RNA polymerase and transcription termination factor of human mitochondria

Abstract

PART 1 The recombinant protein of the catalytic subunit of human mitochondrial RNA polymerase (h-mtRP) was overexpressed in Escherichia coli., and was purified to near homogeneity. The recombinant h-mtRP shows the same characteristics of the partially purified native enzymes from human mitochondria. The recombinant h-mtRP with recombinant h-mtTFA lacks a promoter-dependent transcription activity, pointing to another component(s) being required for promoter-dependent transcription. The promoter-specific binding of recombinant h-mtRP was observed using the electrophoretic mobility shift assay. The binding affinity of h-mtRP for LSP promoter was much greater than that for the HSP promoter, and was independent to the presence of the mtTFA. The binding of mtTFA to the promoters was also not dependent on the presence of the h-mtRP. The tertiary complexes containing both the mtTFA and h-mtRP bound to promoter sequences were observed. The h-mtRP is shown to possess the novel ability to direct robust but promiscuous transcription with mtTFA on duplex DNA templates with 3’-adenosine protruding ends. Analysis of reaction products indicates that h-mtRP, only with mtTFA, is capable of extending the 3’-termini of duplex DNA having 3’-adenosine-protruding DNA ends by ribonucleotides addition. Based on these findings, it is proposed that h-mtTFA activates the h-mtRP by helping to add the following RNA chains to the 3’-adenosine-terminus of the nascent RNA chain. The missing component for the promoter-directed transcription would be a protein to facilitate the h-mtRP to melt the DNA and to start the initial RNA synthesis. PART2 Transcription termination of human mitochondrial genome requires the action of a site-specific DNA-binding protein that binds to a single location of the entire human mitochondrial genome. A specific DNA-binding protein (mTERF) for the termination sequence was recently been identified and cloned. To study the functional characteristi...