Poster abstracts
Poster number 36 submitted by Xiao Ma
SELECTIVITY mechanisms of aminoacyl-tRNA trans-editing factors
Xiao Ma (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Eric M.Danhart, Marina Bakhtina, Will Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Lexie Kuzminshin, Brianne Sanford (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Marija Kouti, Ronald Micura (Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University), Karin Musier-Forsyth, Kotaro Nakanishi, Mark P. Foster (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for charging cognate amino acids onto their corresponding tRNAs, which are then delivered to the ribosome by EF-Tu. The structural features of the aaRS 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 leading to mistranslation, especially for the smaller and isometric amino acids. In addition to the selectivity and proofreading capabilities provided by the synthetases, to maintain high-fidelity translation many organisms possess free-standing editing enzymes that function in trans to hydrolyze mischarged tRNAs. Caulobacter crescentus ProXp-ala, a structural homolog of the bacterial prolyl-tRNA synthetase editing domain, can deacylate mischarged Ala-tRNAPro. To determine how ProXp-ala selectively deacylates Ala-tRNAPro over the cognate Pro-tRNAPro, we performed NMR-mapping studies, site-directed mutagenesis experiments, and MD simulations of ProXp-ala bound to an uncharged microhelixPro and a non-hydrolyzable, amide-linked Ala-microhelixPro substrate analog. These studies show 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. Flexibility of helix α2 is also supported by comparison of a new crystal structure of the protein we obtained to a previously published structure. The structures are virtually identical but differ only in the helix α2 orientation. Correspondence between stereochemical properties of mischarged amino acids and the deacylation activity of ProXp-ala shed light on both the role of size and aminoacyl functional groups in the discrimination mechanism. Taken together, these data suggest that size exclusion, chemical and conformational selection contribute to specific deacylation of Ala-tRNAPro by 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. et. al (In revision)
Keywords: trans-editing, NMR, RNA-protein interaction