Mechanism of EF-G-Dependent Translocation In The Ribosome

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Ribosomes carry out the complex task of protein synthesis in all organisms. A fundamental step during protein biosynthesis is the precise and coordinated movement of the tRNA-mRNA complex within the ribosome, called translocation. The long-term objective of this research is to understand the mechanism of translocation of tRNAs from one site to the next within the E.coli ribosome. The specific goal of this project is to determine the role of two well-characterized base pairs between 23S rRNA and P and A site tRNAs in translocation. The first base pair is between the universally conserved bases C74 of P site tRNA and G2252 of 23S rRNA. The second base pair interaction is between universally conserved bases C75 of A site tRNA and G2553 of 23S rRNA. The effect of disrupting these base pairs on the kinetics of translocation will be examined using a rapid kinetic method recently developed in the PI's laboratory. In addition,methods such as site-directed mutagenesis, toeprinting, and chemical probing will be used to characterize the role of these universally conserved base pairs in translocation. 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 functional role of specific interactions within the ribosome is critical for elucidating the molecular mechanism of translation and will provide insights for developing novel antibiotics. This research also serves an important educational need by integrating research and education. Several undergraduate minority students will obtain invaluable research experience in the laboratory.