Résumé
The recognition of the acceptor stem of tRNAGlnis an important element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18). On the basis of known mutations and the crystal structure of the tRNAGln·GlnRS complex, we mutagenized at saturation two motifs in the acceptor end binding domain of GlnRS. Mutants with lowered tRNA specificity were then selected in vivo by suppression of a glutamine-specific amber mutation (lacZ1000) with an amber suppressor tRNA derived from tRNASer
1. The mischarging GlnRS mutants obtained in this way retain the ability to charge tRNAGln, but in addition, they misacylate a number of noncognate amber suppressor tRNAs. The critical residues responsible for specificity are Arg-130 and Glu-131, located in a part of GlnRS that binds the acceptor stem of tRNAGln. On the basis of the spectrum of tRNAs capable of being misacylated by such mutants we propose that, in addition to taking part in productive interactions, the acceptor end binding domain contributes to recognition specificity by rejecting noncognate tRNAs through negative interactions. Analysis of the catalytic properties of one of the mischarging enzymes, GlnRS100 (Arg-130 → Pro, Glu-131 → Asp), indicates that, while the kinetic parameters of the mutant enzyme are not dramatically changed, it binds noncognate tRNAGlumore stably than the wild-type enzyme does (Kdis 1/8 that of the wild type). Thus, the stability of the noncognate complex may be the basis for mischarging in vivo.