Poster abstracts

Poster number 23 submitted by Eric Danhart

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

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:
During translation, aminoacyl-tRNA synthetases are responsible for covalently attaching amino acids to cognate tRNAs. Mistakes in this process lead to errors in protein synthesis, and accumulation of such errors can be deleterious to cells. Prolyl-tRNA synthetase (ProRS) mischarges tRNA^Pro with alanine and cysteine. Proline residues, due to their unique structural properties, are usually highly conserved in protein sequences; thus, errors in translation of Pro codons are not well-tolerated. To ensure high fidelity of this key step in protein synthesis, multiple proofreading mechanisms exist. Most bacterial ProRSs possess a cis-editing domain (INS) to hydrolyze Ala-tRNA^Pro. Cys-tRNA^Pro and Ala-tRNA^Pro in some organisms, however, must be cleared by freestanding, trans-editing domains. These proteins, the INS superfamily, are responsible for hydrolyzing misacylated tRNA^Pro in all three domains of life. The bacterial protein ProXp-ala is responsible for the deacylation of Ala-tRNA^Pro, but the structure and mechanism of this enzyme remains unknown. Mutagenesis studies have identified acceptor stem elements of tRNA^Pro that are critical for ProXp-ala activity. Due to this localized specificity, a microhelix that mimics the acceptor stem of tRNA^Pro was designed, charged with Ala, and found to be a substrate for ProXp-ala. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze chemical shift perturbations and map the interaction surface for this microhelix^Pro on ProXp-ala. Additionally, a hydrolysis-resistant, charged Ala-microhelix^Pro analog is allowing us to determine regions on the protein that specifically involved in alanine recognition. These results provide structural insights into the interaction between ProXp-ala and mischarged tRNA^Pro and thus allow us to make hypotheses regarding the mechanism of error recognition by other trans-editing enzymes.

Keywords: NMR, tRNA, protein-RNA interaction