Transcription of supercoiled mitochondrial DNA by bacterial RNA polymerase.

Abstract

Mitochondrial DNA isolated from cysts of Artemia salina (brine shrimp) was found to have a closed‐circular structure 5.1 μm long and a buoyant density of 1.697 g/cm3. Escherichia coli RNA polymerase can transcribe this DNA. RNA synthesis is greatly stimulated by the σ subunit and is influenced by the presence of KCl. After formation of an initial complex between the enzyme and the mitochondrial DNA only a limited synthesis of RNA took place in the presence of rifampicin.The products obtained with holo enzyme and core enzyme in the presence of excess nucleoside triphosphates have a mean molecular weight of 1.5 × 106 and greater than 3 × 106 respectively, as determined by acrylamide‐agarose gel electrophoresis. Addition of the termination factor ϱ to the reaction mixture reduced the molecular weight of the products to 1.0 × 106 at a KCl concentration of 0.05 M but not at 0.1 M KCl.Under limiting UTP concentrations, a few discrete RNA bands of shorter size (about 8 × 105, 105 and 3 × 104 mol. wt) are synthesized. Addition of ϱ factor to the reaction mixture caused the disappearance of the higher‐molecular‐weight band (8 × 105) while the other RNA bands remained.Little self‐annealing was observed upon heating the [3H]RNA synthesized in vitro at 70 °C. On the other hand, when the [3H]RNA was heated in the presence of unlabeled mitochondrial rRNA, about 54% of the labeled product was resistant to degradation by a mixture of T1 and pancreatic RNase. In hybridization‐competition experiments, annealing of the [3H]RNA synthesized in vitro was reduced by 65% upon addition of unlabeled mitochondrial rRNA. RNA‐RNA annealing is thought to be the main cause for this competition reaction since similar results were obtained by simultaneous or step‐wise hybridization procedures. It is concluded that transcription in vitro of closed circular‐mitochondrial DNA from Artemia by E. coli RNA polymerase proceeds mainly on the anti‐rRNA strand.