Limited set of amino acid residues in a class Ia aminoacyl-tRNA synthetase is crucial for tRNA binding.

The aim of this work was to characterize crucial amino acids for the aminoacylation of tRNA A r g by yeast arginyl-tRNA synthetase. Alanine mutagenesis was used to probe all the side chain mediated interactions that occurbetween tRNA A r g 2 ICG and ArgRS. The effects of the substitutions were analyzed in vivo in an ArgRS-knockout strain and in vitro by measuring the aminoacylation efficiencies for two distinct tRNA A r g isoacceptors. Nine mutants that generate lethal phenotypes were identified, suggesting that only a limited set of side chain mediated interactions is essential for tRNA recognition. The majority of the lethal mutants was mapped to the anticodon binding domain of ArgRS, a helix bundle that is characteristic for class Ia synthetases. The alanine mutations induce drastic decreases in the tRNA charging rates, which is correlated with a loss in affinity in the catalytic site for ATP. One of those lethal mutations corresponds to an Arg residue that is strictly conserved in all class la synthetases. In the known crystallographic structures of complexes of tRNAs and class Ia synthetases, this invariant Arg residue stabilizes the idiosyncratic conformation of the anticodon loop. This paper also highlights the crucial role of the tRNA and enzyme plasticity upon binding. Divalent ions are also shown to contribute to the induced fit process as they may stabilize the local tRNA-enzyme interface. Furthermore, one lethal phenotype can be reverted in the presence of high Mg 2 + concentrations. In contrast with the bacterial system, in yeast arginyl-tRNA synthetase, no lethal mutation has been found in the ArgRS specific domain recognizing the Dhu-loop of the tRNA A r g . Mutations in this domain have no effects on tRNA A r g aminoacylation, thus confirming that Saccharomyces cerevisiae and other fungi belong to a distinct class of ArgRS.