Talk Abstracts

Talk on Wednesday 03:00-03:15pm submitted by Eric Danhart

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

Eric M. Danhart (Ohio State Biochemistry Program), Brianne Sanford, Oscar Vargas-Rodriguez, Daniel McGowan, Marina Bakhtina (Department of Chemistry and Biochemistry, The Ohio State University), Lexie Kuzmishin (Ohio State Biochemistry Program), Marija Kosutic, Ronald L. Micura (Institute 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)

Abstract:
Aminoacyl-tRNA synthetases (ARS) 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 tRNA^Pro with Ala; this aberrant product is hydrolyzed by a cis-editing domain (INS) in most bacteria. However, some bacteria lacking the INS domain encode a homologous free-standing trans-editing domain known as ProXp-ala that functions to clear Ala-tRNA^Pro. Previous studies showed that specific nucleotides in the acceptor stem of tRNA^Pro (G72 and A73) are critical for ProXp-ala activity, and that a small RNA stem-loop containing these elements, microhelix^Pro, 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 microhelix^Pro and with a non-hydrolyzable, amide-linked Ala-microhelix^Pro mimic. We observe similar but significantly stronger chemical shift perturbations in the presence of the charged microhelix, which also displays 5-fold higher affinity for binding to ProXp-ala, as measured by analytical ultracentrifugation (AUC). The largest chemical shift perturbations were mapped to three main 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) that we propose is the G72/A73 interacting domain. Site-directed mutagenesis and AUC studies are consistent with the critical nature of residues 80-83 for substrate binding. Mutation of conserved active site residues K45 and N46 also caused severe losses in activity. 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-microhelix^Pro that involves induced-fit binding, specific protein-RNA contacts, and key contributions from the Ala moiety.

Keywords: NMR, tRNA, dynamics