The process of protein biosynthesis can be subdivided into three steps: initiation, elongation and termination. Translocation, the movement of the ribosome along mRNA, is one of two reactions during the elongation step which requires a non-ribosomal factor and the hydrolysis of GTP. The translocation factor, found in all living cells, is known as EF-2 in eucaryotes and EF-G in procaryotes. Our long-term goal is to determine the mechanism of the translocation reaction during protein synthesis elongation. This proposal focuses on the structure and function of EF-22 in the yeast Saccharomyces cerevisiae. A major goal of this proposal is to map the functional domains of EF-2 in addition to the methods of protein chemistry, we will make extensive use of recombinant DNA methodology. We will clone and sequence the gene or genes which encode EF-2. The structure and functions of this protein will also be probed using both random and site-directed mutagenesis. In addition, we will evaluate the potential for crystallization of EF-2 with a long-term goal of determining the crystal structure of this protein by X-ray diffraction analysis. In addition to characterization of the structure of EF-2 we will determine whether this protein is subject to covalent regulation in yeast. For example, yeast EF-2 may be regulated by phosphorylation, as it is in mammalian systems. A variety of other potential regulatory mechanisms will also be evaluated. If yeast EF-2 is subject to covalent regulation, we will initiate studies of the characteristics and roles of these regulatory processes.
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