Formation of a functional mitochondrion requires the coordination of genetic information encoded in both the nuclear and mitochondrial genome. Expression of genes in both compartments must respond to different growth states of the cell. Genes encoded in the nucleus must be selectively expressed, processed and transported into the mitochondrion. The mitochondrial encoded polypeptides of multisubunit enzymes must be synthesized in concert with the polypeptide transported from the cytoplasmic ribosomes. A pivotal step in the control of the process is the synthesis of mitochondrial encoded transfer RNAs, ribosomal RNAs and messenger RNAs. In yeast (Saccharomyces cerevisiae) multiple discrete transcripts are initiated from the circular mitochondrial genome. A conserved nonanucleotide sequence appears to be a component of the transcription promoter. The yeast mitochondrial RNA polymerase which interacts with this promoter has been only partially characterized. We have preliminary evidence that the selective yeast mitochondrial RNA polymerase is multisubunit and that it has some kinetic properties similar to prokaryotic RNA polymerase. We propose to purify the enzyme responsible for selective transcription of mitochondrial DNA, to determine its polypeptide subunit composition, to establish whether the individual subunits are encoded in the nucleus or mitochondrion and to characterize its interaction with mitochondrial promoters. The promoters used will be both natural and in vitro synthesized variants. We also propose to use the purified RNA polymerase to clonally isolate the genes which encode it. Assays for the RNA polymerase polypeptides and probes from the isolated genes will be used to determine the role that the enzyme plays in regulation of yeast mitochondrial gene products. Understanding mitochondrial biogenesis and the regulation of mitochondrial gene expression is critical to a molecular description of respiration and energy production by all eukaryotic cells; the power of modern yeast molecular genetics make this understanding a feasible proposal.
Showing the most recent 10 out of 14 publications
