Poster abstracts

Poster number 113 submitted by Xiao Ma

Selectivity and editing mechanisms of an aminoacyl-tRNA trans-editing factor

Xiao Ma (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Eric M. Danhart, Marina Bakhtina, William A. Cantara, (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Alexandra B. Kuzmishin, Brianne L. Sanford (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Marija Kosutic, Ronald Micura (Institute of Organic Chemistry, Center for Molecular Biosciences, Leopold Franzens University), Yuki Goto, Hiroaki Suga (Department of Chemistry, Graduate School of Science, The University of Tokyo), Kotaro Nakanishi, Mark P. Foster, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for charging amino acids onto their cognate tRNAs. The structural features of the aaRSs catalytic domain provide a high degree of selectivity for attachment of the correct amino acid; however, given the similar stereochemical properties of many of the amino acids, errors in tRNA charging can occur, especially for the smaller and isometric amino acids. Many organisms possess editing enzymes that function in trans to hydrolyze mischarged tRNAs and maintain high fidelity in protein synthesis. ProXp-ala, a structural homolog of the editing domain found in most bacterial prolyl-tRNA synthetases can deacylate mischarged Ala-tRNAPro. We have provided evidence that Caulobacter crescentus ProXp-ala recognizes features on the acceptor stem of tRNAPro, while three overlapping mechanisms contribute to discrimination between Ala-tRNAPro and Pro-tRNAPro: conformational selection, size exclusion and chemical selection. However, the structural basis for this discrimination is incompletely understood. We are using X-ray crystallography, NMR spectroscopy and enzymatic assays to probe the structural and thermodynamic details of conformational selection by studying the structures of ProXp-ala in complex with uncharged tRNAPro and bound to a non-hydrolyzable Ala-tRNAPro analog. We are also assessing the catalytic role played by the 2’OH of the terminal 76A in tRNAPro. These studies will facilitate our understanding of both substrate recognition and hydrolysis by the proofreading enzyme ProXp-ala.

References:
1. Das M., Vargas-Rodriguez O., Goto Y., Novoa, E., Pouplana L., Suga H., Musier-Forsyth K., (2014). Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation. Nucl. Acids Res. 42 (6):3943-3953.

2. Ling J, Reynolds N, Ibba M (2009) Aminoacyl-tRNA synthesis and translational quality control. Annu Rev Microbiol 63:61–78.

3. Danhart, E. M., Bakhtina, M., Cantara, W. A., Kuzmishin, A. B., Ma, X., Sanford, B. L., Košutić, M., Goto, Y., Suga, H., Nakanishi, K., et al. (2017) Conformational and chemical selection by a trans-acting editing domain. Proc. Natl. Acad. Sci. 114, 6774–6783.

Keywords: conformational selection, NMR, RNA-protein interaction