Poster abstracts
Poster number 33 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), Karin Musier-Forsyth, Kotaro Nakanishi, Mark P. Foster (Department of Chemistry, Graduate School of Science, The University of Tokyo)
Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for charging amino acids onto their cognate tRNAs. The structural features of the aaRSs catalytic domains 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 to maintain high fidelity translation. ProXp-ala, a structural homolog of the editing domain from ProRS, can deacylate the mischarged tRNA, Ala-tRNAPro. Foster and Musier-Forsyth groups collaborated and have provided evidence that 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. To validate these hypotheses we plan to use X-ray crystallography, NMR spectroscopy and enzymatic assays to: (1) Determine the high-resolution three-dimensional structure of ProXp-ala in complex with tRNAPro to detail recognition between ProXp-ala and the acceptor stem of tRNAPro. (2) Determine the structure of ProXp-ala bound to a non-hydrolyzable Ala-tRNAPro analog in order to reveal the mechanism of Ala/Pro discrimination. (3) We will perform deacylation experiments using native and model substrates to test the catalytic role played by the 2'OH of the terminal 76A in tRNAPro. The high-resolution structures and deacylation assays will facilitate our understanding of both substrate recognition and editing mechanism of ProXp-ala.
References:
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: trans-editing, NMR, RNA-protein interaction