Poster abstracts

Poster number 74 submitted by Byung Ran So

Mechanism of Editing of Mis-charged Cys-tRNA^Pro by Bacterial YbaK

Byung Ran So (Chemistry, Ohio State University), Songon An (Chemistry, University of Minnesota), Sandeep Kumar (Chemistry, Ohio State University), Christopher Hadad (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. 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. However, relatively little is known about the catalytic mechanism of YbaK or other free-standing editing domains at the molecular level. 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 in ProRS and YbaK families, as well as residues identified from substrate docking studies and molecular dynamics simulations. Additionally, to probe the function of the tRNA hydroxyl groups in substrate recognition and catalysis, we tested YbaK activity using dA76 variants of Cys-tRNA. Taken together, the results of these studies allow us to propose a mechanism for Cys-tRNA^Pro editing by YbaK involving stabilization of substrate functional groups by conserved residues within the substrate-binding site and cysteine-thiolactone formation.

Keywords: Aminoacyl-tRNA synthetase, YbaK, editing