Poster abstracts
Poster number 88 submitted by Xiao Ma
STRUCTURAL basis for selectivity by 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 Kuzmishin, Brianne Sanford (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Ziwei Liu, Bradley C. Howard (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, 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 on their corresponding tRNAs, which are then shuttled to the ribosome by EF-Tu so that the amino acid can be incorporated into the elongating peptide chain during protein translation. The structural features of the aaRSs catalytic domains provide a high degree of selectivity for the correct amino acid; however, given the similar chemical properties of many of the amino acids, errors in tRNA charging can occur, thus leading to the mistranslation of the genetic code. In addition to the selectivity provided by the synthetases, in many organisms, there are editing enzymes that function in trans to hydrolyze mischarged tRNAs to maintain the high fidelity of translation. ProXp-ala, a homolog of the editing domain from ProRS, can deacylate the mischarged tRNA, Ala-tRNAPro. To determine how ProXp-ala is selective for Ala-tRNAPro and doesn’t deacylate Pro-tRNAPro or Ala-tRNAAla, NMR and site-directed mutagenesis mapping are performed with uncharged microhelixPro and a non-hydrolyzable, amide-linked Ala-microhelixPro substrate analog. Relaxation experiments show that an α-helix, exhibiting dynamics on the ps-ns timescale, is less mobile with Ala-microhelixPro, but remains dynamics when bound to the uncharged microhelixPro. We propose that conformational selection by ProXp-ala allows substrate sampling and Ala-tRNAPro-specific induced-fit binding via a flexible helix. Another ProRS editing enzyme, Yeak, has been shown in vitro to deacylate multiple mischarged tRNAs (Ser-tRNAAla/Lys/Thr and Thr-tRNALys/Ile/Val). To identify the interaction between Yeak and its multiple substrates, we are using X-ray crystallography and NMR to identify the structural basis for broad selectivity of this generalist enzyme.
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.Liu Z., Vargas-Rodriguez O., Goto Y., Novoa, E., Pouplana L., Suga H., Musier-Forsyth K., (2015). Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation. PNAS May 12, 2015 vol. 112 no. 196027-6032
Keywords: trans-editing, NMR, RNA-protein interaction