Aminoacyl-tRNA Substrate and Enzyme Backbone Atoms Contribute to Translational Quality Control by YbaK

Abstract

Amino acids are covalently attached to their corresponding transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases. Proofreading mechanisms exist to ensure that high fidelity is maintained in this key step in protein synthesis. Prolyl-tRNA synthetase (ProRS) can misacylate cognate tRNA^Pro with Ala and Cys. The <i>cis</i>-editing domain of ProRS (INS) hydrolyzes Ala-tRNA^Pro, whereas Cys-tRNA^Pro is hydrolyzed by a single domain editing protein, YbaK, <i>in trans</i>. Previous studies have proposed a model of substrate-binding by bacterial YbaK and elucidated a substrate-assisted mechanism of catalysis. However, the microscopic steps in this mechanism have not been investigated. In this work, we carried out biochemical experiments together with a detailed hybrid quantum mechanics/molecular mechanics study to investigate the mechanism of catalysis by Escherichia coli YbaK. The results support a mechanism wherein cyclization of the substrate Cys results in cleavage of the Cys-tRNA ester bond. Protein side chains do not play a significant role in YbaK catalysis. Instead, protein backbone atoms play crucial roles in stabilizing the transition state, while the product is stabilized by the 2′-OH of the tRNA