Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase

Abstract

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNAVal. Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3′-terminus of tRNAVal is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNAVal at the aminoacylation and editing sites, and by probing tRNAVal for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNAVal and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS. This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.