The bacterial YbaK protein is a Cys-tRNAPro and Cys-tRNACys deacylase

Bacterial prolyl- tRNA synthetases and some smaller paralogs, YbaK and ProX, can hydrolyze misacylated Cys- tRNA(Pro) or Ala- tRNA(Pro). To assess the significance of this quality control editing reaction in vivo, we tested Escherichia coli ybaK for its ability to suppress the E. coli thymidylate synthase thyA: 146CCA missense mutant strain, which requires Cys- tRNAPro for growth in the absence of thymine. Missense suppression was observed in a ybaK deletion background, suggesting that YbaK functions as a Cys- tRNAPro deacylase in vivo. In vitro studies with the full set of 20 E. coli aminoacyltRNAs revealed that the Haemophilus influenzae and E. coli YbaK proteins are moderately general aminoacyltRNA deacylases that preferentially hydrolyze CystRNAPro and Cys- tRNACys and are also weak deacylases that cleave Gly- tRNA, Ala- tRNA, Ser- tRNA, Pro- tRNA, and Met- tRNA. The ProX protein acted as an aminoacyltRNA deacylase that cleaves preferentially Ala- tRNA and Gly- tRNA. The potential of H. influenzae YbaK to hydrolyze in vivo correctly charged Cys- tRNACys was tested in E. coli strain X2913 ( ybaK (+)). Overexpression of H. influenzae ybaK decreased the in vivo ratio of CystRNA(Cys) to tRNACys from 65 to 35% and reduced the growth rate of strain X2913 by 30% in LB medium. These data suggest that YbaK- mediated hydrolysis of aminoacyltRNA can influence cell growth.