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

摘要

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threomine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA~(val). Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3'-terminus of tRNA~(val) is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA~(val) at the aminoacylation and editing sites, and by probing tRNA~(val) for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNA~(val) 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.