Mechanism Of Ef-G-Dependent Translocation In The Ribosome

University Of California-San Diego, La Jolla CA

Investigators

Abstract

One of the fundamental steps during translation is the precise and coordinated movement of the mRNA-tRNA complex within the ribosome, called translocation. The long-term goal of this research is to understand the molecular mechanism of translocation by the ribosome. Previous studies in the PI's laboratory showed that specific ribose 2'-hydroxyl groups and non-bridging phosphate oxygen atoms in the A and P site tRNAs are important for translocation. The goal of the current project is to identify 2'-hydroxyl groups and non-bridging phosphate oxygen atoms in the mRNA that are required for translocation. Additionally, the current project will directly examine the role of the universally conserved bases in 16S rRNA that interacts with the mRNA-tRNA complex in translocation. The research will use fluorescence-based pre-steady state kinetic analysis, site-directed mutagenesis of critical residues, and biochemical assays to study the mechanism of translocation. These experiments will provide information about the dynamics of ribosome structure that cannot be easily acquired by X-ray crystallography. Bacterial ribosomes are the target for inactivation by several classes of antibiotics. Antibiotics such as erythromycin, viomycin, thiostrepton and spectinomycin specifically inhibit translocation. Understanding the mechanism of translation will provide insights for developing novel antibiotics. This project also serves an important educational need by integrating research and teaching. The research will promote teaching and training in advanced biophysical methods. Several undergraduate minority students, graduate students and postdoctoral students will obtain invaluable research experience in the laboratory.