Poster abstracts

Poster number 103 submitted by Alana Weaver

Measuring the Binding Affinity of the Trans-Editing Enzyme, ProXp-x, to Deacylated Bacterial tRNAs

Hannah Skene (Biology Department and Chemistry Department, Earlham College), Kyle Moore (Biology Department and Chemistry Department, Earlham College), Alana Weaver (Biology Department and Chemistry Department, Earlham College), Urwashi BK (Biology Department and Chemistry Department, Earlham College), Lexie Kuzmishin Nagy, Ph.D. (Biology Department and Chemistry Department, Earlham College)

Abstract:
ProXp-x is a trans-editing enzyme in Rhodopseudomonas palustris bacteria that ensures protein synthesis accuracy. ProXp-x removes incorrect amino acids from tRNAs, but the mechanism by which ProXp-x finds and separates incorrectly formed aminoacyl-tRNAs is unknown. Aminoacyl-tRNAs are important in protein synthesis to deliver the correct amino acids to the ribosome. Cells can survive without ProXp-x, but they are more likely to be susceptible to environmental stressors, like D-amino acids. We hypothesize that ProXp-x prevents D-amino acid toxicity in Rhizobia (soil-dwelling bacteria). This study will aminoacylate tRNA molecules with D-amino acids and L-amino acids to measure ProXp-x’s binding affinity for these molecules and the kinetics of removing amino acids from tRNA. To investigate, ProXp-x will be purified using fast protein liquid chromatography with a nickel affinity column, followed by size exclusion chromatography (SEC). tRNA was synthesized via in vitro transcription and purified via gel electrophoresis. The resulting tRNA and ProXp-x were used in electrophoretic mobility shift assays to assess how ProXp-x finds tRNAs and differentiates between tRNAs charged with D-amino acids and L-amino acids. The electrophoretic mobility shift assays did not show binding between ProXp-x and the different deacylated tRNAs, even with increasing protein concentration. These results suggest that the binding affinity between ProXp-x and decylated tRNAs is much lower than predicted. Future directions include performing more EMSAs to view the protein concentration range in which binding affinity can be measured.

Keywords: aminoacyl-tRNA, trans-editing, D-amino acid