A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNA(Leu)

Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accu rately selecting and activating cognate amino acids for aminoacylation of t he correct tRNA. Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection. These two sieves rely on different stra tegies for amino acid recognition to significantly enhance the accuracy of aminoacylation. We have performed alanine scanning mutagenesis in a conserv ed threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's C P1 domain that is hypothesized to contain a putative editing active site. C haracterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid fro m being hydrolyzed after aminoacylation of the tRNA. Mutation of this threo nine to an alanine eliminates discrimination of the cognate amino acid in t he editing active site. This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.