Poster abstracts
Poster number 81 submitted by Alezandra Hernandez
Plant-exclusive domain of trans-editing enzyme ProXp-ala confers dimerization and enhanced tRNA binding
Jun-Kyu Byun (Department of Chemistry and Biochemistry, The Ohio State University), John A. Vu (Department of Chemistry and Biochemistry, The Ohio State University), Siou-Luan He (Department of Horticulture and Crop Science and Center for Applied Plant Sciences, The Ohio State University), Jyan-Chyun Jang (Department of Horticulture and Crop Science and Center for Applied Plant Sciences, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University), Alezandra M. Hernandez Marquez (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Faithful translation of the genetic code is critical for the viability of all living organisms. The trans-editing enzyme ProXp-ala, encoded by bacteria and eukaryotes, prevents Pro to Ala mutations during translation by hydrolyzing misacylated Ala-tRNAPro that has been synthesized by prolyl-tRNA synthetase. Plant ProXp-ala sequences contain a conserved C-terminal domain (CTD) that is absent in other organisms; the origin, structure, and function of this extra domain are unknown. To characterize the plant-specific CTD, we performed bioinformatics and computational analyses that were consistent with a conserved α-helical structure. We also expressed and purified wild-type Arabidopsis thaliana (At) ProXp-ala in Escherichia coli, as well as variants lacking the CTD or containing only the CTD. Circular dichroism spectroscopy confirmed a loss of α-helical signal intensity upon CTD truncation. Size-exclusion chromatography with multi-angle laser light scattering revealed that wild-type At ProXp-ala was primarily dimeric and CTD truncation abolished dimerization in vitro. Bimolecular fluorescence complementation assays in At protoplasts support a role for the CTD in homodimerization in vivo. The deacylation rate of Ala-tRNAPro by At ProXp-ala was significantly reduced in the absence of the CTD, and kinetic assays indicated that the reduction in activity is primarily due to a tRNA binding defect. Overall, these results broaden our understanding of eukaryotic translational fidelity to the plant kingdom. Our studies revealed that the plant-specific CTD plays a significant role in substrate binding and canonical editing function. Through its ability to facilitate protein-protein interactions, the CTD may also provide expanded functional potential to trans-editing enzymes in plants. Ongoing studies using mutant and transgenic plants are aimed at further exploring the function and interacting partners of At ProXp-ala in vivo.
Keywords: aminoacyl-tRNA, aminoacyl-tRNA synthetase, Arabidopsis thaliana