Poster abstracts

Poster number 59 submitted by Byung Ran So

Mechanism of Editing of Mis-charged Cys-tRNAPro by Bacterial YbaK

Byung Ran So (Chemistry, Ohio State University), Meredith Qualley (Chemistry, Ohio State University), Karin Musier-Forsyth (Chemistry and Biochemistry, Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or “charging”) specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. For example, prolyl-tRNA synthetase (ProRS) mis-activates alanine and deacylates mischarged Ala-tRNA^Pro using an editing active site that is distinct from the site of amino acid activation. A free-standing protein (YbaK) with homology to the ProRS editing domain is present in most bacteria. YbaK has been shown to possess hydrolytic editing activity against mischarged Cys-tRNA^Pro. Previously, we demonstrated that the strictly conserved K46 residue in the putative substrate-binding pocket is critical for Cys-tRNA^Pro editing activity, and that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS/YbaK/tRNA ternary complex. To further characterize the trans-editing mechanism of YbaK, we performed extensive Ala-scanning mutagenesis of conserved residues, as well as residues identified from substrate docking studies and molecular dynamics simulations. Fluorescence anisotropy and isothermal titration calorimetry binding experiments were also performed to probe ternary complex formation with homologous ProRS and tRNA species. Taken together, the results of these studies allow us to propose a mechanism of catalysis by YbaK involving conserved residues within the substrate-binding region, and to begin to define the stoichiometry and interactions between the three binding partners.

Keywords: Prolyl-tRNA synthetase, YbaK