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PubMed1994

Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase.

To elucidate the mode of action of pseudomonic acid, we have compared the deduced amino acid sequences of isoleucyl-tRNA synthetases (ILeRS) from wild-type Escherichia coli strain MC4100, a pseudomonic acid-resistant mutant (strain PS102) of MC4100, and a pseudomonic acid-producing strain, Pseudomonas fluorescens. Compared with the wild-type enzyme, the deduced amino acid sequence of E. coli mutant ileS gene in strain PS102 shows a single amino acid substitution of leucine for phenylalanine at residue 594 of the IleRS. This mutational alteration in IleRS of an E. coli pseudomonic acid-resistant mutant resides in a region of the enzyme in close proximity to one of the consensus sequences of class I aminoacyl-tRNA synthetases, the KMSKS sequence between residues 602 and 606 of the E. coli IleRS. DNA sequence of the cloned ileS gene predicts that the P. fluorescens IleRS consists of 943 amino acids with 54% identity with the E. coli IleRS. The P. fluorescens ileS gene and the wild type and PS102 alleles of E. coli ileS were cloned into an expression vector, pEXPCR, and the sensitivities of E. coli DH5 alpha cells harboring each of these plasmids were compared. The cells harboring the P. fluorescens ileS were found to be most resistant to pseudomonic acid, while the transformants expressing the PS102 IleRS were more resistant than those containing the wild-type E. coli IleRS. IleRS purified from the wild-type E. coli was specifically cleaved by trypsin between Lys605 and Ser606 in the region of K602MSKS606. The protection of the IleRS from the trypsin digestion was found with pseudomonic acid or ATP, but not with isoleucine or tRNA(1Ile). Based on these results, we propose that pseudomonic acid binds to IleRS in the vicinity of the KMSKS sequence that is an ATP-binding subsite, and that pseudomonic acid is a bifunctional inhibitor with characteristics of both isoleucine and ATP, for example, an analog of isoleucyladenylate.

Adenosine Triphosphate↗

PubMed1994

Role of the CCA terminal sequence of tRNA(Val) in aminoacylation with valyl-tRNA synthetase.

All known tRNAs have a universal CCA sequence at the 3'-terminal. To study the role of this terminal sequence in the aminoacylation process, base substitutions were introduced into a transcript of Escherichia coli valine tRNA and the effects on the aminoacylation activity with valyl-tRNA synthetase were evaluated. Substitution of the terminal adenosine residue at position 76 by C or U caused a 5-7-fold decrease of valine charging activity in Vmax/Km, while substitution by G resulted in about a 300-fold decrease. In addition, these mutations gave rise to an appreciable level of misaminoacylation with threonine. ATP hydrolysis activity during threonylation was lower in the terminal adenosine mutants than in the wild-type. Mutations introduced at positions 75 and 74 also caused threonylation instead of reducing valylation, albeit to a much smaller extent. These results indicate that the CCA sequence, especially the base portion of the terminal adenosine residue, plays an important role not only in amino-acylation efficiency with valine but also in preventing misaminoacylation by hydrolyzing misactivated threonyl-tRNA(Val).

Acylation↗

PubMed1993

Purification of glutamine tRNA synthetase from Saccharomyces cerevisiae. A monomeric aminoacyl-tRNA synthetase with a large and dispensable NH2-terminal domain.

Glutamine tRNA synthetase from Saccharomyces cerevisiae has been purified to homogeneity and shown to be a monomer of 91 kDa. The size of the polypeptide agrees with that predicted from the previously reported translated DNA sequence. Mild tryptic digestion removes an amino-terminal domain and releases a fragment of 65 kDa which begins at Ser205. This tryptic fragment is similar in size and sequence to Escherichia coli glutamine tRNA synthetase and shows a modest increase from the full-length yeast enzyme in the Km values for glutamine and ATP and no difference in the kcat for aminoacylation or the Km for tRNA. Thus, the removal of the NH2-terminal domain appears to indirectly affect the ATP- and glutamine-binding sites in the nucleotide-binding fold domain to which the NH2-terminal domain is fused. A monoclonal antibody directed against the NH2-terminal domain of the full-length enzyme has little effect upon the aminoacylation activity. Therefore, over 200 amino acids of the NH2 terminus of the full-length enzyme form a domain that operationally has only a modest influence on the catalytic core of the protein. These studies reinforce the concept that eukaryotic synthetases have quasi-independent domains not found in their prokaryotic counterparts which may confer a function distinct from aminoacylation.

Amino Acid Sequence↗

PubMed1993

[High molecular weight forms of aminoacyl-tRNA synthetases in animals. II. High molecular weight aminoacyl-tRNA synthetase complexes in lower animals].

High-molecular-weight aminoacyl-tRNA synthetase complexes have been found in hen, frog and carp liver cytosol as well as in cytosol from snails and Paramecium. The complexes from vertebrates and snails contained 13-15 aminoacyl-tRNA synthetases, revealed mass of about 4,000 kDa and sedimentation constant about 19 S. The complex from Paramecium containing 11 aminoacyl-tRNA synthetases, revealed a mass of about 1,600 kDa and sedimentation constant 15 S. The results of the present study support the view that the high-molecular-weight synthetase complexes are common entities in the animal kingdom.

Amino Acyl-tRNA Synthetases↗

PubMed1995

tRNA selection by a class II aminoacyl-tRNA synthetase: the role of accessory domains and inter-domain communication in RNA recognition.

We have used a secondary site suppression approach to investigate the basis of tRNA selection by the E. coli histidyl-tRNA synthetase. This enzyme recognizes a unique G-1:C73 base pair located in the acceptor stems of prokaryotic histidyl-tRNAs. A genetic selection system was constructed in which growth on glycerol was dependent on histidine specific amber suppression of a triose phosphate isomerase (tpi) gene containing an amber codon at His 95. Three independent revertants linked to hisS were isolated and sequenced, and the resulting mutant proteins were characterized biochemically. These studies are interpreted in light of the x-ray structure of the E. coli HisRS adenylate complex, and show that the C-terminal domain and its interactions with the catalytic domain play a biologically significant role in tRNA selection.

Binding Sites↗

PubMed2002

Genetic code origin: are the pathways of type Glu-tRNA(Gln) --> Gln-tRNA(Gln) molecular fossils or not?

A logical-evolutionary analysis is conducted to clarify whether or not pathways of type Glu-tRNA(Gln) --> Gln-tRNA(Gln) are molecular fossils of the mechanism that gave rise to the evolutionary organization of the genetic code. The result of this analysis is that these pathways are most likely a manifestation of this mechanism. This provides strong evidence in favor of the coevolution theory of genetic code origin, as this theory is based on the amino acid biosynthetic transformation taking place on tRNA-like molecules which imprinted the genetic code structuring. Comments on the different interpretations of these pathways found in the literature are also provided.

Evolution, Molecular↗

PubMed1984

The effect of aminoacyl- or peptidyl-tRNA at the A-site on the arrangement of deacylated tRNA at the ribosomal P-site.

Photoreactive derivatives of E. coli tRNAPhe bearing arylazido groups on guanine residues (azido-tRNA) were used for affinity labelling of E. coli ribosomes in the region of the P-site when the A-site was either free or occupied by aminoacyl- or peptidyl-tRNA. Corresponding complexes of azido-tRNA with ribosomes and poly(U) were obtained both nonenzymatically and with the use of elongation factors. UV-irradiation of the complexes resulted in labelling of ribosomal proteins (preferentially of 30 S subunit). Proteins S9 and S21 were labelled only when the A-site was free; S14 - only when it was occupied; S11, S13, S19 - in both cases; S5, S7, S12, S20 - in some states.

Affinity Labels↗

PubMed1985

An antisuppressor mutant of Saccharomyces cerevisiae deficient in isopentenylated tRNA has reduced delta 2-isopentenylpyrophosphate: tRNA-delta 2-isopentenyl transferase activity.

We have previously reported the isolation and initial characterization of a mutation in Saccharomyces cerevisiae, designated mod5-1, that reduces the capacity of altered tyrosine tRNAs to suppress ochre nonsense mutations. The mutation results in the virtual elimination of the modified tRNA nucleoside, N6-delta 2-(isopentenyl) adenosine, normally found adjacent to the anticodons of certain tRNA species. We demonstrate here that MOD5 codes for delta 2-isopentenylpyrophosphate: tRNA-delta 2-isopentenyl transferase, or a protein that regulates its synthesis.

Alkyl and Aryl Transferases↗

PubMed2005

Genes coding for SecG and Leu2-tRNA form an operon to give an unusual RNA comprising mRNA and a tRNA precursor.

The secG gene encoding the SecG subunit of the SecYEG translocon and the leuU gene encoding Leu2-tRNA are very closely located on the Escherichia coli chromosome. A secG-leuU disruptant was not viable unless secG-leuU was induced from a plasmid, indicating that leuU is an essential gene since secG is dispensable at 37 degrees C. A mutant strain in which the promoter region for secG was replaced with cat revealed the same phenotype as the secG-leuU disruptant, indicating that leuU was expressed from the secG promoter. When the secG-leuU locus was placed on a high copy plasmid, an RNA comprising both mRNA for SecG and a precursor for Leu2-tRNA was detected on a Northern blot. Moreover, a secG-leuU transcript was amplified by RT-PCR using the total RNA fraction prepared from wild type E. coli cells but not from the secG-leuU and the secG promoter disruptants, indicating that secG-leuU forms an operon. Thus, the expression of Leu2-tRNA requires expression of the upstream secG gene. The gene structure of secG-leuU was conserved among Gram-negative bacteria, although the sequences separating the two genes were quite diverse. The physiological significance of this unusual gene organization is discussed.

Amino Acid Sequence↗

PubMed1989

Time-resolved absorption, circular dichroism, and emission of tRNA. Evidence that the photo-cross-linking of 4-thiouridine in tRNA occurs from the triplet state.

The time-resolved optical density (TROD) and time-resolved circular dichroism (TRCD) spectra of the lowest triplet state of 4-thiouridine (4t-Urd) in aqueous solutions of tRNA are reported. The TROD spectrum is consistent with the triplet state being primarily in the thione tautomer. The intersystem crossing yield to the triplet is 0.35 and 0.27 (+/- 10%), respectively, with and without 10(-2) Mg2+ added to the solution. Upon addition of increasing amounts of I- to solutions of tRNA, the initial triplet yield decreases, the rate of the observed triplet decay increases, and the quantum yield of internal photo-cross-linking decreases for the 4t-Urd chromophore. The results show quantitatively that the near-UV-induced photo-cross-linking reaction in tRNA occurs from the triplet state of 4t-Urd. From the TRCD spectrum the dissymmetry factor (delta epsilon/epsilon) of some of the triplet-triplet absorption bands is shown to be significantly larger than for any of the ground-state absorption bands. Two CD transitions are seen in the triplet-triplet spectrum which are obscured in the TROD spectrum by the strong ground-state bleaching signal near 335 nm. This shows that TRCD may be useful, in some cases, in locating electronic transitions that are not observed in TROD spectra.

Chemical Phenomena↗

PubMed1975

Functional differences in protein synthesis between rat liver tRNA and tRNA from Novikoff hepatoma.

Synthesis of ovalbumin in fragmented oviduct magnum explants of immature, estrogen-stimulated chicks has been studied in the presence of exogenous tRNA. tRAN from Novikoff hepatoma specifically inhibited ovalbumin synthesis, determined by precipitation with antisera. In addition, the major protein(s) synthesized in the presence of hepatoma tRNA had higher electrophoretic mobility than ovalbumin, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. tRNAs from rat liver, rooster liver, and hen oviduct did not affect ovalbumin synthesis, although oviduct tRNA is stimulatory during the earlier stages of estrogen stimulation.

Animals↗

PubMed1975

In vitro synthesis of tRNA precursors and their conversion to mature size tRNA.

Two Escherichia coli tRNA gene clusters, tRNA1Tyr (su3+ and su3-) and tRNA2Tyr, tRNA2Gly (su+36), tRNA3Thr, were transcribed in a purified in vitro system. Evidence indicates that the adjacent tRNA genes are transcribed together as a common precursor of large size, which, on incubation with crude cell extracts, yields mature tRNA molecules.

Base Sequence↗

PubMed1986

Functional complementation between mutations in a yeast suppressor tRNA gene reveals potential for evolution of tRNA sequences.

Successive rounds of mutagenesis of a Schizosaccharomyces pombe strain bearing the UGA-reading sup3 tRNASer suppressor have been carried out for two cycles of inactivation and reactivation of the suppressor. The suppressor phenotype at each stage was found to involve different combinations of three mutations, A30, A53, and A67, in the sup3-UGA gene. Single mutations A30 and A53 inactivate the suppressor as does the presence of all three mutations. A67 by itself is phenotypically neutral, but in combination with either A30 or A53 suppressor function is restored. The frequency with which these and other complementation events occur in S. pombe demonstrates a significant potential for nucleotide sequence evolution in tRNA. Differential expression of the S. pombe genes in Saccharomyces cerevisiae suggests that the two yeasts have diverged at the transcriptional and RNA processing level. Processing of the mutant tRNA precursors in S. cerevisiae reveals a hierarchy of structural domains within the tRNA that vary in their importance for RNase P cleavage.

Alleles↗

PubMed1987

Mutants of Escherichia coli formylmethionine tRNA: a single base change enables initiator tRNA to act as an elongator in vitro.

We show that the absence of a Watson-Crick base pair at the end of the amino acid acceptor stem, which is a hallmark of all prokaryotic initiator tRNAs, is one of the key features that prevents them from acting as an elongator in protein synthesis. We generated mutants of Escherichia coli formylmethionine tRNA that have a base pair at the end of the acceptor stem. The mutants generated were C1----T1, which had a U.A base pair, A72----G72, which had a C.G base pair, and the C1A72----T1G72 double mutant, which lacked the base pair. After aminoacylation, the activity of these and other mutant initiator methionyl-tRNAs (Met-tRNAs) in elongation were assayed in a MS2 RNA-directed E. coli protein-synthesizing system and in binding to the elongation factor Tu (EF-Tu). Unlike wild-type initiator tRNA or the T1G72 double mutant, the T1 and G72 mutant Met-tRNAs were active in elongation, the G72 mutant being more active than the T1 mutant. The T1 and G72 mutant Met-tRNAs also formed a ternary complex with elongation factor EF-Tu.GTP, and their relative affinities for EF-Tu.GTP paralleled their activities in elongation. Combination of the T1 or G72 mutation with mutations in the GGG.CCC sequence conserved in the anticodon stem of initiator tRNAs led to a further increase in the activities of these mutant tRNAs in elongation such that one of these mutants was now almost as good an elongator as E. coli elongator methionine tRNA.

Base Sequence↗

PubMed1997

Biosynthesis of archaeosine, a novel derivative of 7-deazaguanosine specific to archaeal tRNA, proceeds via a pathway involving base replacement on the tRNA polynucleotide chain.

Archaeosine is a novel derivative of 7-deazaguanosine found in transfer RNAs of most organisms exclusively in the archaeal phylogenetic lineage and is present in the D-loop at position 15. We show that this modification is formed by a posttranscriptional base replacement reaction, catalyzed by a new tRNA-guanine transglycosylase (TGT), which has been isolated from Haloferax volcanii and purified nearly to homogeneity. The molecular weight of the enzyme was estimated to be 78 kDa by SDS-gel electrophoresis. The enzyme can insert free 7-cyano-7-deazaguanine (preQ0 base) in vitro at position 15 of an H. volcanii tRNA T7 transcript, replacing the guanine originally located at that position without breakage of the phosphodiester backbone. Since archaeosine base and 7-aminomethyl-7-deazaguanine (preQ1 base) were not incorporated into tRNA by this enzyme, preQ0 base appears to be the actual substrate for the TGT of H. volcanii, a conclusion supported by characterization of preQ0 base in an acid-soluble extract of H. volcanii cells. Thus, this novel TGT in H. volcanii is a key enzyme for the biosynthetic pathway leading to archaeosine in archaeal tRNAs.

Amino Acid Sequence↗

PubMed2001

Cleavage of non-tRNA substrates by eukaryal tRNA splicing endonucleases.

Eukaryal tRNA splicing endonucleases use the mature domains of pre-tRNAs as their primary recognition elements. However, they can also cleave in a mode that is independent of the mature domain, when substrates are able to form the bulge-helix-bulge structure (BHB), which is cleaved by archaeal tRNA endonucleases. We present evidence that the eukaryal enzymes cleave their substrates after forming a structure that resembles the BHB. Consequently, these enzymes can cleave substrates that lack the mature domain altogether. That raises the possibility that these enzymes could also cleave non-tRNA substrates that already have a BHB. As predicted, they can do so, both in vitro and in vivo.

Animals↗

PubMed1990

Residual tRNA secondary structure in 'denaturing' 8M urea/TBE polyacrylamide gels: effects on electrophoretic mobility and dependency on prior chemical modification of the tRNA.

Fifteen individual species of tRNA were treated with the chemical modifiers diethylpyrocarbonate, 50% aqueous hydrazine or hydrazine/3 M NaCl. Following purification of the chemically modified material on polyacrylamide gels containing 8 M urea, variant minor bands, in addition to the expected main band, were observed for 12 of the 15 tRNAs. Characterization of the content of chemically altered bases in material recovered from such bands indicated that tRNAs containing modified nucleotides in base-paired stems were excluded from the main band and present, often in enhanced amounts, in the minor variant bands. The persistence of residual secondary structure on 8 M urea gels run at 45 degrees C and the ability of chemically modified bases to alter electrophoretic mobilities warrant caution in designing and interpreting experiments in which chemically modified RNA is isolated on gels prior to further analysis. tRNA(Val) (VAC) was unique in that modified bases in non base-paired regions, according to the cloverleaf model of secondary structure, caused exclusion from the main band. Consequently, we propose a secondary structure for partially denatured tRNA(Val) (VAC), in which these bases are located in double stranded regions of the molecule.

Base Sequence↗

PubMed1998

Compilation of tRNA sequences and sequences of tRNA genes.

Sequences of 3279 sequences of tRNA genes and tRNAs published up to December 1996 are included in the compilation. Alignment of the sequences, which is most compatible with the tRNA phylogeny and known three-dimensional structures of tRNA, is used. Sequences and references are available under http://www.uni-bayreuth. de/departments/biochemie/trna/

Animals↗