CP1 Domain in Escherichia coli Leucyl-tRNA Synthetase Is Crucial for Its Editing Function

Abstract

The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a “coarse sieve”, while the reaction products of amino acids smaller than the cognate substrate will go through a “fine sieve” and be hydrolyzed. This “double-sieve” mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA^Leu by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA^Leu made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function.