In vitro analysis of Physarum polycephalum mitochondrial RNA polymerase

Gene expression in the mitochondria of Physarum polycephalum requires an additional step outside of the Central Dogma of molecular biology termed RNA editing in which non-templated nucleotides are added to RNAs making their sequence different from that of the DNA that coded for them. RNA editing in Physarum has been shown to be closely associated with transcription of these genes as newly synthesized RNAs are the substrate for RNA editing. Owing to its close association with the process of transcription of mitochondrial genes, the process of RNA editing is thought to be cotranscriptional where the non-templated nucleotide is added to the 3' end of the RNA as it is being synthesized from the DNA template. Because it is intimately associated with transcription, it is thought that potentially the RNA polymerase itself, or a factor closely associated with the RNA polymerase may be performing the addition of the non-templated nucleotide. To address whether the mitochondrial RNA polymerase from Physarum is in fact involved in the RNA editing process, the cDNA for the mitochondrial RNA polymerase was cloned and overexpressed in E. coli. Initial characterization of the mitochondrial RNA polymerase was performed using the recombinant mtRNAP, as well as a partially purified mitochondrial cleared lysate containing polymerase activity, in in vitro studies (Chapter 2). Further characterization of the template requirements of the mitochondrial RNA polymerase led to an interesting observation (Chapter 3) whereby the mitochondrial RNA polymerase is capable of adding non-DNA templated nucleotides to the 3' ends of single stranded nucleic acid templates, a process that closely resembles the proposed mechanism for RNA editing in Physarum mitochondria. Extension of analyses of transcription using various DNA templates that were supercoiled (and therefore had no free 3' ends) led to the observation of a potential RNA:DNA hybrid that was being created by the mitochondrial RNA polymerase (Chapter 4). This RNA:DNA hybrid is proposed to serve as a method for determining the location and identity of editing sites, potentially by the mitochondrial RNA polymerase, as it is transcribing the DNA template. Taken together, these data provide new information with respect to transcription in the mitochondria of Physarum polycephalum, as well as lend insights into the mechanism of RNA editing.