valyl-tRNA Synthetase Activity Research Articles

Genetic Code Ambiguity: CELL VIABILITY RELATED TO SEVERITY OF EDITING DEFECTS IN MUTANT tRNA SYNTHETASES*

The rules of the genetic code are established in reactions that aminoacylate tRNAs with specific amino acids. Ambiguity in the code is prevented by editing activities whereby incorrect aminoacylations are cleared by specialized hydrolytic reactions of aminoacyl tRNA synthetases. Whereas editing reactions have long been known, their significance for cell viability is still poorly understood. Here we investigated in vitro and in vivo four different mutations in the center for editing that diminish the proofreading activity of valyl-tRNA synthetase (ValRS). The four mutant enzymes were shown to differ quantitatively in the severity of the defect in their ability to clear mischarged tRNA in vitro. Strikingly, in the presence of excess concentrations of alpha-aminobutyrate, one of the amino acids that is misactivated by ValRS, growth of bacterial strains bearing these mutant alleles is arrested. The concentration of misactivated amino acid required for growth arrest correlates inversely in a rank order with the degree of the editing defect seen in vitro. Thus, cell viability depends directly on the suppression of genetic code ambiguity by these specific editing reactions and is finely tuned to any perturbation of these reactions.

Functional Interaction of Mammalian Valyl-tRNA Synthetase with Elongation Factor EF-1α in the Complex with EF-1H

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.

Phosphorylation of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high Mr complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the alpha, beta, and delta subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the beta and delta subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1 alpha is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1 alpha subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1 alpha shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase.EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1 alpha. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.

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.

Valyl-tRNA synthetase from rabbit liver: I. Purification as a heterotypic complex in association with elongation factor

Valyl-tRNA synthetase occurs as a high molecular mass entity of approximately ≌ 700 kDa in the crude extract from rabbit liver. The enzyme was purified as a heterotypic complex comprising four polypeptides of 140, 50, 35, and 27 kDa in the molar proportions of 1:2:1:1, respectively, as determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Co-purification of these components at each step of the purification supports the conclusion that they are physically associated within the same complex. In addition to valyl-tRNA synthetase activity, which was assigned to the 140-kDa component, the purified complex exhibits a potent Elongation Factor 1 activity, determined by its ability to sustain poly(U)-dependent polyphenylalanine synthesis in the presence of Elongation Factor 2. Our results are essentially in agreement with those from a recent report (Motorin, Y., Wolfson, A., Orlovsky, A., and Gladilin, K. (1988) FEBS Lett. 238, 262–264), according to which the polypeptides other than that assigned to valyl-tRNA synthetase correspond to the subunits of Elongation Factor 1H.

Response of a phage modification factor to enhanced production of its target molecule.

Escherichia coli cells with plasmids bearing the valS gene were constructed from the Clark-Carbon collection. Their increased valyl-tRNA synthetase (EC 6.1.1.9) activity was not matched by commensurate production of a modifying peptide during T4 infection. Thus, phage vs gene expression is set to modify the normal amount of valyl-tRNA synthetase activity.

Molecular cloning and characterization of the gene for Escherichia coli valyl-tRNA synthetase

A ColE1 hybrid, pB2, carrying the structural gene for the valyl-tRNA synthetase, valS, and the pyrBI operon was isolated from the Clarke-Carbon collection. Strain GRB238 with a temperature-sensitive mutation in the valS gene showed a three-fold increase in valyl-tRNA synthetase activity after transformation with pB2. The valS gene was subcloned into pBR322 after partial Sau3A digestion of pB2. The new plasmid, pHOV1 had a 9.4-kb insert in pBR322 and cells carrying pHOV1 showed an eight-fold increase in specific valyl-tRNA synthetase activity. The localization and direction of transcription of valS in pHOV1 was determined by transposon-directed mutagenesis and analysis of protein synthesis in minicells. The valS gene was shown to be transcribed opposite to the direction of DNA replication i.e., counterclockwise.

Synthesis of the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine biosynthetic enzymes in a threonine deaminase regulatory mutant of Escherichia coli K-12

A mutation in the structural gene for threonine deaminase, ilvA538, results in lower than normal levels of the isoleucyl, valyl- and leueyl-tRNA synthetases. Moreover, this regulatory mutation decreases the level of expression of the ilv biosynthetic operons and renders their expression non-responsive to limitations of the branched-chain amino acids. In this paper, we present in vitro evidence for the inhibition of isoleucyl- and valyl-tRNA synthetase activity by threonine deaminase and 2-ketobutyrate, the product of the threonine deaminase reaction, through the formation of a high molecular weight complex of the three molecules. Based on these results, we propose a model to explain the regulation of the isoleucyl- and valyl-tRNA synthetases in which transient inhibition of the synthetase enzyme activities by threonine deaminase and 2-ketobutyrate increases the expression of ileS and valS, the structural genes for isoleucyl- and valyt-tRNA synthetase, respectively. Further, the results suggest that the hyperattenuated expression of the ilv biosynthetic operons is due to an increased rate of complex formation of valyl and isoleucyl-tRNA synthetases and the altered form of threonine deaminase of the ilvA538 mutant strain.

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.

Studies on the valyl-h-tRNA synthetase of chick embryo brain irradiated with 60Co gamma-rays, in vivo.

18-day-old chick embryos (Leghorn) were irradiated in vivo with 1,2,3,4,5 or 7 Gy 60Co gamma-rays (dose rate = 0.9 Gy/min). Twenty-four hours after irradiation the activity of valyl-tRNA synthetase isolated from the brains was determined and compared with that of the non-irradiated control. The aminoacylation activity was found to decrease exponentially as a function of the dose (D37 = 6 Gy). Irradiation caused a more pronounced decrease in valyl-tRNA synthetase activity in 18-day-old than in 14-day-old chick embryos and induced in the valine-dependent ATP-PPi exchange reaction less change than in the valyl-tRNA FORMATion. The exposure of 15-day-old chick embryos to a dose of 4 or 5 Gy induced, respectively, a 40 or 20 per cent increase in enzyme activity of VRS (prepared on day 19 of embryonic life) relative to the control. In these experiments a decrease of Km value for tRNA has been found. The change in the number of sulfhydryl groups was also investigated.

Effect of60Co γ Radiation on the Valyl-tRNA Synthetase Isolated from Chick Embryo Brain

The effect of 60Co gamma irradiation on the activity of valyl-tRNA synthetase isolated from chick embryo brain was studied. The enzyme activity exponentially decreased in the dose range 10--200 krad. The first step of the enzyme action, i.e. the amino acid activation, was found to be less sensitive to irradiation than the whole reaction, the formation of valyl-tRNA, 2-Mercapto ethanol and/or glycerol had a significant radioprotective effect. The lesion caused by radiation in the enzyme was also influenced by its concentration during exposure (dilution effect). According to gel-electrophoretic experiments, no chain rupture occurred in the enzyme molecule. Not even a change in Km was observed; however, the maximum velocity of the reaction was found to decrease with increasing radiation dose.

Subcellular distribution of aminoacyl-tRNA synthetases in various eukaryotic cells.

The total amount, size distribution and binding of aminoacyl-tRNA synthetases to ribosomes in a variety of mammalian and avian cells was studied under standard conditions of sample preparation and assay. Aminoacyl-tRNA synthetases appear to exist in three general forms; 'free' enzyme of about 4-9 S, one or more 'enzyme complexes' of about 18-25 S, and in association with ribosomes. The aminoacyl-tRNA synthetase activity for many individual amino acids was surprisingly similar in cell types chosen to be diverse with respect to differentiation state, transformation, and growth rate. Total activity for all amino acids varied about 4-fold, based on a constant volume of cells. Embryonic tissues had a comparatively high proportion of total synthetase activity associated with ribosomes, whereas this value was relatively low for mouse liver. Distinctive distribution patterns with common and variable features were observed for individual enzymes. The only aminoacyl-tRNA synthetases found not to be associated in significant amounts with either 18-25 S enzyme complexes or ribosomes in any of the cell types examined were the enzymes for alanine, histidine, and serine. All cell types evidenced 18-25-S synthetase activity for arginine, aspartic acid, glutamine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, and valine, although in quite variable porportions of the total activity observed for these amino acids. For example, of the valyl-tRNA synthetase activity not associated with ribosomes, 35% and 100% were found to sediment at 18-25 S in Friend leukemia cells and mouse liver respectively. All cells had two easily distinguishable peaks of arginyl tRNA synthetase activity at 4-9S and 18-25S respectively; however, the relative proportion of enzyme activity in the peaks differed between cell types. Phenylalanyl-tRNA synthetase was not observed to occur in an 18-25-S complex in any of the cell types examined but was bound to ribosomes in variable but generally relatively high proportions. Numerous other specific differences are described. No underlying physiological or biochemical principle has been recognized to account for the specific distribution patterns observed. However, they may reflect variations in cellular architecture that may be related to regulation of protein synthesis.

A gene of bacteriophage T4 controlling the modification of host valyl-tRNA synthetase

Two hydroxylamine-induced mutants of bacteriophage T4 defective in modification of host valyl-tRNA synthetase have been isolated by assay of crude extracts for the activity that is characteristic of the wild-type virus. The mutations define a single gene that is situated between the rI and e genes on the T4 genetic map. This new gene is designated vs for valyl-tRNA synthetase. One of the mutations may be of the missense type since it results in the production of a valyl-tRNA synthetase activity that has unusual ureainactivation properties. The other appears to be an amber mutation since the viral enzyme can only be found after infection of cells that are permissive for amber mutations. No differences in growth properties were found between wild type and amber mutant strains on the nonpermissive host. We conclude that the bacteriophage T4 valyl-tRNA synthetase is not essential for viability under prevailing laboratory conditions.

Temporal appearance of bacteriophage T4-modified valyl tRNA synthetase in Escherichia coli

Bacteriophage T4-induced modification of Escherichia coli vlayl-tRNA synthetase (EC 6.1.1.9) requires: synthesis of a phage-gene specified tau factor, addition of the factor to host valyl-tRNA synthetase to produce a urea-stable enzyme, and interaction of the modified enzyme with tRNA to produce a more rapidly sedimenting valyl-tRNA synthetase activity on sucrose density gradients. This report demonstrates that the coincident, chloramphenicol-sensitive appearance of urea-stable and rapidly sedimenting valyl-tRNA synthetase activity are immediate early phage functions. It implies that once the tau factor is synthesized, further interactions are stoichiometric rather than catalytic. The potential for valyl-tRNA synthetase modification accumylates when E. coli is infected with T4 PHAGE IN THE PRESENCE OF CHLORAMPHINICOL AND IS EXPRESSED DURING THE RESUMPTION OF PROTEIN SYNTHESIS WHEREAS FURTHER RNA synthesis is inhibited by rifampicin. The modification phenomenon occurs similarly in several strains of E. coli and represents a novel virus-host interaction.

Effects of ionizing radiations on the activity of valyl-tRNA synthetase isolated from rat liver and Morris hepatoma 7777 (author's transl)

The effects of ionizing radiations on valyl-tRNA synthetase (L-valine tRNA ligase (AMP), E.C.6.1.1.9.), 100-fold purified from rat liver and Morris hepatoma 7777, are reported. The results showed that: 1) the enzymatic activity is strongly inhibited by radiations; 2) the enzymatic inactivation is much more marked in dilute than in concentrated solutions; 3) the inactivation of enzyme is linked to oxidation of sulphydryl groups indispensable to the catalytic activity of the enzyme; 4) no substantial difference in relation to inactivation by radiation was observed, between rat liver and Morris hepatoma 7777 enzymes.

Unusual valyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

Escherichia coli strain NP2907 was isolated as a spontaneous mutant of strain NP29, which possesses a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase. The valyl-tRNA synthetase of the new mutant, unlike that of its immediate parent, retains enzymatic activity in vitro but differs from the wild-type enzyme in stability and apparent K(m) for adenosine triphosphate. The new mutant locus, valS-102, cotransduces with pyrB at the same frequency as does the parental locus, valS-1. Cultures of strain NP29 cease growth immediately in any medium when shifted from 30 to 40 C. The new mutant grows normally at 30 C, and upon a shift to 40 C growth quickly accelerates exactly as for normal cells. Exponential growth, however, cannot be sustained at 40 C. At a point characteristic for each medium, growth becomes linear with time. This transition occurs almost immediately in rich media and after 1.5 generations in glucose minimal medium. Net synthesis of valyl-tRNA synthetase ceases in the new mutant as soon as the temperature is raised to 40 C, irrespective of the growth medium. We conclude that it is the amount of valyl-tRNA synthetase activity that limits the rate of growth in the linear phase at 40 C. This property of the mutant makes it possible to evaluate the in vivo efficiency of this enzyme at different growth rates and thereby to determine the concentration that is necessary for a given rate of protein synthesis. The results of our measurements indicate that cells of E. coli growing in minimal medium normally possess a functional excess of valyl-tRNA synthetase with respect to protein synthesis and to repression of threonine deaminase.

Regulation of the biosynthesis of valyl-tRNA synthetase in yeast

When a wild type yeast is grown first in the presence of valine, then transferred to a medium without valine, there is a 2-fold increase in the specific activity of valyl-tRNA synthetase after this shift-down. The addition of cycloheximide at the time of the transfer prevents this increase in specific activity. If valine is added again 2.5 h after the shift in one half of the culture, there is a decrease in specific activity of the enzyme, while in the other half, where no valine has been added, the increase in specific activity is maintained. Addition of valine to exponentially growing cells is followed by a 25–30% decrease in valyl-tRNA synthetase activity. The addition, at the time of the shift-down, of α-aminobutyric acid, an analog of valine which is activated by valyl-tRNA synthetase but not charged on tRNA Val, does not interfere with the increase in specific activity of the enzyme caused by the removal of valine from the medium. But the addition of α-amino-β-chlorobutyric acid, an analog which is activated and charged on tRNA Val, prevents the increase in specific activity of valyl-tRNA synthetase after the shift-sdown. It is possible to grow yeast in the presence of valine and 2H 2O and to perform the shift to a medium containing H 2O but no valine. Under these conditions, the enzymes synthesized before the shift are heavier than those made after the shift and they can be separated upon centrifugation in CsCl. These experiments have shown that removal of valine from the medium causes a specific increase in the amounts of newly synthesized valyl-tRNA synthetase. Of the 2 analogs, only α-amino-β-chlorobutyric acid (which can be charged on tRNA Val) prevents this increase, which suggests that valyl-tRNA is a key-component of the repression mechanism.

Phage-induced modification of valyl-tRNA synthetase.

Infection of Escherichia coli: with T4 bacteriophage causes the appearance of a new valyl-tRNA synthetase activity associated with a molecule that, compared to the host enzyme, exhibits a greater resistance to denaturation by heat or urea, a larger molecular volume, a higher rate of sedimentation in sucrose gradients, a greater net positive charge, and a greater ability to charge yeast tRNA. No evidence has been found for similar changes in synthetase activity for the other amino acids. Appearance of the new activity requires continued protein synthesis and results from a modification of the preexisting host enzyme rather than de novo synthesis of a totally new enzyme. By 20 minutes after infection at 30°C, all of the host enzyme has been converted into the new form. Phage mutants have been isolated that fail to effect a normal conversion. The properties of these mutants suggest that conversion involves the addition, to the host enzyme, of a protein specified by the phage genome. Drastic reduction of phage-induced activity in one of these mutants does not interfere detectably with phage development in a normal host, suggesting that the presence of the new activity is not essential for normal phage production.

Modification of valyl tRNA synthetase by bacteriophage in Escherichia coli

Infection of Escherichia coli with T4 bacteriophage is known to bring about the appearance of a new valyl tRNA synthetase activity distinguished by its behavior during hydroxylapatite fractionation, by its sedimentation in sucrose gradients and, in certain temperature-sensitive bacterial mutants, by its increased stability. Studies were made to determine the origin of this new activity. Its formation was accompanied at all times by a proportional decrease in the original valyl tRNA synthetase activity; 20 minutes after infection at 30 °C no original enzyme remained and no further increase in the phage-induced activity occurred. The addition of chloramphenicol at any time completely blocked both the gain of new and the loss of old activity. An increase of 2.6% in the buoyant density of valyl tRNA synthetase could be achieved by growth of the cells in 80% D 2O medium, permitting resolution of heavy and light enzyme by equilibrium centrifugation with cesium chloride. By appropriate transfer of cells between deuterium-labeled and unlabeled media it was possible to demonstrate that the new enzyme activity brought about by phage infection in both mutant and wild cells consists of polypeptide chains synthesized prior to infection. From these results it was concluded that T4 infection causes a chloramphenicol-sensitive conversion of host valyl tRNA synthetase into a new, possibly dimeric form.

Effect of phage on amino acid activation

Infection of Escherichia coli NP2 with T-even phage strains is known to cause the appearance of a new valyl tRNA synthetase activity. In the present work mutant hosts possessing altered activating enzymes for phenylalanine, glycine, or histidine were employed to detect possible phage-induced modifications in the translating systems for these amino acids. The results establish that T4 has an absolute requirement for the phenylalanyl tRNA synthetase of its host, probably in an unmodified form, and that this enzyme is responsible for the incorporation of most, if not all, phenylalanine residues into phage protein. Other data suggest, less rigorously, that a similar conclusion holds for glycine and histidine.