Hydrolytic action of aminoacyl-tRNA synthetases from Baker's yeast: "chemical proofreading" preventing acylation of tRNA^Ile with misactivated valine

Abstract

Phe-tRNAPhe-C-C-A, Val-tRNAVal-C-C-A, and Ile-tRNAIle-C-C-A, which accept their amino acid on the 2'-OH of the 3'-terminal adenosine, are hydrolyzed readily by their aminoacyl-tRNA synthetase. If the 3'terminal adenosine in these tRNAs is replaced by either 3'-deoxyadenosine or formycin, little if any hydrolysis can be observed. Correspondingly Ser-tRNASer-C-C-A which accepts serine on the 3'-OH of the 3'-terminal adenosine is hydrolyzed by seryl-tRNA synthetase, whereas Ser-tRNASer-C-C-2'dA and Ser-tRNASer-C-C-F are not. Tyr-tRNATyr-C-C-A and all modified Tyr-tRNATyr-C-C-N, which can accept tyrosine on either the 2'OH or the 3'-OH of the 3'terminal adenosine, are not hydrolyzed by tyrosyl-tRNA synthetase. The data can be rationalized assuming that hydrolysis takes place only if the amino acid is bound to the nonaccepting OH and hence is not positioned at the amino acid binding site upon formation of the complex between aminoacyl-tRNA and aminoacyl-tRNA synthetase. In the formycin-carrying tRNA, the amino acid bound to the nonaccepting OH seems to be inaccessible to the enzymatic groups responsible for hydrolysis. Val-tRNAIle-C-C-3'dA and Ile-tRNAIle-C-C-3'DA cannot be hydrolyzed by isoleucyl-tRNA synthetase. Val-tRNAIle-C-C-A is hydrolyzed by the enzyme five times more rapidly than Ile-tRNAIle-C-C-A. Whereas Ile-tRNAIle-C-C-F is absolutely stable, Val-tRNAIle-C-C-F si hydrolyzed immediately. As shown by the earlier finding that valine misactivated by isoleucyl-tRNA synthetase cannot be permanently transferred to tRNAIle-C-C-A but to tRNAIle-C-C-3'dA, the 3'-OH is essential for preventing transfer of misactivated valine. It thus appears that valine is hydrolyzed off Val-tRNAIle-C-C-N if it is bound to the accepting 2'-OH in the binding site for isoleucine. A hypothesis is offered attempting to explain the experimental observations in mechanistic terms. We consider the hydrolytic action of the aminoacyl-tRNA synthetases as a general mechanism of "chemical proofreading" in the protein biosynthesis.

Threonyl‐tRNA synthetase from yeast Aminoacylation of tRNA on its non‐accepting 3′‐terminal hydroxyl group and its behaviour in enzyme‐catalyzed deacylation

“…Thr-tRNAThr-c-C-A is rapidly hydrolysed in the presence of the enyzme with a turnover number of 0.19 rain -1 ( fig.2a). From this value it can be seen that this enzyme is somewhat less efficient in the deacylation reaction than some other synthetases (e.g., phenylalanine-, valyl-and isoleucyl-tRNA synthetases with a turnover number in this reaction of 0.67, 0.77 and 1.8 rain-', respectively [5] ) but it should be noted that the curve in fig.2a was obtained, at pH 7.6, and not at the pH optimum of threonyl-tRNA synthetase (pH 8.5) in order to minimise the non-enzymatic breakdown of the Thr-tRNAThLc-C-A. .OH H Fig.4.…”

“…The AMP/PPfindependent hydrolysis of preformed Thr-tRNAThr-c-C-N species by free threonyltRNA synthetase was studied by the method in [5].…”

“…For analytical purposes, in 100 gi incubation volume, the extent of aminoacylation was followed by the falter disc method as in [12]. For preparative aminoacylation, in 1 ml total vol., the aminoacyiated tRNA was isolated as in [5].…”

“…The hydrolysis of aminoacyl.tRNA by free aminoacyl-tRNA synthetase in the absence of other substrates is well established [1][2][3][4][5], eq. (1):…”

“…In the case of the phenomenologically similar breakdown of aminoacyl-tRNA under aminoacylating conditions, evidence has been obtained for the hydro- lysis of the ester bond after transacylation of the aminoacyl residue from the accepting [6] terminal ribose hydroxyl group to the non-accepting vicinal hydroxyl group according to scheme 1 [5]. Whether this pathway also applies to the hydrolysis of aminoacyl-tRNA by free enzyme remains to be determined, but it is in any event of mechanistic interest to study the role of the 3'-terminal ribose hydroxyl groups of tRNA on the free enzyme catalyzed reaction.…”