DNA-dependent RNA polymerases isolated from yeast mitochondria

Abstract

Purified preparations of yeast mitochondria yield three species of DNA-dependent RNA polymerases. These enzymes have been separated and purified to homogeneity for analysis of their properties and for comparison with the properties of nuclear preparations of yeast RNA polymerases. Three enzymes have been separated by DEAE-Sephadex chromatography of each fraction. Both nuclear and mitochondrial preparations yield three components with nearly identical elution properties. The distributions of enzyme activity on DEAE-Sephadex chromatography differ with the three nuclear peaks, being found in ratios (uncorrected for the effect of increasing salt concentration) of 8:85:7 and the mitochondrial peaks in ratios of 8:32:60 at late log phase of growth under optimized conditions in which protease inhibitors and an antioxidant were included. The type of mitochondrial enzymes in 3-day-old cells differed from those grown to late logarithmic phase. It has been established that the enzymes of the mitochondrial preparation are associated with the membrane fraction. While extraction with 0.5 m KCl solubilizes considerable enzyme activity, greatly enhanced yields of enzyme MIII are obtained by addition of the antioxidant 2,6-di- t-butyl-4-hydroxymethyl phenol during enzyme extraction. Inhibition of protease activity has also been shown to have a major effect on the yield and distribution of enzymes obtained from mitochondrial preparations. The mitochondrial preparations of yeast polymerases are generally similar but not identical to corresponding nuclear polymerases in subunit molecular weights, inhibitor sensitivities, and in DNA template dependence. Comparative studies of nuclear and mitochondrial polymerases clearly establish that differences do exist among the isolated enzymes of these classes. It has not been ruled out to date that these enzymes may be derived in part or in total from the same cytoplasmic subunit pool, nor has it been established that any of these enzymes function in mitochondria in vivo.