Mechanism of EF-G-Dependent Translocation in the Ribosome

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0078322JosephOne of the fundamental steps in the elongation cycle of protein synthesis is the iterative movement of the tRNA-mRNA complex in 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. Ribosomes are composed of RNA and proteins. Results from phylogenetic sequence comparison, genetic studies and biochemical experiments indicate a functional role for ribosomal RNAs in translation. The specific goals of this proposal are to identify functional groups within tRNAs in the A-site and P-site that are required for translocation.Previous research in the PI's laboratory has shown that ribosomes containing a full-length tRNA bound to the P-site and an analog of the anticodon stem-loop of tRNA bound to the ribosomal A-site are translocated. This minimal analog of tRNA will be used to identify essential ribose 2'-hydroxyls and non-bridging phosphate oxygens required for translocation from the A-site. Other experiments from the PI's laboratory indicate that tRNAs with a break in the phosphodiester backbone are translocated from the P-site. Specific modifications within the smaller fragments of the tRNA can be incorporated by chemical synthesis. This presents a unique opportunity to study the role of specific functional groups within P-site tRNA that are required for translocation. Biochemical methods such as toeprinting and chemical probing will be used in addition to functional group substitution studies to identify interactions that are required for the movement of the tRNAs in the ribosome. These studies addressing functional interactions between tRNAs and the ribosome will be especially important once high-resolution crystal structures of the ribosome become available.