Poster abstracts

Poster number 40 submitted by Jun-Kyu Byun

Role of Unique C-terminal Domain in a Plant Aminoacyl-tRNA Trans-editing Protein

Jun-Kyu Byun (Department of Chemistry and Biochemistry), Sio-Luan He (Department of Horticulture and Crop Science and Center for Applied Plant Sciences), William A. Cantara (Department of Chemistry and Biochemistry), Jyan-Chyun Jang (Department of Horticulture and Crop Science and Center for Applied Plant Sciences), Karin Musier-Forsyth (Department of Chemistry and Biochemistry)

Abstract:
Some aminoacyl-tRNA synthetases (aaRSs) are error-prone, mispairing noncognate amino acids with cognate tRNAs. Many aaRSs have evolved quality control mechanisms to prevent mistranslation. Bacterial prolyl-tRNA synthetases (ProRSs) mischarge noncognate Ala onto tRNAPro and possess an insertion (INS) domain that can deacylate this mischarged tRNA and avoid mistranslation. Some bacteria and all eukaryotes lack an INS domain and instead, encode a free-standing trans-editing domain homolog, ProXp-ala. Sequence alignments revealed that all plant ProXp-ala contain a conserved C-terminal domain (CTD) of unknown structure and function that is missing from all other eukaryotic ProXp-ala. The CTD is predicted to fold into an all -helical domain. To determine the function of the CTD, we prepared a truncated Arabidopsis thaliana (At) ProXp-ala variant (ΔC-ProXp-ala). The in vitro Ala-tRNAPro deacylation rate of ΔC-ProXp-ala was decreased 16-fold relative to wild-type (WT) ProXp-ala. Electrophoretic mobility shift assays suggest that this decrease is primarily due to a tRNA binding defect. Size-exclusion chromatography revealed that WT At ProXp-ala adopts several oligomeric states, while ΔC-ProXp-ala is exclusively monomeric. The CTD may therefore function to enhance tRNA binding and/or induce multimerization in vitro. In vivo studies using split-YFP constructs support ProXp-ala homodimerization and show that the CTD is required for strong self-interaction. Yeast two-hybrid experiments to semi-quantitatively determine the role of each domain of ProXp-ala in self-interaction are underway. This work is expected to reveal the structure-function relationship of plant ProXp-ala in quality control of protein synthesis.

Keywords: aminoacyl-tRNA synthetase, trans-editing , plants