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)
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