Poster abstracts
Poster number 72 submitted by Alexandra Kuzmishin
Characterization of the interaction between a trans-editing domain and its aminoacyl-tRNA substrate
Alexandra Kuzmishin (Department of Chemistry and Biochemistry, The Ohio State Biochemistry Program, The Center for RNA Biology, The Chemistry-Biology Interface Program, The Ohio State University, Columbus, OH), Eric Danhart (Department of Chemistry and Biochemistry, The Ohio State Biochemistry Program, The Center for RNA Biology, The Chemistry-Biology Interface Program, The Ohio State University, Columbus, OH), Brianne Sanford, Marina Bakhtina (Department of Chemistry and Biochemistry, The Center for RNA Biology, The Ohio State University, Columbus, OH), Ronald Micura (Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University, Innsbruck, Austria), Mark Foster, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Center for RNA Biology, The Ohio State University, Columbus, OH)
Abstract:
Aminoacyl-tRNA synthetases (ARSs) catalyze the esterification of specific amino acids to their cognate tRNA substrates to form aminoacyl-tRNAs. ARSs can also misactivate and mischarge tRNAs with smaller or similarly-sized non-cognate amino acids. Prolyl-tRNA synthetase (ProRS) mischarges Ala and Cys onto tRNAPro in vitro. Many synthetases, including most bacterial ProRSs, exhibit proofreading activities to correct errors due to mischarging and thus prevent amino acid misincorporation into proteins. Bacteria that encode ProRS with a functional editing domain (INS) are able to deacylate Ala-tRNAPro via post-transfer editing. Some bacteria lack a functional INS domain and instead encode a free-standing INS-homolog, ProXp-ala, that deacylates Ala-tRNAPro via post-transfer editing in trans. The tRNAPro recognition elements for Caulobacter crescentus ProXp-ala have been identified, although the sites of interaction on ProXp-ala are unknown. NMR chemical shift perturbation studies were used to map regions of interaction between ProXp-ala and an uncharged microhelix derived from the acceptor stem of tRNAPro. Binding to a stably mischarged Ala-microhelixPro substrate analog was also investigated for the first time. To establish the significance of the identified regions for substrate binding and catalysis, ProXp-ala variants have been generated via site-directed mutagenesis of 27 residues located throughout the protein structure. Analytical ultracentrifugation was used to screen the variants for relative changes in binding affinity to microhelixPro and deacylation assays have been used to establish the effect on catalytic activity. Based on these studies, we have generated a comprehensive model for substrate binding and recognition by a trans-editing domain.
Keywords: aminoacyl-tRNA synthetase, ProRS, ProXp-ala