Extraction and Purification of DNA-Dependent RNA Polymerase from Rat Liver Nuclei

The enzyme RNA polymerase is difficult to extract from most mammalian cells due to its firm association with chromatin. By a combination of intensive ultrasonic treatment and extraction of the nuclei with 0.75 M NaCl at 0° and pH 8.4 for 1.5 hours approximately 80% of the enzyme can be extracted in a soluble, DNA-dependent form. The extracted enzyme has been purified by DEAE-cellulose chromatography and centrifugation on sucrose gradients (5–20%, w/v) containing 10% glycerol. A purification of approximately 250-fold has been achieved in the peak fraction of the gradient. The enzyme requires all four nucleoside triphosphates, mercaptoethanol, a DNA template and either Mg++ or Mn++, the latter being the preferred ion. Enzyme activity can be stimulated by (NH4)2SO4. No direct stimulation of enzyme activity by cortisol phosphate could be observed.