Knowledge Bank

Aminoacyl-tRNA synthetases (aaRSs) catalyze bonds between tRNAs and cognate amino acids. The formation of cognate aminoacyl-tRNAs is critical for cell viability, as translating noncognate aa-tRNAs leads to amino acid misincorporation and alterations in protein structure and function. While aaRSs make errors in most organisms, proofreading mechanisms also exist; however, our understanding of this machinery is poorly understood, particularly outside of prokaryotes. Unlike Escherichia coli (Ec), which possess a designated editing domain on prolyl- tRNA synthetase (ProRS), some bacteria like Caulobacter crescentus (Cc), multicellular eukaryotes like humans (Homo sapiens, Hs), and plants like Arabidopsis thaliana (At), encode a Pro separate editing enzyme called ProXp-ala that hydrolyzes non-cognate Ala-tRNA . Sequence alignments have revealed that the homologs in plants all possess a C-terminal extension absent in other species. This C-terminal domain (CTD) has a conserved sequence with no known structure or function to date. This work seeks to elucidate the structure and function of this highly conserved CTD of At ProXp-ala. Bioinformatics analyses and homology modeling studies revealed that the plant-specific CTD may be an oligomerization motif. Full-length wild type (WT) and truncated C- terminal domain (∆CTD) At ProXp-ala and At tRNAPro have been expressed and purified from E. coli and their storage conditions were optimized. Homology models predicted that the CTD plays a role in protein oligomerization, which was supported by size-exclusion chromatography with multiple angle laser light scattering experiments. In vitro kinetic deacylation assays demonstrated a 19-fold decrease in activity without the CTD and a positive correlation between activity and enzyme concentration for the ∆CTD variant. These data suggest that the CTD plays a significant role in the activity of At ProXp-ala and are consistent with a model in which truncation results in Pro impaired binding of the ∆CTD mutant to tRNA assays were performed to test this hypothesis but require further optimization. Comparisons of plant and human ProXp-ala activity revealed similar deacylation rates between the human enzyme and ∆CTD At ProXp-ala. If the At CTD does indeed improve tRNA binding, then appending this domain to human ProXp-ala is expected to similarly improve its substrate binding activity and future work will address this hypothesis. The emphasis of this work on translational fidelity in plants is a novel direction in aaRS research that will significantly expand our understanding of eukaryotic editing mechanisms. Our comparative study of plant and human ProXp-ala has implications as to the potential limitations of human translational machinery and may inform future studies on the mechanisms of human disease related to sub-optimal translational fidelity.