Substrate Divergence in Aminoacyl-tRNA Biosynthesis

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DESCRIPTION (provided by applicant): Accurate translation of the genetic code into functional polypeptides relies on critical events in molecular recognition. The aminoacyl-tRNA synthetases (AARSs) are at the heart of this process. These enzymes catalyze specific tRNA aminoacylation reactions. Their products, the aminoacyl-tRNAs, convert the nucleic acid genetic code into proteins within the ribosome. With only a few exceptions, each of the AARSs is exquisitely specific for a single cognate amino acid and a single class of tRNA isoacceptors. Glutamyl-tRNA synthetase (GluRS) is an unusual exception to this specificity rule; some GluRSs aminoacylate both tRNA(Glu), the cognate substrate, and the "non-cognate" tRNA(Gln); in these cases, GluRS is termed non-discriminating (GluRS-ND). The pathogenic bacterium Helicobacter pylori relies on two GluRSs - a discriminating GluRS-D (GluRS1) and a second GluRS (GluRS2) that is neither discriminating nor non-discriminating. This Hp GluRS2 misacylates tRNA(GIn) with glutamate but does not readily aminoacylate "cognate" tRNA(Glu). It is proposed that these two GluRSs represent intermediates in the evolution of a modern GluRS-D and a possible future, bacterial glutaminyl-tRNA synthetase (GInRS). Thus, GIuRS1 and GluRS2 can be used to experimentally probe the evolution of substrate specificity. The research described in this proposal will define the molecular differences that result in the fine-tuned tRNA specificities of these two enzymes, while concomitantly delineating the differences between GluRS-NDs and GluRS-Ds. Additionally, experiments will directly probe the amino acid specificity of GluRS2 to determine how this enzyme selects glutamate over glutamine, asking whether or not it can be converted into a functional, bacterial GInRS. In total, this proposal will provide detailed molecular insight into the subtle differences between the substrate specificities of closely related AARSs, while providing direct insight into how the evolution of these enzymes might have proceeded.