Poster abstracts

Poster number 68 submitted by Byung Ran So

Distinct mechanisms used by interacting editing domains involving steric exclusion and chemistry ensure accurate decoding of proline codons

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), Thomas J. Magliery (Chemistry and Biochemistry, Ohio State University), Karin Musier-Forsyth (Chemistry and Biochemistry, Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases attach specific amino acids to cognate transfer RNAs (tRNAs), an essential step in accurate decoding of genetic information. Prolyl-tRNA synthetases (ProRSs) are known to mischarge tRNA^Pro with the smaller amino acid Ala, and with Cys, which is the same size as Pro. Quality control in translation is partly ensured by an editing domain (INS) present in most bacterial ProRSs that hydrolyzes Ala-tRNA^Pro but not Cys-tRNA^Pro. The latter is cleared by a free-standing INS domain homolog, YbaK. To understand the molecular basis of the distinct substrate specificities of these homologous editing domains, we have investigated the mechanism of hydrolysis of Cys-tRNA^Pro by YbaK. Our data support a substrate-assisted mechanism of catalysis wherein hydrolysis is mediated by the substrate side chain thiol. We have previously reported that YbaK and ProRS interact in vitro. In new work, we now show that the ProRS·YbaK complex demonstrates enhanced proofreading activity relative to the YbaK protein alone. The complex competes efficiently with Ef-Tu for Cys-tRNA^Pro, whereas YbaK alone does not, implying that YbaK functions before the substrate is released from ProRS. To probe protein-protein interactions in vivo, a split-GFP reassembly technique was employed and results support our hypothesis that ProRS, YbaK, and tRNA form a complex in the cell. Taken together, we propose that the ProRS INS and YbaK domains interact in vivo and function via distinct mechanisms involving steric exclusion and thiol-specific chemistry, respectively, thus collaborating to ensure accurate decoding of Pro codons.

Keywords: YbaK, aminoacyl-tRNA synthetase, substrate-assisted catalysis