Structure of the yeast valyl-tRNA synthetase gene (VASI) and the homology of its translated amino acid sequence with Escherichia coli isoleucyl-tRNA synthetase

The VASI gene encoding the valyl-tRNA synthetase from yeast was isolated and sequenced. The gene-derived amino acid sequence of yeast valyl-tRNA synthetase was found to be 23% homologous to the Escherichia coli isoleucyl-tRNA synthetase. This is the highest level of homology reported so far between two distinct aminoacyl-tRNA synthetases and is indicative of an evolutionary relationship between these two molecules. Within these homologous sequences, two functional regions could be recognized: the HIGH region which forms part of the binding site of ATP and the KMSKS region which is recognized as the consensus sequence for the binding of the 3'-end of tRNA (Hountondji, C., Dessen, Ph., and Blanquet, S. (1986) Biochemie (Paris) 68, 1071-1078). Secondary structure predictions as well as the presence of both HIGH and KMSKS regions, delineating the nucleotide-binding domain and the COOH-terminal helical domain in aminoacyl-tRNA synthetases of known three-dimensional structure, suggest that the yeast valyl-tRNA synthetase polypeptide chain can be folded into three domains: an NH2-terminal alpha-helical region followed by a nucleotide-binding topology and a COOH-terminal domain composed of alpha-helices which probably carries major sites in tRNA binding.     

The valyl-tRNA synthetase from Bacillus stearothermophilus has considerable sequence homology with the isoleucyl-tRNA synthetase from Escherichia coli

We report the DNA sequence of the valS gene from Bacillus stearothermophilus and the predicted amino acid sequence of the valyl-tRNA synthetase encoded by the gene. The predicted primary structure is for a protein of 880 amino acids with a molecular mass of 102,036. The molecular mass and amino acid composition of the expressed enzyme are in close agreement with those values deduced from the DNA sequence. Comparison of the predicted protein sequence with known protein sequences revealed a considerable homology with the isoleucyl-tRNA synthetase of Escherichia coli. The two enzymes are identical in some 20-25% of their amino acid residues, and the homology is distributed approximately evenly from N-terminus to C-terminus. There are several regions which are highly conservative between the valyl- and isoleucyl-tRNA synthetases. In one of these regions, 15 of 20 amino acids are identical, and in another, 10 of 14 are identical. The valyl-tRNA synthetase also contains a region HLGH (His-Leu-Gly-His) near its N-terminus equivalent to the consensus HIGH (His-Ile-Gly-His) sequence known to participate in the binding of ATP in the tyrosyl-tRNA synthetase. This is the first example of extensive homology found between two different aminoacyl-tRNA synthetases.     

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.     

Structure of the yeast isoleucyl-tRNA synthetase gene (ILS1). DNA-sequence, amino-acid sequence of proteolytic peptides of the enzyme and comparison of the structure to those of other known aminoacyl-tRNA synthetases

The ILS1 gene encoding for cytoplasmic isoleucyl-tRNA synthetase from Saccharomyces cerevisiae was subcloned from a 5.4-kb insert of the shuttle vector YEp13 to M13mp8 and M13mp9. Nucleotide sequence analysis of a 4.3-kb BamHI-HpaI fragment revealed a single open reading frame from which we deduced the amino-acid sequence of the enzyme. Independently obtained amino-acid sequence information from ten tryptic peptides of the purified enzyme confirmed the gene-derived structure. The enzyme is comprised of 1073 amino-acids consistent with earlier determinations of its molecular mass. The codon usage of ILS1 is typical of abundant yeast proteins. A significant homology to E. coli isoleucyl- and valyl-tRNA synthetases as well as to yeast valyl-tRNA synthetase was detected. The characteristic amino-acid residues of the aminoacyl-adenylate site and of the potential binding site of the 3'-end of tRNA found in other synthetases are present in the structure.     

Analysis of the structure of T4 bacteriophage-modified valyl-tRNA synthetase by limited proteolysis and isoelectric focusing.

The new form of valyl-tRNA synthetase (EC 6.1.1.9) that appears immediately after infection of Escherichia coli with bacteriophage T4 was purified and subjected to mild proteolysis using five different proteases. The inactivation of aminoacylation activity was both more extensive and rapid than that obtained with valyl-tRNA synthetase purified from uninfected E. coli. The addition of bulk tRNA from E. coli B protected the phage-specific form of valyl-tRNA synthetase from proteolysis, but ATP and valine did not exhibit a similar protective effect. The characteristic property of phage-modified valyl-tRNA synthetase, resistance to denaturation by 4 M urea, remained unaffected during treatment with trypsin. This suggested that the phage-specific factor tau, known to be associated with the synthetase in phage-infected cells, was protected from proteolysis in the synthetase-tau complex. Comparison by isoelectric focusing of normal valyl-tRNA synthetase, the phage-specific form of this enzyme, and phage enzyme from which tau had been removed, revealed no differences in the isoelectric points of these three molecules. Based on these results a model was drawn for the structural changes occurring in valyl-tRNA synthetase after association with the phage factor tau.     

Site-directed mutagenesis reveals transition-state stabilization as a general catalytic mechanism for aminoacyl-tRNA synthetases

Some aminoacyl-tRNA synthetases of almost negligible homology do have a small region of similarity around four-residue sequence His-Ile(or Leu or Met)-Gly-His(or Asn), the HIGH sequence. The first histidine in this sequence in the tyrosyl-tRNA synthetase, His-45, has been shown to form part of a binding site for the gamma-phosphate of ATP in the transition state for the reaction as does Thr-40. Residue His-56 in the valyl-tRNA synthetase begins a HIGH sequence, and there is a threonine at position 52, one position closer to the histidine than in the tyrosyl-tRNA synthetase. The mutants Thr----Ala-52 and His----Asn-56 have been made and their complete free energy profiles for the formation of valyl adenylate determined. Difference energy diagrams have been constructed by comparison with the reaction of wild-type enzyme. The difference energy profiles are very similar to those for the mutants Thr----Ala-40 and His----Asn-45 of the tyrosyl-tRNA synthetase. Thr-52 and His-56 of the valyl-tRNA synthetase contribute little binding energy to valine, ATP, and Val-AMP. Instead, the wild-type enzyme binds the transition state and pyrophosphate some 6 kcal/mol more tightly than do the mutants. Preferential transition-state stabilization is thus an important component of catalysis by the valyl-tRNA synthetase. Further, by analogy to the tyrosyl-tRNA synthetase, the valyl-tRNA synthetase has a binding site for the gamma-phosphate of ATP in the transition state, and this is likely to be a general feature of aminoacyl-tRNA synthetases that have a HIGH region.     

Mammalian high molecular weight and monomeric forms of valyl-tRNA synthetase

Valyl-tRNA synthetase from rat liver sediments at 15.5 S with a Stokes radius of 90 A, corresponding to a native molecular weight of 585,000. Purification of valyl-tRNA synthetase to homogeneity by a combination of conventional and affinity column chromatography yields a fully active monomeric form of valyl-tRNA synthetase with a sedimentation coefficient of 7.7 S and a Stokes radius of 45 A. The subunit molecular weight of the monomeric valyl-tRNA synthetase is 140,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the presence of 400 mM KCl, the purified monomeric valyl-tRNA synthetase associates to a high molecular weight form. The high molecular weight valyl-tRNA synthetase in the homogenate can be readily converted to the monomeric form by controlled trypsinization. The kinetic parameters of the two forms are nearly identical. The results suggest that the high molecular weight valyl-tRNA synthetase is a homotypic tetramer and converts to the monomeric valyl-tRNA synthetase after the cleavage of a small peptide.     

A novel thiol-dependent arsenite-sensitive valyl-tRNA synthetase activity from yeast

Arsenite strongly inhibits the activation by thiols of a fraction of the valyl-tRNA synthetase in yeast extracts that precipitates in low concentrations of ammonium sulfate. Once activated, however, the enzyme is insensitive to arsenite. It is suggested that arsenite blocks the function of an enzyme-bound hydrogen-transferring agent that mediates reduction of the enzyme and normally serves as part of an oxido-reduction regulatory mechanism. On gel filtration, much of the arsenite-sensitive activity behaves as a complex of about 500,000 molecular weight, whereas the behavior of the arsenite-insensitive activity is consonant with the molecular weight of 130,000 previously reported for yeast valyl-tRNA synthetase.     

Valylation of the two RNA components of turnip-yellow mosaic virus and specificity of the tRNA aminoacylation reaction.

A comparative study of the aminoacylation of the two RNA components of turnip yellow mosaic virus, of yeast tRNAVal, tRNAfMet and of tRNAPhe by purified yeast valyl-tRNA synthetase is reported. Aminoacylations were performed in the presence of pure yeast tRNA nucleotidyltransferase, since 85% of the viral RNA molecules lacked the 3'-adenosine. We find that aminoacylation of the viral RNAs, like tRNA aminoacylation, reflects an equilibrium between the acylation and deacylation reactions. The kinetic parameters of TYM virus RNA valylation resemble the values found for tRNAVal valylation; in particular, there is a strong affinity between the viral RNA and valyl-tRNA synthetase and the rate constant for TYM virus RNA valylation is only slightly lower than that for tRNAVal. This result contrasts with the reduced rates observed in tRNA mischarging, and suggests that the viral RNA could be easily aminoacylated in vivo. Considering the fact that the 3'-terminal sequence of TYM virus RNA has only a few points of resemblance to a tRNA sequence, we propose that there are some structural motifs found in both tRNAVal and TYM virus RNA which are brought in a similar spatial arrangement recognized by valyl-tRNA synthetase.     

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. V. Mutants with increased levels of valyl-transfer ribonucleic acid synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase were selected for growth at 40 degrees C. Of these, a large number still contain the thermolabile valyl-tRNA synthetase. Three of these revertants contained an increased level of the thermolabile enzyme. The genetic locus, valX, responsible for the enzyme overproduction, is adjacent to the structural gene, valS, of valyl-tRNA synthetase. Determination (by radioimmunoassay) of the turnover rates of valyl-tRNA synthetase showed that the increased level of valyl-tRNA synthetase is due to new enzyme synthesis rather than decreased rates of protein degradation.     

Internal flexibility of valyl-tRNA synthetase from E. coli.

The values of the rotational correlation times of the native valyl-tRNA synthetase and the proteolytic modified enzyme are very close to those of the large fragment of molecular weight 70,000 that has a correlation time of 70 nsec, whereas the small proteolytic fragment has a correlation time of 15 nsec. This indicates that there is rotational freedom within the native valyl-tRNA synthetase corresponding to the biochemically active fragment of molecular weight 70,000. The structural model drawn from these results reveals that the valyl-tRNA synthetase is composed of two unequal, quasi-spherical parts covalently linked by a small peptide bridge. Mild tryptic hydrolysis breaks the covalent bridge between these quasi-spherical domains without changing the overall structure of the valyl-tRNA synthetase significantly.     

Phosphorylation of valyl-tRNA synthetase and elongation factor 1 in response to phorbol esters is associated with stimulation of both activities

Valyl-tRNA synthetase from mammalian cells is isolated in a high Mr complex with elongation factor 1 (EF-1). This complex, which represents all of the valyl-tRNA synthetase activity and a significant portion of the EF-1 activity in rabbit reticulocytes, contains five polypeptides identified as valyl-tRNA synthetase and the four subunits of EF-1. In this study, we have examined the potential for regulation of the complex by phosphorylation of these components. The valyl-tRNA synthetase.EF-1 complex has been purified by gel filtration and tRNA-Sepharose chromatography from 32P-labeled rabbit reticulocytes stimulated by phorbol 12-myristate 13-acetate (PMA) and compared to the complex purified from control cells. One- and two-dimensional polyacrylamide gel electrophoresis and autoradiography show that valyl-tRNA synthetase and the alpha, beta and delta subunits of EF-1 are phosphorylated in vivo. Phosphorylation of each of the four proteins is increased 2-4-fold in response to PMA. Phosphorylation of valyl-tRNA synthetase in response to PMA is reproducibly accompanied by a 1.7-fold increase in aminoacylation activity, whereas phosphorylation of EF-1 is associated with a 2.0-2.2-fold stimulation of activity, as measured by poly(U)-directed polyphenylalanine synthesis. These data suggest that stimulation of translational rates in response to PMA is mediated, at least in part, by phosphorylation of valyl-tRNA synthetase and EF-1.     

A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli

The purification of valyl-tRNA synthetase from Bacillus stearothermophilus is described. The protein was greater than 90% homogeneous on polyacrylamide-gel electrophoresis after more than 850-fold purification. It has a molecular weight of 110000, and no evidence was found for the presence of subunit structure. The properties of the purified enzyme were compared with those of purified valyl-tRNA synthetase from Escherichia coli. The thermal stability, pH-stability and dependence of activity on the temperature and pH of the assay are reported. The two enzymes recognize and charge tRNA(Val) from crude tRNA of the mesophile E. coli and of the thermophile B. stearothermophilus, indiscriminately. The gel-filtration method was extended to measure the binding of tRNA to synthetase directly. Binding constants for tRNA(Val) to valyl-tRNA synthetase from B. stearothermophilus were determined between 5 degrees and 60 degrees C.     

Mammalian valyl-tRNA synthetase forms a complex with the first elongation factor

The high-molecular-mass form of valyl-tRNA synthetase is associated with the first elongation factor activity. It includes two polypeptides of about 50 kDa and two others of 40 and 30 kDa, identified as alpha, beta, gamma and delta subunits of eEF-1H. The complex of valyl-tRNA synthetase with eEF-1H is suggested to be a novel form of the first elongation factor.     

Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12.

The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twofold higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

Complete purification and studies on the structural and kinetic properties of two forms of yeast valyl-tRNA synthetase.

Two forms of baker's yease valyl-tRNA synthetase have been purified to apparent homogeneity by classical methods. It was demonstrated that one of the two forms of the enzyme originates from the other by proteolysis, the respective amounts of each form depending on the physiological state of the yeast. The species mainly isolated from exponential growing yeast cells is a monomer of 130,000 daltons molecular weight. In stationary phase cells or in commercial yeast the major species is a degraded monomer of 120,000 daltons molecular weight ; however when the purification is carried out in the presence of phenylmethyl-sulphonyl fluoride, or diisopropylfluorophosphate large amounts of the not - degreded monomer can be obtained. Of great practical usefulness is the fact that large amounts of the native enzyme can be obtained pure after only two chromatographic steps on DEAE-cellulose and hydroxylapatite. The kinetic constants for valine, ATP and tRNAVal were determined, as well as the optimum aminoacylation conditions. It was found that the specific activity of the nondegraded valyl-tRNA synthetase is higher than that of the proteolysed enzyme for the aminoacylation reaction. On the contrary, both forms have the same ATP-pyroposphate exchange activity. The amino acids composition of the native enzyme was established. The tryptic fingerprints of the two valyl-tRNA synthetases were studied. Essentially similar maps were obtained. The number of the spots in the fingerprints indicates that the enzymes contain a high proportion of repeated sequences.     

Valyl-tRNA synthetase from rabbit liver. II. The enzyme derived from the high-Mr complex displays hydrophobic as well as polyanion-binding properties

The preceding paper (Bec, G., Kerjan, P., Zha, X.D., and Waller, J.P. (1989) J. Biol. Chem. 264, 21131-21137) described the purification to apparent homogeneity from rabbit liver, of a heterotypic complex comprising valyl-tRNA synthetase and Elongation Factor 1H. In the present study, valyl-tRNA synthetase was dissociated and separated from the other components of this complex by hydroxylapatite chromatography in the presence of 0.5 M NaSCN. The properties of the homogeneous mammalian enzyme were compared to those of the corresponding enzyme from yeast. Both behaved as monomeric entities, with apparent molecular masses of 140 and 125 kDa, respectively. Furthermore, both displayed strong affinity toward the polyanionic support heparin-Ultrogel, a property not manifested by the corresponding prokaryotic enzyme. However, unlike the yeast enzyme, that of mammalian origin additionally exhibited hydrophobic properties, as reflected by its affinity toward phenyl-Sepharose. A structural model is proposed according to which mammalian valyl-tRNA synthetase has conserved the polycationic N-terminal domain that distinguishes the corresponding lower eukaryotic enzyme from its prokaryotic counterpart, while acquiring a hydrophobic domain most likely responsible for its association to Elongation Factor 1H.