Incorrect Aminoacylations Involving tRNAs or Valyl-tRNA Synthetase from BacilIus stearothermophilus

Abstract

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. I n 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 tRNAVa1 and noncognate 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 tRNA,Met and of yeast tRNA,Met and tRNAPhe. These reactions are characterized by K , 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. I n the presence of dimethylsulfoxide, the rate and the extent of those misacylation reactions are enhanced. I n the case of E . coli tRNAPhe, the misacylation occurs only in the presence of the organic solvent. I n no case however, new aminoacylation errors are induced a t 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 a t 30 "C has been observed a t 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 valyltRNA synthetases from E . coli and from yeast. This observation suggests the existence of a family of tRNAs containing besides of tRNAVB', other tRNAs such as tRNATle, tRNAyet 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.The possibility of the tRNAs to be enzymatically mischarged with a wrong amino acid seems to be a rather general feature as numerous incorrect aminoacylation systems have now been described [i -31 (and references therein). These mischarging reactions have especially been detected in vitro, under various experimental conditions, either in heterologous systems where the enzyme and the tRNA came from different sources, but also in homologous systems, where one pure tRNA species interacts Enzyme. Valyl-tRNA synthetase (EC 6.1.1.9). Eur. J. Biochem. 45 (1974)with one non-cognate pure ...

Interpretation of tRNA-Mischarging Kinetics

“…Phenylalanyl-tRNA synthetase (EC 6.1. I .20); valyluse of purified tRNA's, avoiding the competitive effect due to the cognate tRNA [6], makes the incorrect aminoacylation easier.…”

“…The exact amount of tRNA and of enzyme will be indicated in the text or in the legends to figures. 100-300 pl reaction mixtures were incubated at 37 "C. Aliquots of 20 p1 were taken and the radioactive valyl-tRNA's formed were measured in the usual manner [6].…”

“…Mischarging reactions differ from the correct aminoacylation reactions by a slower rate and by the fact that in most cases they are incomplete [1 -211 (varying from less than 1 % to nearly 100% aminoacylation of the acylable tRNA). They are easier to observe in nonstandard aminoacylation conditions, but it has been shown that it is possible to perform the mischarging of tRNA under classical aminoacylation conditions (see [6] and references therein). In that case, high enzyme concentrations are required.…”

Revisiting the operational RNA code for amino acids: Ensemble attributes and their implications

“…Class-1 enzymes differ from class-2 enzymes in secondary structure, sequence motifs at the active site, the side of the tRNA acceptor stem onto which they dock, and the domains responsible for editing out misacylated amino acids with steric and/or biochemical similarity. Members of class-1 approach the tRNA acceptor stem from the minor groove side, dock on the 59 branch, and acylate at the 29 hydroxyl group of the A76 terminus; whereas those of class-2 approach from the major groove side, dock on the 39 branch, and acylate at the 39-OH of the terminal adenine (Giegé et al 1974;Cavarelli and Moras 1993;Arnez et al 1995;Fersht 1998;Nureki et al 1998;Fukai et al 2000;Beebe et al 2003Beebe et al , 2004. Only PheRS, although a class-2, attaches the amino acid to the 29-OH (Goldgur et al 1997).…”

Stepwise tRNA recognition mechanism and its kinetic consequences

“…The first step may be considered to result from interactions of substrate with enzyme of relatively low specificity. It may be interactions common to some set of tRNA's capable of being aminoacylated with one ARSase [2,3] or all elongator aa-tRNA's interacting with a ribosome-mRNA complex with one codon exposed to translation [4,5]. The existence of these common interactions seems to be reasonable because of the suggested common tertiary structure of tRNA's [6].…”