Poster abstracts

Poster number 14 submitted by Eric Danhart

Structural studies to define a novel ternary complex required for tRNA-Pro editing

Eric M. Danhart (Chemistry and Biochemistry, The Ohio State University), Brianne Sanford (Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Chemistry and Biochemistry, The Ohio State University)

Abstract:
Aminoacyl tRNA synthetases are responsible for covalently attaching amino acids to cognate tRNAs though a two-step aminoacylation reaction. Mistakes in this process lead to errors in protein synthesis, and accumulation of such errors can be deleterious to cells. Multiple proofreading mechanisms exist to ensure high fidelity of this key step in protein synthesis. Prolyl-tRNA synthetase (ProRS) can mischarge tRNA^Pro with alanine and cysteine. Bacterial ProRSs possess a cis-editing domain (INS) to hydrolyze Ala-tRNA^Pro. In contrast, Cys-tRNA^Pro is cleared in trans by a freestanding INS homolog, YbaK. In vivo experiments have shown that editing by YbaK is carried out via a novel YbaK:ProRS:tRNA^Pro ternary complex. To help elucidate the biological role of this interaction, computational modeling approaches were used to generate a model of this ternary complex. Although crosslinking data was used to guide the modeling, more restraints will allow the generation of a higher confidence, more refined model. Using Nuclear Magnetic Resonance (NMR) with different combinations of the binding partners, it is possible to determine specific residues involved in the binding interactions within the complex. After optimizing conditions for multi-dimensional NMR studies of YbaK, experiments were performed in conjunction with both full-length tRNA^Pro and with a non-hydrolyzable substrate analog to map their respective binding sites. Chemical shift perturbations were observed for each, and efforts are underway to assign YbaK spectra to determine which residues are involved in these interactions. These experiments and other structural studies will allow me to better define the ternary complex interface, leading to the development of a higher confidence model.

Keywords: NMR, tRNA, protein