The overall objective of the proposed research is to investigate the mechanism of protein biosynthesis in mammalian mitochondria. The first major focus of our research will be to study the translational elongation factor complex (EF-Tu Tsmt) in detail. EF-TUmt is responsible for promoting the binding of aminoacyl- tRNA to the A-site of the ribosome. It is present in a tight complex with its recycling factor (EF-Tsmt). This complex is tightly associated and, unlike the corresponding complex in Escherichia coli, it is not readily dissociated by either GDP or GTP. The cDNAs for both EF-Tumt and EF-Tsmt have been cloned and expressed in E. Coli. The expressed factors will be used to develop a detailed model for the elongation cycle in mammalian mitochondria. The regions of EF-Tumt and EF-Tsmt that are involved in the interaction between these two proteins will be probed by examining the properties of mutated derivatives of EF-Tumt is able to promote the binding of the structurally unusual mitochondrial aminoacyl-tRNAs to the A-site of the ribosome. In contrast, although E. Coli EF-Tu can form a ternary complex with several mitochondrial aminoacyl-tRNAs, it is unable to promote their binding to the a-site of the ribosome. The regions of EF- Tumt that allow it to use mitochondrial aminoacyl-tRNAs will be examined using chimeric proteins and site-directed mutagenesis. The second major goal of this application is to purify and characterize a new mammalian mitochondrial initiation factor that promotes initiation complex formation in the presence of the mRNA for cytochrome oxidase subunit II. Our long-term goal is to develop an understanding of the mechanism of protein synthesis in mammalian mitochondria and its integration into the complex metabolism of the eukaryotic cell.
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