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 and assembled into the large oligomeric complexes in the inner membrane. The overall objective of this research is to investigate the mechanism of protein biosynthesis in mammalian mitochondria and its integration into the macromolecular metabolism of this organelle. The first objective is focused on polypeptide chain initiation and is designed to provide an understanding of the roles and mechanism of action of two translational initiation factors required for protein biosynthesis in mammalian mitochondria. Studies will be carried out to examine their binding sites on ribosomes and to investigate their interactions with other components of the protein biosynthetic machinery. In addition, the secondary structure of the RNA near the start sites in mitochondrial mRNAs will be probed. The second major objective 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. Soren Thirup (Univ. Aarhus, Denmark) to determine the structures of the ternary complex (EF-Tumt:GTP:aa-tRNA) and several other components of the translational cycle in mammalian mitochondria. These structural insights will lay the foundation for understanding the properties of the unusual tRNAs found in mammalian mitochondria. The third major aim of this project is to examine the interaction of the mitochondrial ribosome with the inner membrane. All of the products of mammalian mitochondrial protein biosynthesis are hydrophobic membrane proteins, which are thought to be synthesized while the ribosome is associated with the inner membrane. The interaction of the ribosome with the transmembrane protein Oxal will be studied and the parameters governing the binding of the ribosome to the inner membrane will be investigated. Well over 100 disease states in humans are associated with alterations in mitochondrial function and gene expression. Understanding protein synthesis in this organelle is essential for developing strategies to combat these diseases.
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