Poster abstracts

Poster number 28 submitted by Oscar Vargas

In vitro characterization of two free-standing homologs of the bacterial prolyl-tRNA synthetase editing domain in Caulobacter crescentus

Oscar Vargas-Rodriguez (Departments of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)

Abstract:
Fidelity during protein synthesis relies on aminoacyl-tRNA synthetases, which are responsible for matching specific amino acids with their cognate tRNAs. Although, synthetases are efficient enzymes, the similar size and volume of many amino acids results in the formation of mismatched amino acid-tRNA pairs. For example, prolyl-tRNA synthetase (ProRS) can mis-activate and mis-acylate tRNA^Pro with cysteine or alanine. However, bacteria have evolved editing mechanisms to ensure the fidelity of the proline codon. In most bacteria, Ala-tRNA^Pro is hydrolyzed by a ProRS catalytic domain distinct from the synthetic active site, the insertion domain (INS), via “post-transfer” editing. In contrast, YbaK, a free-standing homolog of the ProRS INS domain, edits Cys-tRNA^Pro. Interestingly, the complete genomes sequenced to date have revealed that some bacterial ProRSs encode a truncated INS domain that has lost editing capability. However, many of these organisms encode an additional free-standing INS domain homolog. For instance, Caulobacter crescentus (Cc) expresses a truncated ProRS and encodes both YbaK and PrdX. We hypothesize that the latter is a deacylase that hydrolyzes Ala-tRNAPro. We cloned, expressed, purified, and characterized the in vitro activity of Cc ProRS, YbaK, and PrdX and found that Cc ProRS can mis-charge Cys onto tRNAPro, and to a lesser extent mis-acylates Ala. As expected, Cc YbaK showed hydrolytic activity towards Cys-tRNA^Pro. On the other hand, PrdX can deacylate Ala-tRNA^Pro and Ala-tRNA^Ala in vitro. Our data suggest that the fidelity of proline codon translation in Cc relies on the activities of YbaK and PrdX, since Cc ProRS is unable to edit Cys- or Ala-tRNA^Pro. This study provides the first characterization of an organism possessing a ProRS that lacks post-transfer editing capability, but encodes two free-standing domains that collaborate to maintain fidelity during translation.

Keywords: prolyl-tRNA synthetase, YbaK, PrdX