Understanding primary and alternate functions of leucyl-tRNA synthetases from Escherichia coli, Saccharomyces cerevisiae and Mycobacterium tuberculosis.

摘要

The first step during protein synthesis includes activation of amino acids and their esterification to cognate tRNAs by aminoacyl-tRNA synthetases (aaRSs). It is an error prone process and in order to ensure fidelity, the synthetases have evolved two types of proofreading mechanisms which can occur before (pre-transfer) or after (post-transfer) the transfer of amino acid to the tRNA. Editing is supported by a distinct domain called the connective polypeptide 1 (CP1). Leucyl tRNA synthetase (LeuRS), from Escherichia coli edits exclusively via post-transfer editing. However, recently it was shown that point mutations at a critical site, alanine 293 (A293) within the CP1 domain of the E. coli LeuRS aid the enzyme to exhibit an alternate editing pathway in vitro, even when the enzyme's post transfer editing is blocked. Further mutational characterization of the A293 peptide spanning the critical residue revealed rescue of fidelity defects both in vitro and in vivo. These results along with previously published work enabled us to hypothesize that mutagenesis of this small A293 peptide in the CP1 domain stimulated an alternate editing pathway to ensure additional fidelity in E. coli LeuRS.;The CP1 domain, which is essential to amino acid editing, was also implicated in the splicing activity of ymLeuRS. In ymLeuRS a critical tryptophan (W238) at the beginning of the N-terminal beta-strand that continues into the CP1 domain was identified to be a splicing-sensitive site. Herein, we observed that a homologous site in E. coli LeuRS (W223) functionally differentiates for the dual aminoacylation and splicing roles of the protein.;The A293 peptide was identified to influence alternate activities of LeuRS. Yeast mitochondrial LeuRS (ymLeuRS) is an essential protein co-factor for the RNA splicing of group I introns in yeast mitochondria. Diverse LeuRSs from varied origins such as Mycobacterium tuberculosis and human mitochondria complement the ymLeuRS activities. Similarly, we determined that the E. coli LeuRS wild type enzyme complemented the null strains for ymLeuRS. Interestingly, we also detected that at reduced levels of E. coli LeuRS expression in yeast cells, the heterologous synthetase supported protein synthesis, but not RNA splicing. Thus, it is a weak splicing suppressor. Surprisingly, a gain of a secondary function for RNA splicing was exhibited by positive charge substitutions at the critical A293 position, suggesting that this A293 peptide can be adapted for alternate activities. A homologous site in Mycobacterium tuberculosis LeuRS was also found to be splicing-sensitive.