The principal goal of this grant proposal is to obtain information about the control of transcription of mitochondrial DNA. The purpose is to gain better insight to the mechanisms that control mitochondrial biogenesis in both the lower and higher eukaryotes. The work is particularly relevant to cardiac tissue in which metabolism is almost completely dependent upon the aerobic synthesis of ATP generated by mitochondria.
Our specific aims are to identify the transcriptional initiation sites of the yeast mitochondrial genes and to sequence these regions to gain information about possible controlling elements. We wil identify mitochondrial transcriptional initiation sites in grande and petite yeast by using the guanylyltransferase reaction, and map initiation sites by hybridiation to restriction enzyme digests of mtDNA. The extent of processing of the 5' terminus will be determined. The 5' flanking regions of different mitochondrial genes will be sequenced to identify homologies which may be essential for regulation of transcription. We will also develop an in vitro transcription system with mitochondrial DNA and mitochondrial RNA polymerase to characterize factors which regulate transcription. Furthermore, we will isolate mutants of mitochondrial RNA polymerase and of other factors participating in the regulation of mitochondrial transcription. We will also examine the biosynthesis of mitochondrial RNA polymerase, the regulation of its transcription, and its role in the control of mitochondrial development. Gene sequences for this enzyme will be isolated and used to evaluate transcription as related to derepression and repression of mitochondrial development.
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