Talk abstracts

Talk on Friday 03:15-03:30pm submitted by Eric Danhart

Determinants of specific recognition of mischarged Ala-tRNAPro by a bacterial trans-editing domain

Eric M. Danhart (Department of Chemistry and Biochemistry, The Ohio State University), Lexie Kuzmishin, Brianne Sanford, Marina Bakhtina (Department of Chemistry and Biochemistry, The Ohio State University), Marija Kosutic, Ronald L. Micura (2Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to cognate tRNAs. Mistakes in this process lead to errors in protein synthesis that can be deleterious to cells. Prolyl-tRNA synthetase (ProRS) mischarges tRNAPro with Ala; this aberrant product is hydrolyzed by a cis-editing domain (INS) in most bacteria. Bacteria whose ProRS lack the INS domain encode a homologous free-standing trans-editing protein known as ProXp-ala that performs the same function. Enzyme assays show that specific nucleotides in the acceptor stem of tRNAPro are critical for ProXp-ala activity, and a small RNA stem-loop containing these elements, microhelixPro, is a good substrate for Ala deacylation. To define the elements in ProXp-ala that confer acceptor stem specificity, NMR mapping studies were carried out with an uncharged microhelixPro and with a non-hydrolyzable, amide-linked Ala-microhelixPro mimic. We observe similar but significantly stronger chemical shift perturbations (CSPs) in the presence of the charged microhelix, which also displays higher affinity for binding to ProXp-ala. The largest CSPs were mapped to three regions: helix α2 at the top of the active site pocket (aa 27-30), β-strands β2 (aa 43-49) and β6 (aa 128-134) within the active site, and β-strand β4 (aa 80-84). Site-directed mutagenesis and AUC studies are consistent with the critical nature of these residues. Additionally, 15N NMR relaxation experiments revealed that the helix α2 exhibits significant dynamics at the ps-ns timescale. These results allow us to propose a mechanism for recognition of Ala-microhelixPro that involves induced-fit binding, specific protein-RNA contacts, and key contributions from the Ala moiety.

Keywords: tRNA, NMR, RNA-protein interaction