Poster abstracts

Poster number 64 submitted by Daya Kharel

Mimicking deleterious mutations in helix 69 rRNA to understand the consequences on stability and structure

Daya Kharel (Department of Chemistry, Wayne State University), Jun Jiang (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
The bacterial ribosome is the target for a number of antibacterial drugs; however, drug resistance in bacteria has increased significantly in recent years. Therefore, novel methods for overcoming the resistance problem are needed. Towards understanding more clearly the function of individual ribosome components in protein synthesis, in vivo genetic studies on a region called helix 69 (H69) were carried out.1 Mutants A1912G, A1917G, and A1919G strongly inhibited bacterial growth and caused inefficient in vitro translation. Residues A1912 and A1917 of H69 were shown to be essential for ribosome function. In this study, in vitro biophysical studies on H69 were carried out in which the relevant nucleotides from the genetic studies were replaced. The goal was to investigate the effects of functionally deleterious mutants on H69 structure, and to understand how these particular nucleotides play roles to either stabilizing or destabilizing H69. The stabilizing effects as well as pH dependence of the structures of mutant H69 RNAs (A1912G, A1918G, and Ψ1917C) were compared to wild-type H69 (a construct that contains three pseudouridine modifications at positions 1911, 1915, and 1917) by using thermal melting analysis and circular dichroism spectroscopy. The results to be presented correlate with the in vivo genetic studies. Our long-term goal is to use these results in combination with drug-binding studies to better understand drug resistance, and to develop improved compounds that take advantage of the genetic information.

References:
1. Liiv, A.; Karitkina, D.; Maivali, U.; Remme, J., Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis. BMC Mol. Biol. 2005, 6, 18.

2. Hirabayashi, N.; Sato, N. S.; Suzuki, T., Conserved loop sequence of helix 69 in Escherichia coli 23 S rRNA is involved in A-site tRNA binding and translational fidelity. The J. Biol. Chem. 2006, 281, 17203.

Keywords: Ribosome, Helix 69