Poster abstracts
Poster number 15 submitted by Jun-Kyu Byun
Role of Unique C-terminal Domain in an Aminoacyl-tRNA Trans-editing Protein from Arabidopsis thaliana
Jun-Kyu Byun (The Ohio State University ), Marina Bakhtina (The Ohio State University), Karin Musier-Forsyth (The Ohio State University)
Abstract:
Some aminoacyl-tRNA synthetases (aaRSs) are error-prone, mispairing non-cognate 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 Ala-tRNAPro. However, some bacteria and all eukaryotes lack this editing domain and instead, encode a free-standing editing domain homolog, ProXp-ala, that edits Ala-tRNAPro in trans. Here, we report that ProXp-ala from plants has a unique C-terminal extension domain (CTD) of unknown structure that is missing from some bacterial and all eukaryotic ProXp-ala domains. This domain is predicted to encode a long helical segment connected to the catalytic domain via a random coil. Ancient aaRSs are believed to have existed as single-domain enzymes and additional domains reflect selective evolutionary adaptations to enhance specificity and efficiency of aminoacylation, as well as to confer new functions. Similarly, additional domains fused to ProXp-ala may be indicative of enhanced specificity or new functions. To determine the function of the CTD in plant ProXp-ala, we prepared a truncated Arabidopsis thaliana (At) ProXp-ala variant (ΔC-ProXp-ala). Circular dichroism spectroscopy showed that the secondary structure of ProXp-ala is not perturbed upon truncation. In vitro deacylation of At Ala-tRNAPro showed that the truncation resulted in a 16-fold decrease in the deacylation rate relative to wild-type ProXp-ala, primarily due to a tRNA binding defect. We hypothesize that this extra domain may confer strong tRNA binding via specific RNA interactions. Ongoing studies will investigate this hypothesis, as well as the In vivofunction of At ProXp-ala, using available At knockdown strains. Understanding the functional significance of the ProXp-ala CTD will reveal new insights into how plants have uniquely evolved to avoid mistranslation.
References:
1. Das M, Vargas-Rodriguez O, Goto Y, Suga H, Musier-Forsyth K. Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation. Nucleic Acids Research. 2014;42(6):3943-3953.
2. Vargas-Rodriguez O, Musier-Forsyth K. Exclusive Use of trans-Editing Domains Prevents Proline Mistranslation. The Journal of Biological Chemistry. 2013;288(20):14391-14399.
Keywords: aminoacyl tRNA synthetase, Trans editing protein, ProXp-ala