Poster abstracts
Poster number 22 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 and The Ohio State University), William A. Cantara (Department of Chemistry and Biochemistry and The Ohio State University), John Vu (Department of Chemistry and Biochemistry and The Ohio State University), Jawad Abid (Department of Chemistry and Biochemistry and The Ohio State University), Jyan-Chyun Jang (Department of Horticulture and Crop Science and The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and The Ohio State University)
Abstract:
Some aminoacyl-tRNA synthetases (aaRSs) are error-prone, mispairing noncognate amino acids with cognate tRNAs. To prevent mistranslation, many aaRSs have evolved quality control mechanisms. 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. However, 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) that is missing from all other eukaryotic ProXp-ala. The CTD is predicted to fold into a long helical segment connected to the catalytic domain via a random coil. 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. SEC-MALS showed 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. Microscale thermophoresis and kinetic studies are currently underway to determine the effect of tRNA anticodon mutations on ProXp-ala binding. In vivo studies in At using split-GFP constructs do not support homo-oligomerization, but do not rule out the possibility that this domain promotes interactions with other binding partners. Therefore, yeast two-hybrid experiments are planned to identify potential interacting partners. At ProXp-ala disruption strains showed an early germination phenotype. Additional phenotypic experiments are being carried out under a variety of growth conditions to better understand the function of the WT enzyme and the role of the unique CTD in vivo. This work will reveal how plants have uniquely evolved to avoid mistranslation and may lead to the discovery of new ProXp-ala functions.
Keywords: aminoacyl-tRNA synthetase, trans-editing , plants