Multiple RNA polymerase species from rat liver tissue: Possible existence of a cytoplasmic enzyme

Abstract

DNA-dependent RNA polymerase species have been extracted and purified from rat liver tissue. Apart from the established nuclear forms of the enzyme A (or I) and B (or II), an RNA polymerase activity has been purified from the cytosol of the liver cell which has been designated tentatively as C. The observed activity requires the nucleoside triphosphates ATP, GTP, CTP, and UTP, and a DNA template which cannot be substituted by synthetic ribopolymers or RNA. The incorporation of UTP catalyzed by enzyme C is resistent to rifampicin but can be suppressed by actinomycin D, and taken together with the high molecular weight of the enzyme, it is very unlikely that it belongs to a polynucleotide phosphorylase or terminal nucleotidyl transferase activity, elongating preexisting RNA chains. It can be shown that enzyme C does not emanate from a mitochondrial source and is different from both nuclear enzymes A and B by a number of suitable criteria. Phosphocellulose chromatography differentiates enzymes A and C, and chromatography on DEAE-cellulose and hydroxylapatite columns show B and C to be clearly different. In addition, functional tests show that the enzyme found in the cytoplasm is not identical to either of the nuclear enzymes A and B. Titration curves with the specific inhibitor α-amanitin show that the enzyme obtained from the cytosol is basically sensitive to amanitin, although the required concentrations of inhibitor exceed those for the B enzyme by two to three orders of magnitude. This amanitin sensitivity of enzyme C, which is intermediate between that observed for enzymes A and B, seems to clearly differentiate these three components. This observation is substantiated by other functional tests like ionic and template dependence, the capacity of catalyze pyrophosphorolysis, and the ability of a protein factor (23) to stimulate transcription by enzymes A, B, and C.