Poster abstracts
Poster number 79 submitted by Xiao Ma
Selectivity mechanisms of 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 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 mischarged Ala-tRNAPro. To determine how ProXp-ala selectively deacylates Ala-tRNAPro over the cognate Pro-tRNAPro, or Ala-tRNAAla, we performed NMR, site-directed mutagenesis mapping and MD simulations of ProXp-ala with an uncharged microhelixPro and a non-hydrolyzable, amide-linked Ala-microhelixPro substrate analog. Relaxation experiments showed that helix α2, which exhibits dynamics on the ps-ns timescale, is less mobile when bound to Ala-microhelixPro, but remains dynamic when bound to the uncharged microhelixPro. Relaxation dispersion experiments also reveal that this helix is dynamic on the μs-ms timescale. Flexibility of helix α2 is also supported by a new crystal structure (PDB: 5VXB) of the protein obtained in a different crystallographic space group than a previously published structure (PDB: 1VJF); the structures differ only at in helix α2. Correspondence between stereochemical properties of amino acids and the deacylation activity of ProXp-ala sheds light on both the role of size and aminoacyl functional groups in the discrimination mechanism. These suggest that chemical selection, size exclusion and conformational selection contribute to allow ProXp-ala to conduct specific deacylation of Ala-tRNAPro. We expect the high-resolution structure of ProXp-ala in complex with its substrate analogue to further illuminate its mechanism.
References:
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, trans-editing