2013 Rustbelt RNA Meeting
RRM

 

Home

Registration

Agenda

Abstracts

Directions

Poster abstracts

Poster number 26 submitted by Eric Danhart

Biophysical characterization of a trans-editing complex that mediates high-fidelity charging of tRNAPro

Eric M. Danhart (Department of Chemistry and Biochemistry, The Ohio State University), Brianne Sanford (Department of Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of 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 tRNAPro with alanine and cysteine. The YbaK superfamily of proteins is responsible for hydrolyzing misacylated tRNAPro in all three domains of life. Some bacterial ProRSs possess a cis-editing domain (INS) to hydrolyze Ala-tRNAPro. Cys-tRNAPro and Ala-tRNAPro in some organisms, however, must be cleared by freestanding, trans-editing domains. The bacterial protein ProXp-Ala is responsible for the deacylation of Ala-tRNAPro, yet little is known about their interaction. Mutagenesis studies have identified acceptor stem elements of tRNAPro that are critical for ProXp-Ala activity. Due to this localized specificity, a microhelix that mimics the acceptor stem of tRNAPro was designed and found to be a substrate for ProXp-Ala. No such binding information is available for the protein, however. With the use of Nuclear Magnetic Resonance (NMR) spectroscopy, it is possible to identify specific residues of ProXp-Ala involved in the binding interaction with tRNAPro. After optimizing conditions for multi-dimensional NMR studies of ProXp-Ala, experiments were performed in conjunction with both full-length tRNAPro and the microhelix to map their respective binding sites. Chemical shift perturbations were observed for each, and efforts are underway to assign ProXp-Ala spectra to determine which residues are involved in these interactions. Additionally, Isothermal Titration Calorimetry (ITC) has been performed with both full-length tRNAPro and microhelix to determine the thermodynamics of their interaction, most notably binding affinity and stoichiometry. These experiments and other structural studies will allow me to better characterize the editing complex formed by ProXp-Ala and tRNAPro.

Keywords: tRNA, NMR, ITC