminoacylation on the cleavage of tRNAPhe, tRNAVal and tRNAAsp were also studied. Whereas no modification of the cleavage map was observed in the aspartic system, aminoacylation resulted in slight but significant modifications of the hydrolysis pattern for tRNAPhe and tRNaVal in the 3'-terminal region.

Adenosine Triphosphate↗
PubMed1994

Intron-dependent formation of pseudouridines in the anticodon of Saccharomyces cerevisiae minor tRNA(Ile).

We have isolated and sequenced the minor species of tRNA(Ile) from Saccharomyces cerevisiae. This tRNA contains two unusual pseudouridines (psi s) in the first and third positions of the anticodon. As shown earlier by others, this tRNA derives from two genes having an identical 60 nt intron. We used in vitro procedures to study the structural requirements for the conversion of the anticodon uridines to psi 34 and psi 36. We show here that psi 34/psi 36 modifications require the presence of the pre-tRNA(Ile) intron but are not dependent upon the particular base at any single position of the anticodon. The conversion of U34 to psi 34 occurs independently from psi 36 synthesis and vice versa. However, psi 34 is not formed when the middle and the third anticodon bases of pre-tRNA(Ile) are both substituted to yield ochre anticodon UUA. This ochre pre-tRNA(Ile) mutant has the central anticodon uridine modified to psi 35 as is the case for S.cerevisiae SUP6 tyrosine-inserting ochre suppressor tRNA. In contrast, neither the first nor the third anticodon pseudouridine is formed, when the ochre (UUA) anticodon in the pre-tRNA(Tyr) is substituted with the isoleucine UAU anticodon. A synthetic mini-substrate consisting of the anticodon stem and loop and the wild-type intron of pre-tRNA(Ile) is sufficient to fully modify the anticodon U34 and U36 into psi s. This is the first example of the tRNA intron sequence, rather than the whole tRNA or pre-tRNA domain, being the main determinant of nucleoside modification.

Anticodon↗
PubMedDate not supplied

Recent approaches to probe functional groups in ribonuclease P RNA by modification interference.

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of (pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for the pro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA to E. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.

Base Sequence↗
PubMed1993

Tertiary structural analysis of Escherichia coli lysine tRNA.

It has been suggested that Escherichia coli lysine tRNA (E. coli tRNA(Lys)) has an unusual anticodon loop structure, judging from the abnormal CD pattern derived from 5-methylaminomethyl-2-thiouridine (mnm5s2U) located at the first position of the anticodon of the tRNA. Here, the structure of E. coli tRNA(Lys) was analysed by chemical modification and nuclear magnetic resonance (NMR) of the imino protons. The chemical probing approach showed that the anticodon arm region forms the ordinary stem-and-loop structure as expected from the standard clover-leaf structure, because N7 of G30, N3 of C40, N7 of A41 and N7 of A42 were protected under a native condition. By using the heteronuclear 15N-1H single quantum coherence (HSQC) spectra of 15N-labeled E.coli tRNA(Lys) and the analyses of the nuclear Overhauser effects (NOE) between the imino protons of non labeled E.coli tRNA(Lys), the imino protons in the acceptor stem was assigned, which indicates that the acceptor stem exists in an usual form. The tertiary base-base interactions was also detected. Thus, E.coli tRNA(Lys) seems to maintain a similar structure to the ordinary L-shaped tertiary structure, as a whole. The fine structure of the anticodon loop is now being analyzed.

Anticodon↗
PubMed2006

The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase.

BACKGROUND: Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interestingly, it was found that tRNA methylation, one of the most common modifications, can be introduced by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that belong to two structurally and phylogenetically unrelated protein superfamilies: RFM and SPOUT. RESULTS: As a part of a large-scale project aiming at characterization of a complete set of RNA modification enzymes of model organisms, we have studied the Escherichia coli proteins YibK, LasT, YfhQ, and YbeA for their ability to introduce the last unassigned methylations of ribose at positions 32 and 34 of the tRNA anticodon loop. We found that YfhQ catalyzes the AdoMet-dependent formation of Cm32 or Um32 in tRNASer1 and tRNAGln2 and that an E. coli strain with a disrupted yfhQ gene lacks the tRNA:Cm32/Um32 methyltransferase activity. Thus, we propose to rename YfhQ as TrMet(Xm32) according to the recently proposed, uniform nomenclature for all RNA modification enzymes, or TrmJ, according to the traditional nomenclature for bacterial tRNA MTases. CONCLUSION: Our results reveal that methylation at position 32 is carried out by completely unrelated TrMet(Xm32) enzymes in eukaryota and prokaryota (RFM superfamily member Trm7 and SPOUT superfamily member TrmJ, respectively), mirroring the scenario observed in the case of the m1G37 modification (introduced by the RFM member Trm5 in eukaryota and archaea, and by the SPOUT member TrmD in bacteria).

Escherichia coli Proteins↗
PubMed1994

Dual hydrolytic role for Pb(II) ions.

RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds in the 3'-end of tRNA and also cleaves the bond between tRNA and its amino-acid, attached at the 3'-end via an ester bond to the terminal ribose in aminoacyl tRNA. The two reactions proceed at different rates. The rate of deacylation is significantly faster than the rate of cleavage of phosphodiester bonds, with a pH-optimum of 7. This dual hydrolytic role is not seen for other metal ions examined, namely Zn(II), Cd(II) and Mn(II). The rate of the two kinds of hydrolyses by Pb2+ ions is compared with that of other metal-ions. The mechanism of cleavage is investigated further by modification of the 3'-end of tRNA.

Acylation↗
PubMed2002

Identification and characterization of tRNA (Gm18) methyltransferase from Thermus thermophilus HB8: domain structure and conserved amino acid sequence motifs.

BACKGROUND: Transfer RNAs from an extreme thermophile, Thermus thermophilus, commonly possess 2'-O-methylguanosine at position 18 (Gm18) in the D-loop. This modification is post-transcriptionally introduced by tRNA (Gm18) methyltransferase. RESULTS: Partial amino acid sequence data were obtained from purified T. thermophilus tRNA (Gm18) methyltransferase by peptide sequencing and mass spectrometry. The sequence data were used to screen the T. thermophilus genome database currently in progress, resulting in the identification of the corresponding gene. Purified recombinant enzyme showed a strict specificity for methylation at the 2'-OH of G18 in tRNA. Sequence alignment with other known or putative methyltransferases elucidates that tRNA (Gm18) methyltransferases have specific conserved region as well as three consensus motifs found in RNA ribose 2'-O-methyltransferases. The enzyme truncated at its N and C termini by limited tryptic digestion still retained binding activity for S-adenosyl-l-homocysteine, but lost the catalytic activity. CONCLUSION: This is the first report describing the identification of a methyltransferase gene of the trmH family through the analysis of a purified protein. Further, our results indicate that a restricted region(s) in the terminal amino acid residues of T. thermophilus tRNA (Gm18) methyltransferase are responsible for tRNA recognition and a main part of the enzyme is allocated for a catalytic core.

Amino Acid Motifs↗
PubMed1979

Yeast tRNA Leu UAG. Purification, properties and determination of the nucleotide sequence by radioactive derivative methods.

A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu). The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2'-O-Methylated guanosine was characterized using the [gamma-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis. A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units. Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

Adenine↗
PubMed1995

Identification of the elongation factor Tu binding site on 70S E. coli ribosomes by chemical crosslinking.

Elongation factor Tu (EF-Tu), in the presence of Phe-tRNA, GMPPCP, and Poly (U), binds to 70S ribosomes at the recognition (R) site. In order to identify the ribosomal proteins adjacent to the EF-Tu occupying the R site, EF-Tu:Phe-tRNA:GMPPCP:ribosome complexes were crosslinked by modification with 2-iminothiolane and mild oxidation to form disulfide bridges between neighbouring proteins whose endogenous or introduced SH groups were appropriately located. The binding of Phe-tRNA to the ribosome was shown to be largely dependent on the presence of Poly(U). The total protein from the complexes was extracted and separated by two-dimensional gel electrophoresis by non-equilibrium pH gradient electrophoresis (NEpHGE) in the first dimension, followed by gradient SDS gel electrophoresis in the second dimension. Comparison of control samples crosslinked without Poly(U) to those crosslinked with Poly(U) present showed a single crosslinked complex in the region of the gel near EF-Tu. No cross-links in the vicinity of EF-Tu were visible in the absence of Poly(U). The crosslinked proteins in this region were recovered by electroelution, radiolabeled and their identity was confirmed by 2D gel electrophoresis and immunoblot analyses. Two major 50S ribosomal proteins, L7/L12 and L10 were found to be covalently linked to EF-Tu. The isolated crosslinked complex did not contain any protein from the 30S subunit. These results demonstrate that L7/L12 and L10 are the major, if not only, ribosomal protein cross-links to EF-Tu in the R site. In contrast to previous crosslinking results obtained by others, our results define a unique location for the EF-Tu binding site, one compatible with functional data and near that of the EF-G binding site on the ribosome.

Binding Sites↗
PubMedDate not supplied

[Role of zinc ions in the functioning of bovine tryptophanyl-tRNA-synthetase].

By means of atomic absorption spectroscopy up to 0.9 Zn2+ atom per molecule of bovine tryptophanyl-tRNA-synthetase (E. C. 6.1.1.2) was found. Treatment of the enzyme with orthophenanthroline (Zn2+-chelating agent) or prolonged dialysis leading to the removal of bound Zn2+ causes inactivation of the enzyme whereas the addition of Zn2+ reactivates it. Kinetic analysis of the inhibiting action of orthophenanthroline at various concentrations of tryptophan, ATP and tRNA leads to the conclusion that removal of Zn2+ prevents the binding of the ATP molecule to tryptophanyl-tRNA-synthetase. By means of chemical modification it is shown that exposed histidine residues and the carboxylic groups of the enzyme participate in Zn2+ binding. According to circular dichroism data removal of Zn2+ has no influence on the secondary structure although some local alterations of the ternary structure are revealed.

Adenosine Triphosphate↗
PubMed2000

The np 3243 MELAS mutation: damned if you aminoacylate, damned if you don't.

The np 3243 MELAS mtDNA mutation in tRNA(leu(UUR))has been variously proposed as a loss-of-function or as a gain-of-function mutation, based on apparently contradictory studies in cultured cell lines. A new report describing the molecular effects of the mutation in vivo now mirrors this variability. This should prompt a more systematic re-investigation of cells carrying the mutation, in order to separate primary from secondary and pathogenic from compensatory effects, all of which may contribute to disease phenotype. Nuclear genetic and developmental background, mitochondrial haplotype, and epigenetic effects may all influence the pathological outcome. Defects in both base-modification and aminoacylation of the mutant tRNA could play critical roles.

Acylation↗
PubMed1995

Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site.

Previous studies suggest that the mechanism of action of the ribosome in translation involves crucial transfer RNA (tRNA)-ribosomal RNA (rRNA) interactions. Here, a selection scheme was developed to identify bases in 16S rRNA that are essential for tRNA binding to the P site of the small (30S) ribosomal subunit. Modification of the N-1 and N-2 positions of 2-methylguanine 966 and of the N-7 position of guanine 1401 interfered with messenger RNA (mRNA)-dependent binding of tRNA to the P site. Modification of the same positions as well as of the N-1 and N-2 positions of guanine 926 interfered with mRNA-independent binding of tRNA at high magnesium ion concentration. These results suggest that these three bases are involved in intermolecular contacts between ribosomes and tRNA.

Aldehydes↗
PubMed1980

Phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases. Substrate specificity in the ATP/PPi exchange with regard to ATP analogs.

The analogs of ATP have been tested in the ATP/PPi exchange reaction of phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases from baker's yeast. Three compounds are substrates for phenylalanyl-tRNA, seven for lysyl-tRNA, two for isoleucyl-tRNA and five for arginyl-tRNA synthetase. Their Km and V values have been determined. No analog was an inhibitor. (3'-dATP), 3'-Deoxyadenosine 5'-triphosphate which is an inhibitor of the four enzymes in the aminoacylation reaction, becomes a good substrate in the PPi exchange. Additionally lysyl-tRNA synthetase accepts two analogs with modifications at position 6 of the purine and three analogs modified at the ribose moiety as substrates in the PPi exchange, whereas these compounds are inactive or inhibitors in the aminoacylation reaction. In general the enzymes are less specific in the ATP/PPi exchange and the results indicate a more sophisticated proof of the nucleotide moiety upon aminoacylation. This could occur with the aminoacyladenylate intermediate as well as with any other intermediate.

Adenosine Triphosphate↗
PubMed2000

End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae.

All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNA(Trp)) is edited by a C to U modification at the first position of the anticodon. To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNA(Trp)(CCA), tRNA(Ile)(UAU), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile) and tRNA(Lys) were mainly mitochondrial; and tRNA(Trp) and tRNA(Val) were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNA(Ile) underwent importation in vitro into isolated mitochondria more efficiently than 5'-extended precursor tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNA(Trp) was imported in vitro more efficiently than edited tRNA(Trp), suggesting the presence of importation determinants in the anticodon.

Animals↗
PubMed1995

RNA recognition based on a pair of tertiary hydrogen interaction.

The recognition of transfer RNAs (tRNAs) by aminoacyl tRNA synthetases is a critical step in establishing the fidelity of translation. For E. coli cysteine tRNA synthetase, recognition of tRNA(Cys) and discrimination from all other tRNAs is based on the U73 discriminator base, the GCA anticodon, and a G15:G48 tertiary base pair. While the discriminator base and the anticodon sequence are often used by many synthetases as the determinants for tRNA recognition, the dependence on a tertiary interaction in the cognate tRNA for recognition is unique to the cysteine enzyme. Here the structural basis for recognition at the G15:G48 tertiary interaction of E. coli tRNA(Cys) is explored by structural modeling, chemical modifications, and kinetic analysis. The results established an unusual RNA tertiary interaction that provides a plausible mechanism for recognition by cysteine tRNA synthetase.

Anticodon↗
PubMedDate not supplied

Single nucleotide modulation of uridine to pseudouridine rearrangement in transfer RNA catalyzed by pseudouridine synthase I.

E. coli pseudouridine synthase I (PSUI) catalyzes the rearrangement of uridine residues in positions 38, 39 and 40 of tRNA transcripts to pseudouridine. These positions are located in the anticodon stem-loop of the tRNA molecule. Fourteen different E. coli tRNAs are substrates for the enzyme, whereas four other tRNAs which contain uridine in position 38 are not. Investigations were focused on the basis of enzyme differentiation between substrate and non-substrate tRNAs. Comparison of modification reactions with mutant and wild-type tRNA transcripts demonstrates that the presence of a G36 residue modulates modification by PSUI at position 38. In addition to local sequence effects, steady-state kinetic analyses suggest the existence of other recognition elements distinct from the immediate vicinity of modification.

Base Sequence↗