Mechanism of initiation of yeast mitochondrial DNA replication: Role of RNA polymerase

Yeast mitochondrial RNA polymerase was purified and resolved into 2 distinct fractions. Peak A was found to be nonspecific and exhibited characteristics of the core polymerase, whereas peak B exhibited characteristics of the holoenzyme.In vitro replication assays were carried out, using the peak B enzyme, the clonedori sequences and other DNA templates. It was found thatori 2 was the most efficient template for RNA polymerase primed DNA synthesis, as compared to all the other templates studied.     

Specificity factor of yeast mitochondrial RNA polymerase. Purification and interaction with core RNA polymerase

We have identified a mitochondrial protein from Saccharomyces cerevisiae which confers the ability to recognize mitochondrial promoters onto a nonspecifically transcribing mitochondrial core RNA polymerase and we have purified this specificity factor 10,700-fold from a whole cell extract. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fraction followed by elution and renaturation of protein activity shows that the specificity factor is a 43-kDa polypeptide which directs mitochondrial core RNA polymerase to promoters belonging to rRNA-, tRNA-, and protein-encoding genes, as well as to mitochondrial replication origins. Gel filtration and glycerol gradient sedimentation studies indicate that the specificity factor shows little association with core RNA polymerase in the absence of DNA, and that it behaves like a monomeric 43-kDa protein.