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PubMed1989

Identification of the major tRNA(Phe) binding domain in the tetrameric structure of cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast.

Native cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast is a tetramer of the alpha 2 beta 2 type. On mild tryptic cleavage it gives rise to a modified alpha 2 beta 2 form that has lost the tRNA(Phe) binding capacity but is still able to activate phenylalanine. In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated beta subunit. Each purified peptide was unambiguously assigned to a unique stretch of the beta subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing. Together with earlier results from affinity labelling studies the present data show that the Lys 172-Ile 173 bond is the unique target of trypsin under mild conditions and that the N-terminal domain of each beta subunit (residues 1-172) contains the major tRNA(Phe) binding sites.

Amino Acid Sequence↗
PubMed1988

Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase--beta-galactosidase chimeric protein.

The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Phenylalanyl-tRNA synthetases as an example for comparative and evolutionary aspects of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are indispensable components of protein synthesis in all three lines of evolutionary descent, eubacteria, archaebacteria and eukaryotes. Furthermore they are also present in the translational apparatus of the semi-autonomous organelles, mitochondria and chloroplasts, of the eukaryotic cell. Therefore aminoacyl-tRNA synthetases are appropriate objects for comparative molecular biology in order to obtain a comprehensive picture of the evolution of the translational process. The analysis of the phenylalanyl-tRNA synthetase in a large variety of organisms and organelles in this respect is the most advanced. In addition to comparison of quaternary structure, analysis includes functional aspects of accuracy mechanisms (proofreading) and comparison of structural features by means of substrate analogs. Evolutionary relationships are furthermore elucidated using the immunological approach and heterologous aminoacylation.

Amino Acyl-tRNA Synthetases↗
PubMed2024

Self-quenched tRNA reporters for imaging tRNA-derived RNA biogenesis.

tRNA-derived small RNAs (tDRs) are an emerging class of small non-coding RNAs that play crucial roles in various cellular processes. However, there is a paucity of data on their sub-cellular localization due to a lack of tools and reagents to image tDRs. Imaging tDRs remains challenging due to the similar sequences between tDR and its parent tRNA. Here, we describe an innovative tool for studying the formation and localization of tDRs in various biological processes using a self-quenched tDR biogenesis reporter. This method utilizes a full-length tRNA molecule conjugated with both fluorescence and quencher groups at 5'- and 3'- ends. In its intact state, the fluorescence is quenched. Upon cleavage by specific ribonucleases and strand separation, the fluorescence becomes detectable, allowing real-time imaging of tDR biogenesis. This protocol details the design, synthesis, and application of this reporter, including transfection procedures and imaging techniques. The method offers a powerful approach for investigating tDR dynamics in living cells, providing insights into their roles in cellular processes and stress responses.

RNA, Transfer↗
PubMed2001

Ester and hydroxamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases.

The structure activity relationship on a series of ester and hydroxamate analogues of methionyl and isoleucyl adenylate has been investigated through introducing linkers between the 1'-position of ribose and adenine surrogates as methionyl-tRNA, and isoleucyl-tRNA synthetase inhibitors, respectively. The results indicate that ester analogue 23 was found to be a potent inhibitor of Escherichia coli methionyl-tRNA synthetase, and its interaction with the active site was proposed by a molecular modeling study.

Adenosine↗
PubMed2001

Vanilloid and isovanilloid analogues as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases.

As aminoacyl adenylate surrogates, a series of methionyl and isoleucyl phenolic analogues containing bioisosteric linkers mimicking ribose have been investigated. Inhibition of synthesized compounds to the aminoacylation reaction by the corresponding Escherichia coli methionyl-tRNA and isoleucyl-tRNA synthetases indicated that 18 was found to be a potent inhibitor of isoleucyl-tRNA synthetase. A molecular modeling study demonstrated that in 18, isovanillate and hydroxamate served as proper surrogates for adenine and ribose in isoleucyl adenylate, respectively.

Adenine↗
PubMed1998

Influence of FIS on the transcription from closely spaced and non-overlapping divergent promoters for an aminoacyl-tRNA synthetase gene (gltX) and a tRNA operon (valU) in Escherichia coli.

The gltX gene, encoding the glutamyl-tRNA synthetase (GluRS), and the valU operon, whose transcripts contain three tRNAVal/UAC and one tRNALys/UUU, are adjacent and divergently transcribed. It is the only known case of adjacent genes encoding an aminoacyl-tRNA synthetase and a tRNA precursor in Escherichia coli. The gltX promoters (P1, P2 and P3) direct the synthesis of transcripts non-overlapping with and divergent from the one initiated at the valU promoter. We report that their promoter region (250 bp) contains three binding sites for the factor for inversion stimulation (FIS), centred at positions -71, -91 and -112 from the valU transcription initiation site, and that the destruction of any of these sites does not prevent the binding of FIS to the others. As FIS is one of the major positive regulators of stable RNA operons, we have studied its role on gltX and valU transcription. FIS stimulates valU transcription in vitro and about twofold in vivo during steady-state exponential growth. In contrast, gltX transcription is repressed by the presence of FIS in vitro and about twofold in vivo during growth acceleration when a decrease in GluRS concentration was observed. Under all conditions tested, most of the gltX transcripts start at the P3 promoter. Nested deletions of this regulatory region reveal that the FIS-dependent repression of the gltX-P3 promoter is abolished after the removal of the valU promoter, and is not altered by the additional removal of the FIS binding sites; moreover, in vivo transcription from gltX-P1 and/or gltX-P2 present on some of these regulatory region variants is modulated by the nature of the upstream region by FIS and is sometimes stronger than that from gltX-P3. These results show that the strength and the site of gltX transcription initiation are influenced by the upstream region up to and including the valU promoter; furthermore, they indicate that although these adjacent genes are involved in the first step of protein biosynthesis and share cis and trans regulatory elements, their transcription is non-co-ordinate.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Characterization of a unique enzyme complex composed of S-adenosyl-L-methionine-tRNA-methyltransferase and aminoacyl-tRNA synthetase activities.

S-adenosyl-L-methionine-tRNA methyltransferases of a murine leukemia cell line were found to exist in a high molecular weight enzyme complex. Aminoacyl-tRNA synthetase activity always co-chromatographed and co-sedimented with methyltransferase activity in evidence of a unique association of these two groups of enzymes. Molecular weight studies showed a probable molecular weight of 9 X 10(5) daltsons for the intact complex which dissociates to complexes of 6 X 10(5) and 3 X 10(5) daltons. The complexes contain discrete polypeptides of 25,000-90,000 daltons as determined from SDS-gel electrophoresis. High resolution fatty acid analysis showed that only very small amounts of saponifiable lipids were associated with the purified enzyme complex. Similarly very little protein-bound sugars was found within the complex indicating that neither lipids nor sugars were involved in the protein-protein interactions of the complex. Analysis of tRNA methylated in vitro indicated the presence of most methyltransferase activities in the purified complex. Of note was the absence from the complex of the methyltransferase responsible for the production of ribo Tp.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Influences of amino acid, ATP, pyrophosphate and tRNA on binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

Aminoalcohol-AMP esters, structurally related to the assumed intermediates of the amino acid activation reaction, behave as competitive inhibitors both with respect to the amino acid and ATP, when tested in the ATP-(32P) PPi-exchange or the tRNA-charging reaction. However, closer investigation of the binding of norvalinyl adenylate to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 by an equilibrium method shows that only the amino acid is a true competitor, while ATP cannot displace the ester from binding. Pyrophosphate enhances the stability of the ester-enzyme complex whereas tRNA is without detectable influence.

Adenosine Monophosphate↗
PubMed1979

The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase.

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60 leads to 51 leads to 40 leads to 24 kilodaltons. The transition 51 leads to 40 is hindered in tryptophanyl adenylate.enzyme complex. Yeast tRNATrp accelerates the first steps of hydrolysis and decelerates the transition 40 leads to 24. Once tRNATrp is added to the synthetase.adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRNATrp oxidized with NaI04 and tRNATrp lacking the 3'-terminal adenosine. Oxidized tRNATrp (but not tRNATrp without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNATrp with beef pancreas tryptophanyl-tRNA synthetase involving the association of tRNA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate.

Adenosine Monophosphate↗
PubMed1979

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).

Amino Acyl-tRNA Synthetases↗
PubMed1986

Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa.

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate.

Adenosine Triphosphate↗
PubMed1988

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A.

For discrimination between isoleucine and 19 other amino acids by isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from kcat and Km values in amino-acylation of cognate tRNA(Ile)-C-C-A. Factors D are also products of initial discrimination factors I' and proof-reading factors II'; D = I' II'. Factors II' were calculated from AMP formation stoichiometries and factors I' as quotients of D and II'; I' = D/II'. II' is considered as a product of a pre- and post-transfer proof-reading factor (II' = II1II2), I' as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination. With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D = 38,000); other D values in a high range (10,000-20,000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300-700); the remaining amino acids are discriminated with medium D values (1000-10,000). Discrimination factors D observed for isoleucyl-tRNA synthetase from E. coli are on average 2-3 times higher than for the yeast enzyme. Highest values were found in discrimination against Gly, Ala and Val (60,000-72,000), the lowest values for Cys, Arg and Trp (600-3000); the other amino acids exhibit D values between 20,000 and 50,000. Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.

Acylation↗
PubMed1988

ATP-induced activation of the aminoacylation of tRNA by the isoleucyl-tRNA synthetase from Escherichia coli.

The rate of aminoacylation of tRNA catalyzed by the isoleucyl-tRNA synthetase form Escherichia coli has been measured. A steady-state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile-tRNA from the enzyme.

Adenosine Triphosphate↗
PubMed1997

Evolution of the aminoacyl-tRNA synthetase family and the organization of the Drosophila glutamyl-prolyl-tRNA synthetase gene. Intron/exon structure of the gene, control of expression of the two mRNAs, selective advantage of the multienzyme complex.

In Drosophila, glutamyl-prolyl-tRNA synthetase is a multifunctional synthetase encoded by a unique gene and composed of three domains: the amino- and carboxy-terminal domains catalyze the aminoacylation of glutamic acid and proline tRNA species, respectively, and the central domain is made of 75 amino acids repeated six times amongst which 46 are highly conserved and constitute the repeated motifs [Cerini, C., Kerjan, P., Astier, M., Gratecos, D., Mirande, M. & Sémériva, M. (1991) EMBO J. 10, 4267-4277]. The intron/exon organization of the Drosophila gene reveals the presence of six exons among which four are in the 5'-end encoding glutamic acid activity. Only one exon encodes the repeated motifs. A comparison of introns positions, intron classes and intron/exon boundaries in the Drosophila gene and in its human counterpart is compatible with the intron-early hypothesis presiding, at least in part, to the evolution of the synthetases. The full-length fly protein is encoded by a 6.1-kb mRNA which is expressed throughout development. In addition, a shorter transcript encompasses the 3'-end of the cDNA and it is especially abundant in 5-10-h embryos until the first larval stage. Expression of these two mRNAs seems to be controlled by two independent promoters. The 6.1-kb mRNA promoter is probably localized in the 5'-end of the gene. The small mRNA promoter resides in the 4th intron and evidence is provided that the mRNA encodes only the domain corresponding to prolyl-tRNA synthetase and is functional in vivo. Finally, transgenic flies have been established by using constructs corresponding to the three domains of the protein. Overexpression of the repeated motifs leads to a sterility of the flies that suggests a role of these motifs in linking the multienzyme complex to an, as yet, unknown structure of the protein synthesis apparatus.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data.

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Role of the anticodon loop in triggering conformational changes of tryptophanyl-tRNA synthetase by tRNA Trp].

The circular dichroic spectrum of the bovine tRNATrp is altered in complex with homologous tryptophanyl-tRNA synthetase showing the conformational changes of the substrate under the action of enzyme. The conversion of the exposed cytosine into uridine bases in tRNATrp (beef, yeast) does not prevent complex formation but abolishes the tRNA action on the limited proteolysis of the synthetase. It was shown that elimination of this type of tRNA activity is solely attributed to the cytosine leads to uridine conversion in the anticodon loop. A conclusion is made that the anticodon loop of the tRNATrp plays a dominant role in triggering conformational changes of the synthetase under substrate action whereas the CCA-end is not directly involved in this process.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. II. Structural characterization of the polypeptide components and immunological identification of the methionyl-tRNA synthetase subunit.

The extensively purified multienzyme complexes from sheep and rabbit livers containing seven aminoacyl-tRNA synthetases specific for Ile, Leu, Met, Gln, Glu, Lys, and Arg displayed characteristic one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic patterns composed of 11 and 10 major polypeptide components, respectively. Their polypeptide compositions revealed by two-dimensional electrophoresis, including isoelectric focusing in 9 M urea, were not significantly more complex. The isoelectric point of each component from the two complexes fell within the pH range of 6.2 to 7.1, with the notable exception of the common polypeptide of Mr = 43,000 which was distinctly basic. The apparent molecular weight of each component from both complexes was determined by SDS-polyacrylamide gel electrophoresis. Four polypeptides, corresponding to molecular weights of 139,000, 129,000, 43,000, and 38,000 were common to both complexes. The other components from the two complexes displayed similar yet clearly distinct molecular weights. The molar ratios of the polypeptides, estimated by densitometry scanning of stained SDS-polyacrylamide gels, indicated that several components from each complex may be present as more than one copy. Following SDS-polyacrylamide gel electrophoresis, the methionyl-tRNA synthetase component from each complex was identified by the protein blotting procedure, using specific antibodies and 125I-labeled protein A. The unique labeled bands from the complexes of sheep and rabbit precisely matched the major polypeptides of Mr = 103,000 and 108,000, respectively. Mild trypsin treatment of the two native complexes generated fully active forms of methionyl-tRNA synthetase, with molecular weights of 68,000 and 69,500, respectively. The kinetics of proteolysis showed that modification proceeded sequentially through discrete intermediates.

Amino Acyl-tRNA Synthetases↗