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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026832-12
Application #
3274278
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1979-07-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
12
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Veldman, S; Rao, S; Bodley, J W (1994) Differential transcription of the two Saccharomyces cerevisiae genes encoding elongation factor 2. Gene 148:143-7
Phan, L D; Perentesis, J P; Bodley, J W (1993) Saccharomyces cerevisiae elongation factor 2. Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin. J Biol Chem 268:8665-8
Perentesis, J P; Phan, L D; Gleason, W B et al. (1992) Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling. J Biol Chem 267:1190-7
Donovan, M G; Veldman, S A; Bodley, J W (1992) A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants. Yeast 8:629-33
Perentesis, J P; Miller, S P; Bodley, J W (1992) Protein toxin inhibitors of protein synthesis. Biofactors 3:173-84
Donovan, M G; Bodley, J W (1991) Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endogenous kinase. FEBS Lett 291:303-6
Miller, S P; Bodley, J W (1991) Alpha-sarcin cleavage of ribosomal RNA is inhibited by the binding of elongation factor G or thiostrepton to the ribosome. Nucleic Acids Res 19:1657-60
Miller, S P; Bodley, J W (1988) Alpha-sarcin cleaves ribosomal RNA at the alpha-sarcin site in the absence of ribosomal proteins. Biochem Biophys Res Commun 154:404-10
Chen, J Y; Bodley, J W (1988) Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase. J Biol Chem 263:11692-6
Miller, S P; Bodley, J W (1988) The ribosomes of Aspergillus giganteus are sensitive to the cytotoxic action of alpha-sarcin. FEBS Lett 229:388-90

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