The generation.of energy in mammalian mitochondria is accomplished by a series of oligomeric complexes in the inner membrane. The synthesis and assembly of these complexes requires genetic information contained in both the nuclear and mitochondrial genomes. Limited information is available on the mechanism by which the mitochondrially-encoded components in these complexes are synthesized. The overall objective of this research is to investigate the mechanism of protein biosynthesis in mammalian mitochondria. The first objective of is focused on polypeptide chain initiation and contains three parts: (1) An examination of the properties and mechanism of action of the factor (initiation factor 2, IF-2mt) which promotes the binding of the initiator tRNA to the ribosome. We will examine the domain organization of IF-2mt, its interaction with the mitochondrial ribosome and its ability to use the unusual tRNAMet in mitochondria that must serve both as the initiator and as an elongator tRNA. (2) Properties and mechanism of action of a newly discovered mammalian mitochondrial initiation factor distantly related to prokaryotic initiation factor 3. The properties of this factor including its domain organization, interaction with mitochondrial ribosomes and its role in proofreading the AUG and AUA codons used for methionine in mammalian mitochondria will be examined. (3) Investigation into the role of additional factors in the formation of the initiation complex in mitochondria. The second major objective of this application focuses on the properties of the translation elongation factor (EF-Tumt) that promotes the binding of aminoacyl-tRNA to the A-site of the ribosome. A collaborative project has been established with the laboratory of Dr. Jens Nyborg (Univ. Aarhus, Denmark) to determine the structures of the ternary complex (EF-Tumt.GTP.aa-tRNA), of the complex formed between EF-Tumt and its guanine nucleotide exchange factor EF-Tsmt and of the active (GTP-form) of EF-Tumt. EF-Tumt is able to promote the binding of the structurally unusual mitochondrial aa-tRNAs to the A-site of the ribosome. The regions of EF-Tumt that allow it to use mitochondrial aa-tRNAs will be examined using site-directed mutagenesis.
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