Poster abstracts
Poster number 31 submitted by Eric Danhart
Mapping elements that confer specific acceptor stem recognition of Ala-tRNAPro by a bacterial trans-editing domain
Eric M. Danhart (Department of Chemistry and Biochemistry, The Ohio State University), Lexie Kuzmishin, Brianne Sanford, Oscar Vargas-Rodriguez (Department of Chemistry and Biochemistry, The Ohio State University), Daniel McGowan, Marina Bakhtina (Department of Chemistry and Biochemistry, The Ohio State University), Marija Kosutic, Ronald L. Micura (Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University, Innsbruck, Austria), 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 tRNAPro with Ala, which 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-tRNAPro. Previous studies identified acceptor stem elements of tRNAPro (G72 and A73) that are critical for ProXp-ala activity. In fact, a microhelix that mimics the acceptor stem of tRNAPro 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 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). Four main regions of interaction were identified: a %beta-strand (aa 43-49) within the active site, a region (aa 68-70; 80-84) that we propose is the G72/A73 interacting domain, a dynamic helix at the top of the active site pocket (aa 27-30), and amino acids proximal to this helix (aa 128-134). Mutagenesis and AUC studies are consistent with the critical nature of conserved residues R80 and F83 for substrate binding. Mutation of conserved active site residues K45 and N46 also caused severe (>10-fold) losses in activity, whereas mutation of conserved residues V29, H130, and N134 resulted in more modest decreases (3- to 6-fold). The combined results of these studies allow us to propose a mechanism for Ala-microhelixPro recognition that involves induced fit binding, specific protein-RNA interactions, and key contributions from the Ala moiety.
Keywords: tRNA, NMR, AUC