Poster abstracts

Poster number 42 submitted by Ziwei Liu

tRNA synthetase, post-transfer editing

Ziwei Liu (Ohio State Biochemistry Program)

Abstract:
Most bacterial prolyl-tRNA synthetase (ProRS) possesses an editing domain (INS), which hydrolyzes the mischarged Ala-tRNAPro. In addition to this in cis editing domain, several freestanding INS domain homologs, such as YbaK, ProXp-ala and ProXp-y are found in various species. Among these proteins, YbaK and ProXp-ala are shown to deacylate Cys-tRNAPro and Ala-tRNAPro separately. We found that ProXp-y has ability to edit Ser- and Thr-tRNA species. In vitro deacylation assays showed that none of the mischarged Ala-, Leu- or Cys- tRNAPro is the substrate of Escherichia coli (E.coli) ProXp-y alone. To explore the role of ProXp-y, we performed halo and growth curve assays for an E.coli proxp-y null strain and compared its behavior with that of the wild-type (wt) strain under high concentrations of 20 natural amino acids individually. Both of these in vivo assays suggested that proxp-y null strain showed growth defect under Ser condition. The slow growth phenotype of proxp-y null strain can be rescued by addition of Ala. Upon overexpression of Ec AlaRS, the null strain displays a more severe growth phenotype in the presence of Ser. In vitro assays confirmed robust deacylation of Ser-tRNAThr and Ser-tRNAAla, which are known to form due to misacylation by ThrRS and AlaRS. In vitro assays also showed that ProXp-y deacylates Thr-tRNAVal and Thr-tRNALys with similar efficiency as Ser-tRNAs. Taken together, these data suggest that ProXp-y may deacylate tRNAs other than tRNAPro and may collaborate with AlaRS to avoid Ala-to Ser mistranslation and with LysRS to avoid Lys-to-Thr mistranslation, as E.coli AlaRS and LysRS have no post-transfer editing domain that is responsible for mischarged Ser- and Thr-tRNAs. In ongoing studies, immunoprecipitation method will be used to identify the RNA and protein partners of ProXp-y. Crystallization trays will be set up to for ProXp-y protein alone and with substrate candidates to obtain the three-dimensional structures.

Keywords: tRNA synthetases