Poster abstracts

Poster number 41 submitted by Arundhati Kavoor

E. coli Alanyl-tRNA Synthetase Maintains Proofreading Activity and Translational Accuracy under Oxidative Stress

Arundhati Kavoor (MCDB, OSU), Dr. Paul Kelly (MCDB, OSU)

Abstract:
Aminoacyl-tRNA synthetases (aaRS) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, therefore, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species (ROS) can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in E. coli AlaRS, unlike in other aaRSs studied to date, this does not affect proofreading activity against the non-cognate substrates serine and glycine, and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as ROS “sinks” to protect catalytically-critical sites from oxidative damage. Combined, our results indicate E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.

Keywords: aminoacyl-tRNA synthease, tRNA, oxidative stress