Poster abstracts

Poster number 44 submitted by Antonia Duran

NMR-based structure determination of the tRNA trans-editing enzyme ProXp-ala

Antonia D. Duran (Department of Chemistry and Biochemistry, The Ohio State University), Xiao Ma, Eric M. Danhart (Department of Chemistry and Biochemistry, The Ohio State 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:
ProXp-ala is a key component of the translational machinery in all three Domains of life. This enzyme helps to maintain the fidelity of proline codon translation through aminoacyl-tRNAPro proofreading. In the first step of tRNA aminoacylation, the cognate aminoacyl-tRNA synthetase (aaRS) binds and activates an amino acid in the enzyme’s synthetic active site. If a non-cognate amino acid passes this first selection step and is charged onto the tRNA, a distinct aaRS editing active site may recognize the mischarged tRNA and deacylate it. Alternatively, this editing reaction may be carried out by a separate enzyme that deacylates the mischarged tRNA in trans. ProXp-ala is responsible for editing Ala mischarged onto tRNAPro. Since trans-editing domains such as ProXp-ala bind their substrates after release from the synthetase, they must recognize not only the mischarged amino acid, but also the specific tRNA. Previous studies showed that Caulobacter crescentus ProXp-ala distinguishes tRNAPro from tRNAAla, in part, based on the unique tRNAPro acceptor stem base pair C1:G72. Previous crystallographic and NMR data revealed a role for conformational selection by the ProXp-ala α2 helix in Ala- versus Pro-tRNAPro substrate discrimination. The α2 helix makes lattice contacts in the crystal, which is a caveat of the crystallography data. NMR-based solution structure determination of the free ProXp-ala domain will allow a more accurate description of the position of the α2 helix in the absence of the substrate and will set the stage for structure determination of ProXp-ala bound to Ala-tRNAPro. Results obtained to date toward NMR structure determination of the free Caulobacter crescentus ProXp-ala domain will be presented.

Keywords: protein NMR