tRNA Aminoacylation Reaction Research Articles

Valyl-tRNA synthetase from Bacillus stearothermophilus. Purification and binding with the substrates L-valine and ATP.

Valyl-tRNA synthetase (L-valine: tRNAval ligase (AMP forming); EC 6.1.1.9) (VRS) was purified from Bacillus stearothermophilus NCA 1503 by streptomycin treatment, ammonium sulfate fractionation, and batch adsorption on DEAE-Sephadex A-50, then column chromatographies on hydroxylapatite, DEAE-Toyopearl 650S, BUTYL-Toyopearl 650S, and Sephacryl S-300 superfine. The enzyme preparation, purified approximately 1100-fold, was homogeneous on polyacrylamide gel disc electrophoresis and consisted of a single chain polypeptide. The molecular weight was about 100, 000 and 110, 000 by SDS-polyacrylamide gel electrophoresis and Sephacryl S-300 gel filtration, respectively. Enzyme activity in tRNA aminoacylation reaction increased as the temperature increased up to 52°C at pH 7.6 and pH 8.5. pH-optima of the aminoacylation activity were 8.5 at 30°C and 8.0 at 52°C under our conditions. Ligand-induced decrease of the enzyme protein fluorescence was observed and used as a probe for studying the binding of substrates (L-valine and ATP) to VRS. Substrate bindings were also studied by equilibrium dialysis; two moles of L-valine or ATP were bound to a mole of enzyme when each substrate was examined separately. These two binding equilibria of L-valine or ATP seemed equivalent when each substrate is bound alone.

Kinetic mechanism of arginyl-tRNA synthetase from human placenta

1. 1. Like arginyl-tRNA synthetases from other organisms, human placental arginyl-tRNA synthetase catalyzes the arginine-dependent ATP-PP i exchange reaction only in the presence of tRNA. 2. 2. We have investigated the order of substrate addition and product release of this human enzyme in the tRNA aminoacylation reaction by using initial velocity experiments and dead-end and product inhibition studies. 3. 3. The kinetic patterns obtained are consistent with a random Ter Ter sequential mechanism, instead of the common Bi Uni Uni Bi ping-pong mechanism for all other human aminoacyl-tRNA synthetases so far investigated in this respect.

Comparative study of the effect of ochratoxin a analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells

Ochratoxin A (OTA), a naturally occurring mycotoxin of Aspergillus and Penicillium species, consists of a 5 ' chlorinated dihydromethyl isocoumarin linked to l,ß-phenylalanine by an α-amide bond. 8 analogues of OTA were prepared in which the phenylalanine was always substituted by another amino acid. The effects of these analogues on yeast tRNA amino acylation reaction and on growth and protein synthesis of hepatoma culture cells were compared with those of OTA. In addition, Ochratoxin B (OTB) and ochratoxin α (OTα) were examined. All the analogues of OTA had inhibitory effects in the 3 test systems, although to a lesser degree than OTA. The degree of inhibition depended on the kind of substituted amino acid, the tyrosine, valine, serine and alanine analogues being most effective, in contrast to the proline analogue. OTB and OTα were ineffective.

Dimeric glutamyl-tRNA synthetases from wheat. Kinetic properties and functional structures.

The Michaelis constants in the tRNA aminoacylation reaction have been studied for the three dimeric glutamyl-tRNA synthetases C, P and E. The values were found to be: for tRNA, 0.20 microM, and 0.44 microM; for glutamic acid, 10 microM, 83 microM and 83 microM; for MgATP, 0.46 mM, 0.38 mM and 0.26 mM. MgATP concentrations higher than 2 mM induce pronounced inhibition. The presence of the cognate tRNA is required for [32P]PPi-ATP isotopic exchange. In the absence of tRNA no hyperbolic saturation of the enzymes by glutamic acid occurs in our experimental conditions. Analysis of the enzymic activity as a function of enzyme concentration leads to the conclusion that the active forms are dimers which are in equilibrium with inactive monomers. The values of the dissociation constants Kd were found to be 43 nM, 53 nM and 87 nM for glutamyl-tRNA synthetases C, P and E respectively.

The mechanism of inhibition of valyl-tRNA synthetase by S-adenosylhomocysteine

S-Adenosylhomocysteine inhibits ATP-PP i exchange and tRNA aminoacylation reactions catalysed by pure lupin valyl-tRNA synthetase. The reactions catalysed by phenylalanyl-tRNA, tyrosyl-tRNA, seryl-tRNA, arginyl-tRNA and tryptophanyl-tRNA synthetases are not inhibited by S-adenosylhomocysteine. The inhibition is proportional to first power of S-adenosylhomocysteine concentration. S-Adenosylhomocysteine is a competitive inhibitor of the lupin valyl-tRNA synthetase with respect to ATP and valine in the ATP-PP i exchange reaction, noncompetitive with respect to ATP and valine and uncompetitive with respect to tRNA in the tRNA aminoacylation reaction. Inhibition constant for S-adenosylhomocysteine determined from the reciprocal of K m for tRNA vs. the inhibitor concentration (at saturating ATP and not saturating valine concentration) is 0.35 mM. Inhibition constants for S-adenosylhomocysteine determined from the ATP-PP i exchange are 0.21 mM at saturating valine concentration with variable ATP and 0.33 mM at saturating ATP concentration with variable valine. The data are consistent with the binding of S-adenosylhomocysteine to the valyl adenylate site on the enzyme.

Purification and some properties of alanyl- and leucyl-tRNA synthetases from baker's yeast

Alanyl- and leucyl-tRNA synthetases from baker's yeast were purified to homogeneity in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. Both consist of single polypeptide chains of 118 000 and 125 000 daltons, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions. The monomeric structure of leucyl-tRNA synthetase differs from the dimeric one obtained previously in the absence of protease inhibitors. This illustrates the sensitivity of the synthetases to proteolytic actions and indicates that native structures can only be obtained under optimal protecting conditions. Alanyl- and leucyl-tRNA synthetases differ with respect to pH optimum (6.5 and 8.5, respectively), Michaelis constant for amino acid (1 mM and 0.03 mM, respectively) and in the rate-limiting step for the tRNA aminoacylation reaction. Whereas the catalytic step itself was rate-limiting for alanyl-tRNA synthetase, a step occurring after this was rate-limiting for leucyl-tRNA synthetase.

Kinetics of formation of tryptophanyl-adenylate by tryptophanyl-tRNA synthetase from beef pancreas

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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.

On dealing with anomalies in the transfer RNA aminoacylation reaction in partially purified systems

Apparent differences in tRNA and aminoacyl-tRNA synthetase complements in tissues undergoing differentiation have frequently been used to support theories of translational control. The validity of at least some of these studies must now be questioned because of anomalies in the tRNA aminoacylation reaction which can lead to incomplete aminoacylation of tRNA. Incomplete acylation of a tRNA mixture could result in different relative amounts of acylated isoaccepting species if acylation rates were not identical for all species. Using common methods of analysis, this situation could lead to misestimation of relative levels of isoacceptors or an inability to detect the presence of minor species. Bonnet and Ebel [Bonnet, J., and Ebel, J. (1972) Eur. J. Biochem. 31, 335] used a highly purified valyl-tRNA and valyl-tRNA synthetase from yeast to demonstrate the presence of four reactions that occur simultaneously in that system. Herein, I apply the findings of Bonnet and Ebel to a mammalian system in a manner which is representative of attempts to study relative tissue proportions of tRNA isoacceptors. Total complements of tRNAs and the aminoacyl-tRNA synthetases have been partially purified from rabbit liver according to the methods of Yang and Novelli [Yang, W. K., and Novelli, G. D. (1971) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, p. 44, Academic Press, New York], probably the most commonly used procedures in the literature. Reaction conditions for tRNA acylation are shown to be modifiable so as to influence the extent of tRNA acylation. Procedures for optimizing the extent of tRNA acylation in such systems are demonstrated, and the unfavorable influence of Tris buffer, a factor not discussed by Bonnet and Ebel, is shown. Finally, examples of altered ratios of isoaccepting species in samples incompletely acylated due to suboptimal reaction conditions are provided.

The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.

Interpretation of tRNA-mischarging kinetics.

Incorrect tRNA aminoacylation reactions are characterized by very slow reaction rates and by the fact that in most cases they are incomplete. In a previous study some of us explained the incompleteness of the correct aminoacylation reactions of tRNA, which can be encountered under certain experimental conditions (for instance low enzyme concentration or high ionic strength) by an equilibrium between the aminoacylation and the deacylation reactions [J. Bonnet and J.P. Ebel (1972) Eur. J.Biochem. 31, 335-344]. In the present report we bring evidence that the incorrect valylation of yeast tRNAfMet by yeast valyl-tRNA synthetase studied under standard experimental conditions, can also be described by a kinetic rate law including the rate equations of the aminoacylation and of the various deacylation reactions. In particular we show that the incomplete mischarging plateaus reflect the existence of an equilibrium between the valylation reaction on the one hand and the spontaneous and enzymic deacylation of valyl-tRNAfMet and the reverse of the valylation reaction on the other hand. However, when the valyl-tRNA synthetase concentration is not very high the reverse reaction of the amino-acylation does not play a predominant part in the establishment of the plateau. These interpretations have been extended to other mischarging systems: valylation of yeast tRNAPhE by yeast valyl-tRNA synthetase and mischarging of tRNAfMet and tRNA2Val from yeast by yeast phenylalanyl-tRNA synthetase. Unusual mischarging kinetics have been discussed. Furthermore, and as in correct systems, we found that during the mischarging of tRNAfMet one ATP is hydrolyzed per tRNA charged with valine. We conclude that the correct and the incorrect amino-acylation of tRNA behave kinetically in a similar way.

Histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium. Interaction with substrates and ATP analogues.

Structural requirements for substrate binding to histidyl-tRNA synthetase from Salmonella typhimurium have been investigated using ATP analogues. Ki values and the relative binding affinity of the enzyme for these analogues have been determined in the tRNA aminoacylation reaction. The enzyme is highly specific for ATP: no binding was found for GTP, CTP, TTP and UTP. dATP is a very poor substrate for acylation of tRNA, with a Km 40-fold higher than that of ATP. Binding of adenosine 5'-triphosphate requires interactions of the amino group of adenosine and the sugar moiety; the 2' and the 5' positions of the ribose appear to be essential for recognition; the phosphate groups enhance the binding. AMP is a noncompetitive inhibitor with ATP. The interaction of histidyl-tRNA synthetase, a dimeric enzyme, with histidine and ATP was examined by fluorescence measurements at equilibrium and by equilibrium dialysis. Binding with L-histidine is significantly tighter at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 7.5 by equilibrium dialysis and is 1 mol ATP/mol enzyme and, variably, close to 2 or 1 mol histidine/mol enzyme.

Histidyl-tRNA synthetase from Salmonella typhimurium: specificity in the binding of histidine analogues.

The topography of the active site of histidyl-tRNA synthetase has been investigated by determining Ki values for a variety of structural analogues of histidine, using the ATP-PPi exchange and tRNA aminoacylation reactions. Using these kinetic constants it has been possible to have a measure of the relative binding affinity of the enzyme for the histidine analogues. The following conclusions have been drawn: (a) the enzyme is stereospecific in the formation of aminoacyl-tRNA complexes, since the D-isomer of histidine does not influence the two reactions; (b) the carboxyl group is not required for binding; (c) bulky derivatives of the carboxyl group prevent the molecules from binding to the enzyme; (d) the amino group permits a good binding affinity; (e) the length of the ring side chain plays a very important role as point of attachment to the enzyme; (f) the kinds of heteroatoms on the ring determine the inhibitory properties of the analogues.

Incorrect aminoacylations involving tRNAs or valyl-tRNA synthetase from Bacillus stearothermophilus.

Numerous misacylations occur on heterologous systems containing unfractionated tRNAs from yeast or from Bacillus stearothermophilus and pure valyl-tRNA synthetase from B. stearothermophilus or from yeast, when special aminoacylation conditions are used. In the homologous system and in heterologous systems where the unfractionated tRNAs and the enzyme originate from prokaryotic organisms (B. stearothermophilus and Escherichia coli), the errors are seldom. This phenomenon is explained by competition effects between the cognate tRNAVal and non-cognate tRNAs for the valyl-tRNA synthetase. But using pure tRNA species, errors can be observed in such systems, even under classical assay conditions; in particular it was shown that the valyl-tRNA synthetase from B. stearothermophilus catalyses the misacylation of E. coli tRNAIle and tRNAtMet and of yeast tRNAtMet and tRNAPhe. These reactions are characterized by Km values slightly increased as compared to the value obtained in the cognate system and by V values decreased by a factor of about 40 to 3000 compared to the cognate tRNA species. In the presence of dimethylsulfoxide, the rate and the extent of those misacylation reactions are enhanced. In the case of E. coli tRNAPhe, the misacylation occurs only in the presence of the organic solvent. In no case however, new aminoacylation errors are induced at high temperature (50–75 °C) in the presence of the thermostable valyl-tRNA synthetase from B. stearothermophilus; only an increase of the rates of the aminoacylation reactions which already occur at 30 °C has been observed at higher temperature. Thus organic solvent and heat must have distinct effects on the essential parameters determining the specificity of the tRNA aminoacylation reactions. Also it has been observed that the most easily misacylated tRNA species by valyl-tRNA synthetase from B. stearothermophilus are the same as those which are misacylated by the valyl-tRNA synthetases from E. coli and from yeast. This observation suggests the existence of a family of tRNAs containing besides of tRNAVal, other tRNAs such as tRNAIle, tRNAtMet and tRNAPhe, and which are likely to be related from a phylogenic point of view. Moreover these tRNA species have also been found to be easily misacylated by other aminoacyl-tRNA synthetases namely the enzymes specific for isoleucine and phenylalanine, thus suggesting more generally the existence of interacting tRNA-aminoacyl-tRNA synthetase families.

Anomalies in the tRNA-aminoacylation reaction which could lead to misinterpretation of evidence for tRNA changes during development and aging

Anomalies in the tRNA-aminoacylation reaction are described and discussed. These include enzyme-dependent tRNA-acylation plateau values at low enzyme to tRNA ratios in the presence of Tris buffer, and the attainment of tRNA-acylation maxima at high enzyme to tRNA ratios. The enzyme-dependent plateaus are believed to represent the steady state resultant of simultaneously occurring enzymatic and non-enzymatic deacylation processes with the acylation reaction. The failure to maintain a level plateau at high enzyme to tRNA ratios is ascribed to another competing reaction — the hydrolysis of ATP to AMP and pyrophosphate (PPi) which is not dependent upon aminoacyl-tRNA formation. Tris buffer appears to accelerate non-enzymatic deacylation of tRNA thus leading to plateau values representing (at low enzyme to tRNA ratios) incomplete charging of tRNA. The relative balance of these competing reactions may be favorably manipulated by avoiding Tris-type buffers and by altering the enzyme to tRNA ratio to obtain an optimal acylation plateau. If increasing the enzyme concentration (keeping the tRNA concentration constant) leads to increasing tRNA acylation plateaus, this is evidence that one is operating below the optimal ratio; if increasing enzyme concentration leads to decreasing plateaus or to charging maxima (no plateau) it is evidence that one is operating above the optimal ratio. Since these precautions have not ordinarily been considered, some reports of changes in tRNA and/or aminoacyl-tRNA synthetase complements during development and aging may be inaccurate.

Structure-activity relationship in tryptophanyl-transfer ribonucleic acid synthetase from beef pancreas. Role of -SH groups in the activity of the enzyme.

The titration of the – SH groups of beef pancreas tryptophanyl‐tRNA synthetase by N‐ethylmaleimide and the consequent effect on the exchange and transfer reactions of the enzyme were examined.The 16 sulfhydryl groups of the native dimeric enzyme do not react homogeneously at pH 7, with 1 mM reagent. They can be divided into three classes with decreasing reactivities. The reaction of the first class (6 – SH groups) has no effect on activity. The reaction of the second class (4 – SH groups) brings about an increase of Km(app) for tryptophan both in the exchange and in the transfer reactions of the enzyme. The activity of the enzyme is lost during reaction of the third class (6 ‐ SH groups). This is evidenced by the decrease of the V of the [32P]PPi‐ATP isotope exchange and tRNA aminoacylation reactions and by the decrease in the amount of the tryptophanyladenylate · enzyme complex isolatable on Biogel P30. The destruction of the active site by alkylation of the sulfhydryl groups of class III can be the consequence of a structural change of the enzyme induced by the chemical modification. During the alkylation of the first two classes of ‐ SH groups the Km(app) for ATP, PPi and tRNA are not altered significantly.The presence of tryptophan or tryptophanyladenylate reduces the rate of titration of the ‐SH groups of class II and class III and in a parallel fashion the rates of increase of Km(app) for tryptophan and of decrease of V. The titration of the –SH groups of class I is not affected by the presence of substrates. The alkylation in the presence of tryptophanyladenylate demonstrates that class II is not homogeneous and that the two molecules of adenylate bound to the dimeric enzyme specifically protect two –SH groups out of the four of this class.

Factors determining the specificity of the tRNA aminoacylation reaction: Non-absolute specificity of tRNA-aminoacyl-tRNA synthetase recognition and particular importance of the maximal velocity

It is generally believed that the specificity of tRNA aminoacylation results solely from a specific recognition between the aminoacyl-tRNA synthetase and the cognate tRNA. In fact, this specificity is not absolute: this is supported by the following observations (1) the existence of tRNA mischarging in homologous systems under usual aminoacylation conditions, (2) the existence of inhibitions produced by « non-cognatetRNA species in correct aminoacylation reactions, (3) the lack of specificity of AMP- and PPi- independent aminoacyl-tRNA synthetase catalysed deacylation of aminoacyl-tRNA species, (4) the isolation of complexes between aminoacyl-tRNA synthetases and non-cognate tRNA species. The affinities between aminoacyl-tRNA synthetases and non-cognate tRNA species, estimated by the Km measurements in mischarging reactions, have been found only diminished by 1 or 2 orders of magnitude as compared to the values found in specific systems, whereas the Vmax values for mischarging have been found diminished by 3 or 4 orders of magnitude. This suggests that tRNA aminoacylation depends more upon the maximal velocity of the reaction than upon the recognition between aminoacyl-tRNA synthetase and tRNA. Furthermore, we found that the recognition of a tRNA by an aminoacyl-tRNA synthetase does not seem to require the 3′ terminal part of the amino acid acceptor stem. As the importance of this part of the tRNA molecule during the aminoacylation process has been well established, it is possible that it is involved in determining the Vmax of the aminoacylation reaction, probably by positioning the 3′ terminal adenosine in the catalytic site of the enzyme. In conclusion, it appears that the specificity of the tRNA aminoacylation reaction proceeds through two discrimination mechanisms: the first one, measured by the Km, acts at the recognition level; the second one, which is more effective, is measured by the Vmax values. Competition phenomena have been observed between cognate and non-cognate tRNA species. They enhance the specificity of the tRNA aminoacylation, but their contribution to the specificity is low compared to that brought by Km and Vmax. Finally we found that a more rapid enzymatic deacylation of mischarged tRNA species (as compared to correctly charged ones) cannot be considered as a general mechanism for correction of misaminoacylation. Il est généralement admis que la spécificité de l'aminoacylation des tRNA résulte seulement de la reconnaissance entre l'aminoacyl-tRNA synthétase et le tRNA correspondant. En fait, les observations suivantes montrent que cette reconnaissance n'est pas absolue: (1) l'existence de charges incorrectes dans des systèmes homologues et dans les conditions habituelles d'aminoacylation, (2) la mise en évidence d'inhibitions produites par des tRNA non spécifiques dans des réactions d'aminoacylation correcte, (3) le manque de spécificité de la désacylation d'aminoacyl-tRNA catalysée par une aminoacyl-tRNA synthétase, en absence d'AMP et de PP1, (4) l'isolement de complexes entre une aminoacyl-tRNA synthétase et des tRNA non spécifiques. Pour un enzyme donné, l'affinité d'un tRNA non spécifique, mesurée par le Km de la réaction d'aminoacylation, diminue de 1 à 2 ordres de grandeur par rapport à l'affinité du tRNA spécifique, alors que la vitesse maximum d'une aminoacylation incorrecte est de 3 à 4 ordres de grandeur inférieure à celle de la réaction correcte. De plus, nous avons trouvé que la reconnaissance d'un tRNA par une aminoacyl-tRNA synthétase ne semble pas requérir la partie 3′ terminale du bras accepteur. Comme l'importance dans l'aminoacylation de cette région du tRNA a été bien établie, il est vraisemblable qu'elle détermine pour une grande part la Vmax de la réaction d'aminoacylation, sans doute par l'intermédiaire du positionnement de l'adénosine 3′ terminale dans le site catalytique de l'enzyme. En conclusion, il apparaît que la spécificité de l'aminoacylation procède de deux mécanismes de discrimination: le premier, mesuré par le Km, intervient au niveau de la reconnaissance, le second, plus efficace. au niveau de la vitesse de la réaction d'aminoacylation. Des phénomènes de compétition dans la réaction d'aminoacylation ont été observés entre des tRNA spécifiques et des tRNA non spécifiques. Ils augmentent la spécificité de l'aminoacylation, mais leur contribution est faible comparée à celle apportée par la Vmax et le Km. Enfin, nous avons constaté que la désacylation enzymatique plus rapide des faux aminoacyl-tRNA par rapport aux tRNA correctement chargés ne peut être considérée comme un mécanisme général de correction des fausses aminoacylations.