Studying Lysine Acetylation of Aminoacyl‐tRNA Synthetases in Escherichia coli

Abstract

Aminoacyl‐tRNA synthetases (AARSs) generate aminoacylated tRNAs for ribosomal protein synthesis, and play key roles in maintaining high fidelity of translation. A series of acetylome studies have shown that the family of AARSs is one of the protein families which have both the most acetylation sites and the highest acetylation levels. However, those putative acetylation targets have been less studied, mostly because of the challenge to produce homogeneously acetylated proteins at specific positions which needs the knowledge of specific acetyltransferases for target lysine residues. To overcome this issue, we applied the genetic code expansion strategy to co‐translationally incorporate acetyllysine at the target positions directly. We totally generated 25 site‐specifically acetylated variants of four AARSs. Kinetic analyses were performed to biochemically characterize the impact of site‐specific acetylation on AARS functions, including both tRNA aminoacylation and editing activities. The results showed that impacts of acetylation were different for class I and class II AARSs, and also varied among the same class of AARSs. Combined with quantitative proteomic studies, we proposed that the impact of acetylation on AARS editing activities could be more significant than that on tRNA aminoacylation activities. To the best of our knowledge, this is the first work to show the association of protein acetylation with the AARS editing function. Furthermore, using threonyl‐tRNA synthetase (ThrRS) as a representative, both in vivo and in vitro studies were performed to characterize the acetylation and deacetylation processes of AARSs. Although nonenzymatic acetylation and CobB‐dependent deacetylation were concluded, our results also indicated existence of additional acetyltransferases and deacetylases for AARSs.Support or Funding InformationThis work was supported by Oak Ridge Associated Universities, Arkansas Biosciences Institute, and the University of Arkansas.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.