The overall objective of the proposed research is to examine the mechanism of protein biosynthesis in animal mitochondria and to compare this system to those of prokaryotes and the eukaryotic cell cytoplasm. The first major focus of our work will be to study the steps that occur during the elongation cycle in bovine mitochondria. The translation elongation factor required to promote the binding of aminoacyl-tRNA to mitochondrial ribosomes has recently purified in this laboratory. This factor (EF-Tu/mt) is present in a very tight complex with its recycling factor (EF-Ts/mt). The properties of this complex (EF-Tu-Ts/mt) will be examined and the details of the elongation cycle in animal mitochondria will be investigated. The basis for the tight interaction between EF-Tu/mt and EF-Ts/mt will be studied. In addition, the genes for EF-Tu/mt and EF-Ts., will be cloned, sequenced and attempts will be made to over-express these proteins in E. coli providing the material needed for detailed structure/function studies. The second major focus of the proposed work will be to investigate the process of chain initiation in the mitochondrial system. The first animal mitochondrial translational initiation factor has recently been purified in our laboratory. This factor (IF-2mt) is responsible for binding the initiator tRNA to ribosomes. Partial amino acid sequence for IF-2mt will be obtained and used for the isolation of a cDNA clone of for this factor. The gene for IF-2mt will be sequenced and attempts will be made to over-express this factor in E. coli. The properties of IF-2mt will be studied and its mechanism of action investigated. Our longterm goal is develop an understanding of the mechanism of mitochondrial translation and its integration into the complex metabolism of the animal cell.
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