The objectives of this application continue to be an understanding of the biogenesis, regulation and function of the complexes of the respiratory chain in the mitochondria of the yeast Saccharomyces cerevisiae. As in the past, special emphasis will be placed on the role of mitochondrial genes, their expression and products in these processes. To this end the following specific aims will be pursued: 1. A study of the structure, origin and mode of action of a protein implicated in the processing of the transcripts of the mitochondrial genes for both, apocytochrome b (cob) and subunit I of cytochrome oxidase (oxi3). This will be accomplished by a combination of genetic and biochemical (protein purification and immunochemical) techniques. 2. A test of the function of this protein by complementing with it suitable mitochondrial preparations of trans-recessive mutants to render them competent for splicing, by demonstrating conversion of precursor to mature mRNA, and the ability to synthesize cytochrome b and SUI of cytochrome oxidase. We also intend to use the antibody described above for the isolation of the hypothetical splicing complex and examine its composition and other functions. 3. An extension of these studies and the model to oxi3 by the isolation and characterization of cis-dominant, as well as trans-recessive mutants in introns 1, 2 and 3 of this gene. Following the procedures developed with cob, the nature and consequence of the mutational lesions will be explored by establishing a) the nature of the resulting transcriptional blocks; b) the nature, relationship and possible function in RNA processing of novel polypeptides accumulating in some of these mutants; c) sequencing the appropriate segment of the DNA of representative examples of the various mutant classes; d) the possibility of demonstrating requirements for the splicing reaction in vitro. 4. An inquiry into the function of cytochrome b in influencing the nature, properties and kinetics of assembly of Complex III of wild-type vs. cells containing modified forms of the cytochrome due to specific, known mutations in the cob gene (large deletions, truncated forms of apocytochrome b of any desired chain length, and missense mutants at different positions of the polypeptide chain).
| Shaw, L C; Lewin, A S (1997) The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria. Nucleic Acids Res 25:1597-604 |
| Shaw, L C; Thomas Jr, J; Lewin, A S (1996) The Cbp2 protein suppresses splice site mutations in a group I intron. Nucleic Acids Res 24:3415-23 |
| Shaw, L C; Lewin, A S (1995) Protein-induced folding of a group I intron in cytochrome b pre-mRNA. J Biol Chem 270:21552-62 |
| Ritchings, B W; Lewin, A S (1992) Mutational evidence for competition between the P1 and the P10 helices of a mitochondrial group I intron. Nucleic Acids Res 20:2349-53 |
| Partono, S; Lewin, A S (1988) Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria. Mol Cell Biol 8:2562-71 |
