Purification and characterization of the isoleucyl-tRNA synthetase component from the high molecular weight complex of sheep liver: a hydrophobic metalloprotein

Native isoleucyl-tRNA synthetase and a structurally modified form of methionyl-tRNA synthetase were purified to homogeneity following trypsinolysis of the high molecular weight complex from sheep liver containing eight aminoacyl-tRNA synthetases. The correspondence between purified isoleucyl-tRNA synthetase and the previously unassigned polypeptide component of Mr 139 000 was established. It is shown that dissociation of this enzyme from the complex has no discernible effect on its kinetic parameters. Both isoleucyl- and methionyl-tRNA synthetases contain one zinc ion per polypeptide chain. In both cases, removal of the metal ion by chelating agents leads to an inactive apoenzyme. As the trypsin-modified methionyl-tRNA synthetase has lost the ability to associate with other components of the complex [Mirande, M., Kellermann, O., & Waller, J. P. (1982) J. Biol. Chem. 257, 11049-11055], the zinc ion is unlikely to be involved in complex formation. While native purified isoleucyl-tRNA synthetase displays hydrophobic properties, trypsin-modified methionyl-tRNA synthetase does not. It is suggested that the assembly of the amino-acyl-tRNA synthetase complex is mediated by hydrophobic domains present in these enzymes.     

Expression of the aminoacyl-tRNA synthetase complex in cultured Chinese hamster ovary cells. Specific depression of the methionyl-tRNA synthetase component upon methionine restriction

Cultured Chinese hamster ovary cells were subjected to amino acid restriction to examine its effects on the level of expression of the nine aminoacyl-tRNA synthetase components of the multienzyme complex which was previously characterized (Mirande, M., Le Corre, D., and Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289). Lowering the methionine concentration in the medium from 100 to 1 microM led to growth arrest, rapid deacylation of tRNAMet, and progressive 2-fold elevation of the methionyl-tRNA synthetase level, as assessed by specific activity measurements and immunotitration. The levels of the other eight aminoacyl-tRNA synthetases were not affected. Total methionine deprivation led to the additional derepression of the leucyl- and isoleucyl-tRNA synthetase components, whereas the corresponding tRNAs remained fully acylated. These pleiotropic responses to total methionine restriction were abolished in the presence of 2 mM methioninol, suggesting that amino acid transport systems may play a role in the regulation of aminoacyl-tRNA synthetase expression. The effect of total deprivation of arginine, glutamine, isoleucine, leucine, lysine, or proline from the culture medium on the level of expression of the corresponding aminoacyl-tRNA synthetases was also examined. In all cases, no elevation of the level of the corresponding synthetase was observed. The behavior of methionyl-tRNA synthetase from Chinese hamster ovary cells displaying a 2-fold increased level of the enzyme due to methionine restriction was examined in detail. Failure to detect a free form of the enzyme by gel filtration, as well as the finding that the isolated complex displayed twice the amount of methionyl-tRNA synthetase relative to the other components, indicates that this multienzyme structure can accommodate at least one additional copy of one of its components.     

Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Valyl-tRNA synthetase gene of Escherichia coli K12. Primary structure and homology within a family of aminoacyl-TRNA synthetases

The DNA nucleotide sequence of the valS gene encoding valyl-tRNA synthetase of Escherichia coli has been determined. The deduced primary structure of valyl-tRNA synthetase was compared to the primary sequences of the known aminoacyl-tRNA synthetases of yeast and bacteria. Significant homology was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. In pairwise comparisons the highest level of homology was detected between the homologous valyl-tRNA synthetases of yeast and E. coli, with an observed 41% direct identity overall. Comparisons between the valyl- and isoleucyl-tRNA synthetases of E. coli yielded the highest level of homology detected between heterologous enzymes (19.2% direct identity overall). An alignment is presented between the three branched-chain aminoacyl-tRNA synthetases (valyl- and isoleucyl-tRNA synthetases of E. coli and yeast mitochondrial leucyl-tRNA synthetase) illustrating the close relatedness of these enzymes. These results give credence to the supposition that the branched-chain aminoacyl-tRNA synthetases along with methionyl-tRNA synthetase form a family of genes within the aminoacyl-tRNA synthetases that evolved from a common ancestral progenitor gene.     

Initiation of protein synthesis by folate-sufficient and folate-deficient Streptococcus faecalis R: partial purification and properties of methionyl-transfer ribonucleic acid synthetase and methionyl-transfer ribonucleic acid formyltransferase

The initiation of protein synthesis by Streptococcus faecalis R grown in folate-free culture occurs without N-formylation or N-acylation of methionyl-tRNA(f) (Met). Methionyl-tRNA synthetase and methionyl-tRNA formyltransferase were partially purified from S. faecalis grown under normal culture conditions in the presence of folate (plus-folate); the general properties of the enzymes were determined and compared with the properties of the enzymes purified from wild-type cells grown in the absence of folate (minus-folate). S. faecalis methionyl-tRNA synthetase displays optimal activity at pH values between 7.2 and 7.8, requires Mg(2+), and has an apparent molecular weight of 106,000, as determined by gel filtration, and 127,000, as determined by sucrose density gradient centrifugation. The K(m) values of plus-folate methionyl-tRNA synthetase for each of the three substrates in the aminoacylation reaction (l-methionine, adenosine triphosphate, and tRNA) are nearly identical to the respective substrate Michaelis constants of minus-folate methionyl-tRNA synthetase. Furthermore, both plus- and minus-folate S. faecalis methionyl-tRNA synthetases catalyze, at equal rates, the aminoacylation of tRNA(f) (Met) and tRNA(m) (Met) isolated from either plus-folate or minus-folate cells. S. faecalis methionyl-tRNA formyltransferase displays optimal activity at pH values near 7.0, is stimulated by Mg(2+), and has an apparent molecular weight of approximately 29,900 when estimated by sucrose density gradient centrifugation. The K(m) value of plus-folate formyltransferase for plus-folate Met-tRNA(f) (Met) does not differ significantly from that of minus-folate formyltransferase for minus-folate Met-tRNA(f) (Met). Both enzymes can utilize either 10-formyltetrahydrofolate or 10-formyltetrahydropteroyltriglutamate as the formyl donor; the Michaelis constant for the monoglutamyl pteroyl coenzyme is slightly less than that of the triglutamyl pteroyl coenzyme for both transformylases. Tetrahydrofolate and uncharged tRNA(f) (Met) are competitive inhibitors of both plus- and minus-folate S. faecalis formyltransferase; folic acid, pteroic acid, aminopterin, and Met-tRNA(m) (Met) are not inhibitory. These results indicate that the presence or absence of folic acid in the culture medium of S. faecalis has no apparent effect on either methionyl-tRNA synthetase or methionyl-tRNA formyltransferase, the two enzymes directly involved in the formation of formylmethionyl-tRNA(f) (Met). Therefóre, the lack of N-formylation of Met-tRNA(f) (Met) in minus-folate S. faecalis is due to the absence of the formyl donor, a 10-formyl-tetrahydropteroyl derivative. Although the general properties of S. faecalis methionyl-tRNA synthetase are similar to those of other aminoacyl-tRNA synthetases, S. faecalis methionyl-tRNA formyltransferase differs from other previously described transformylases in certain kinetic parameters.     

Purification of the yeast mitochondrial methionyl-tRNA synthetase. Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes

Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNA(Met). Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes. Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.     

A stereochemical and positional isotope-exchange study of the mechanism of activation of methionine by methionyl-tRNA synthetase from Escherichia coli

Methionyl-tRNA synthetase from Escherichia coli catalyses the activation of [18O2]methionine by adenosine 5'-[(R)-alpha 17O]triphosphate with inversion of configuration at P alpha. Furthermore methionyl-tRNA synthetase does not catalyse positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the absence of methionine or in the presence of the competitive inhibitor, methioninol, which eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved. These observations require that methionyl-tRNA synthetase catalyses the activation of methionine by an associative 'in-line' nucleotidyl transfer mechanism. A kinetic study of positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the presence of methionine, Mg2+ and methionyl-tRNA synthetase showed that torsional equilibration (18O exchange into the P alpha--O--P beta bridge) occurs faster than tumbling (18O exchange into P gamma by rotation about the C2 axis of Mg[18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E X Met-AMP X Mg[18O2]PPi complex.     

Purification and NMR studies of [methyl-13C]methionine-labeled truncated methionyl-tRNA synthetase

A procedure for the rapid purification of a truncated form of the Escherichia coli methionyl-tRNA synthetase has been developed. With this procedure, final yields of approximately 3 mg of truncated methionyl-tRNA synthetase per gram of cells, carrying the plasmid encoding the gene for the truncated synthetase [Barker, D.G., Ebel, J.-P., Jakes, R., & Bruton, C.J. (1982) Eur. J. Biochem. 127, 449], can be obtained. The catalytic properties of the purified truncated synthetase were found to be identical with those of the native dimeric and trypsin-modified methionyl-tRNA synthetases. A rapid procedure for obtaining milligram quantities of the enzyme is necessary before the efficient incorporation of stable isotopes into the synthetase becomes practical for physical studies. With this procedure, truncated methionyl-tRNA synthetase labeled with [methyl-13C]methionine was purified from an Escherichia coli strain auxotrophic for methionine and containing the plasmid encoding the gene for the truncated methionyl-tRNA synthetase. Both carbon-13 and proton observe-heteronuclear detect NMR experiments were used to observe the 13C-enriched methyl resonances of the 17 methionine residues in the truncated synthetase. In the absence of ligands, 13 of the 17 methionine residues could be resolved by carbon-13 NMR. Titration of the synthetase, monitoring the chemical shifts of resonances B and M (Figure 3), with a number of amino acid ligands and ATP yielded dissociation constants consistent with those derived from binding and kinetic data, indicating active site binding of the ligands under the conditions of the NMR experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly

Eukaryotic aminoacyl-tRNA synthetases are usually organized into high-molecular-weight complexes, the structure and function of which are poorly understood. We have previously described a yeast complex containing two aminoacyl-tRNA synthetases, methionyl-tRNA synthetase and glutamyl-tRNA synthetase, and one noncatalytic protein, Arc1p, which can stimulate the catalytic efficiency of the two synthetases. To understand the complex assembly mechanism and its relevance to the function of its components, we have generated specific mutations in residues predicted by a recent structural model to be located at the interaction interfaces of the N-terminal domains of all three proteins. Recombinant wild-type or mutant forms of the proteins, as well as the isolated N-terminal domains of the two synthetases, were overexpressed in bacteria, purified and used for complex formation in vitro and for determination of binding affinities using surface plasmon resonance. Moreover, mutant proteins were expressed as PtA or green fluorescent protein fusion polypeptides in yeast strains lacking the endogenous proteins in order to monitor in vivo complex assembly and their subcellular localization. Our results show that the assembly of the Arc1p-synthetase complex is mediated exclusively by the N-terminal domains of the synthetases and that the two enzymes bind to largely independent sites on Arc1p. Analysis of single-amino-acid substitutions identified residues that are directly involved in the formation of the complex in yeast cells and suggested that complex assembly is mediated predominantly by van der Waals and hydrophobic interactions, rather than by electrostatic forces. Furthermore, mutations that abolish the interaction of methionyl-tRNA synthetase with Arc1p cause entry of the enzyme into the nucleus, proving that complex association regulates its subcellular distribution. The relevance of these findings to the evolution and function of the multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases is discussed.     

Sedimentation behaviour of aminoacyl-tRNA synthetases from mixed lysates of yeast and rabbit liver

The subcellular distribution of five aminoacyl-tRNA synthetases from yeast, including lysyl-, arginyl- and methionyl-tRNA synthetases known to exist as high-molecular-weight complexes in lysates from higher eukaryotes, was investigated. To minimize the risks of proteolysis, spheroplasts prepared from exponentially grown yeast cells were lysed in the presence of several proteinase inhibitors, under conditions which preserved the integrity of the proteinase-rich vacuoles. The vacuole-free supernatant was subjected to sucrose density gradient centrifugation. No evidence for multimolecular associations of these enzymes was found. In particular, phenylalanyl-tRNA synthetase activity was not associated with the ribosomes, whereas purified phenylalanyl-tRNA synthetase from sheep liver, added to the yeast lysate prior to centrifugation, was entirely recovered in the ribosomal fraction. A mixture of lysates from yeast and rabbit liver was also subjected to sucrose gradient centrifugation and assayed for methionyl- and arginyl-tRNA synthetase activities, under conditions which allowed discrimination between the enzymes originating from yeast and rabbit. The two enzymes from rabbit liver were found to sediment exclusively as high-molecular-weight complexes, in contrast to the corresponding enzymes from yeast, which displayed sedimentation properties characteristic of free enzymes. The preservation of the complexed forms of mammalian aminoacyl-tRNA synthetases upon mixing of yeast and rabbit liver extracts argues against the possibility that failure to observe complexed forms of these enzymes in yeast was due to uncontrolled proteolysis. Furthermore, this result denies the presence, in the crude extract from liver, of components capable of inducing artefactual aggregation of the yeast aminoacyl-tRNA synthetases, and thus indirectly argues against an artefactual origin of the multienzyme complexes encountered in lysates from mammalian cells.     

Caenorhabditis elegans evolves a new architecture for the multi-aminoacyl-tRNA synthetase complex

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains.     

A complex from cultured Chinese hamster ovary cells containing nine aminoacyl-tRNA synthetases. Thermolabile leucyl-tRNA synthetase from the tsH1 mutant cell line is an integral component of this complex

The size distribution of the 20 aminoacyl-tRNA synthetases from wild-type Chinese hamster ovary (CHO) cells and from the mutant cell line tsH1, containing a temperature-sensitive leucyl-tRNA synthetase, was determined by gel filtration. Nine aminoacyl-tRNA synthetases, specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine and proline, which coeluted as high-Mr entities (Mr approximately 1.2 X 10(6)), were further co-purified to yield a multienzyme complex, the polypeptide composition of which was identical to that previously determined for the complex from rabbit liver. Immunoprecipitates obtained from crude extracts of wild-type and tsH1 mutant cells, using specific antibodies directed to the lysyl-tRNA or methionyl-tRNA synthetase components of the complex, displayed the same polypeptide compositions as that of the purified complex, thereby establishing the heterotypic nature of this complex. Although the activity of leucyl-tRNA synthetase from the mutant cells, grown at a permissive temperature, was low compared to that from the wild-type, the polypeptide of Mr 129 000, corresponding to this enzyme, was present in similar amounts and occurred exclusively as a component of the high-Mr complex. Finally, we report that attempts to demonstrate phosphorylation of the components of the complex from cultured CHO, HeLa and C3 cells were unsuccessful.     

Variations in activity of aminoacyl-tRNA synthetases as a function of development in Bacillus subtilis

Extracts from Bacillus sublilis cells at various stages of growth and spores were assayed for aminoacyl-tRNA synthetase and methionyl-tRNA transformylase activity. There was no major change in any synthetase activity or in methionyl-tRNA transformylase activity during the sporulation cycle, which implies that these are not sporulation induced enzymes. However, extracts from B. subtilis cultures showed a burst of activity of aminoacyl-tRNA synthetases during exponential growth.Preparations from dormant spores possessed the same kinds of aminoacyl-tRNA synthetase activities as vegetative cells for all the amino acids which were studied. Spores also contained methionyl-tRNA transformylases. These findings suggest that spores ought to be able to aminoacylate tRNA and formylate the initiator. N-formylmethionyl-tRNA, immediately upon germination.     

Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver

The functional interaction of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the Km values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyl-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Ile-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.     

Role of 5SrRNA as a positive effector of some aminoacyl-tRNA synthetases in macromolecular complexes, with specific reference to methionyl-tRNA synthetase.

1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. coli 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PPi exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H]isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H]tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H]glutamic acid, [3H]glycine, [3H]valine, [3H]alanine, and [3H]tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Synthesis of Methionyl-, Prolyl-, and Threonyl-Transfer Ribonucleic Acid Synthetases of Escherichia coli

Proline- and threonine-restricted growth caused a three- to fourfold derepression of the differential rate of synthesis of the prolyl- and threonyl-transfer ribonucleic acid (tRNA) synthetases, respectively. Similarly, there was approximately a 24-fold derepression in the rate of synthesis of methionyl-tRNA synthetase during methionine restriction. Addition of the respective amino acids to such derepressed cultures resulted in a repression of synthesis of their cognate synthetases. These results support previous findings and further strengthen the idea that the formation of aminoacyl-tRNA synthetases is regulated by some mechanism which is mediated by the cognate amino acids.     

Sulphur metabolism in Paracoccus denitrificans. Purification, properties and regulation of cysteinyl-and methionyl-tRNA synthetase.

Cysteinyl- and methionyl-tRNA synthetases (EC 6.11.-) were purified 1200- and 1000-fold, respectively, from sonic extracts of Paracoccus denitrificans strain 8944, and kinetics, substrate specificity and regulatory properties were determined using the ATP-PPi exchange reaction. Both enzymes had pH optima of approx. 8 and were inhibited by sulphydryl-group reagents. Cysteinyl-tRNA synthetase catalysed L-selenocysteine- and alpha-aminobutyric acid-dependent ATP-PPi exchange and methionyl-tRNA synthetase catalysed L-homocysteine-, L-selenomethionine- and norleucine-dependent ATP-PPi exchange. Both enzymes were inhibited by O-acetylserine. Cysteinyl-tRNA synthetase activity was stimulated by methionine and methionyl-tRNA synthetase activity was stimulated by sulphide, cysteine, and cysteic acid.     

Multiple forms of arginyl- and lysyl-tRNA synthetases in rat liver: a re-evaluation

The size distribution of lysyl- and arginyl-tRNA synthetases in crude extracts from rat liver was re-examined by gel filtration. It is shown that irrespective of the addition or not of several proteinase inhibitors, lysyl-tRNA synthetase was present exclusively as a high-Mr entity, while arginyl-tRNA synthetase occurred as high- and low-Mr forms, in the constant proportions of 2:1, respectively. The polypeptide molecular weights of the arginyl-tRNA synthetase in these two forms were 74000 and 60000, respectively. The high-Mr forms of lysyl- and arginyl-tRNA synthetases were co-purified to yield a multienzyme complex, the polypeptide composition of which was virtually identical to that of the complexes from rabbit liver and from cultured Chinese hamster ovary cells. Of the nine aminoacyl-tRNA synthetases, specific for lysine, arginine, methionine, leucine, isoleucine, glutamine, glutamic and aspartic acids and proline, which characterize the purified complex, each, except prolyl-tRNA synthetase, was assigned to the constituent polypeptides by the protein-blotting procedure, using the previously characterized antibodies to the aminoacyl-tRNA synthetase components of the corresponding complex from sheep liver.     

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).