In organello footprinting. Analysis of protein binding at regulatory regions in bovine mitochondrial DNA

Detailed study of mammalian mitochondrial transcriptional and replicative mechanisms is currently limited to in vitro analyses, requiring isolation and reassembly of functional components to mimic the in vivo process. To complement these studies and begin understanding the intracellular function of mitochondrial DNA (mtDNA) regulatory regions, we developed in organello footprinting to investigate protein-DNA interactions within the mitochondrion. Using a methylation protection technique and mitochondria isolated from bovine brain tissue, we analyzed five domains believed critical for regulating mtDNA function. Near equivalent protein-DNA interactions were seen upstream of both light and heavy strand RNA start sites at positions consistent with those observed for human mitochondrial transcription factor 1 DNA binding in vitro. Downstream of the 16 S rRNA gene, a high level of protein occupancy was detected within the bovine homologue of the human tridecamer sequence required for attenuation of transcription in vitro. Immediately upstream of the origin of heavy strand replication, a novel site of protein-mtDNA interaction was observed within conserved sequence block 1, suggesting its potential as a regulatory element involved in initiation of heavy strand DNA synthesis. Unlike regions associated with the origin of heavy strand replication, protein-mtDNA interaction was not detected at the origin of light strand replication or within flanking tRNA genes.