Altered characteristics of mammalian RNA polymerase following solubilization from nuclei

Unlike the bacterial enzyme, mammalian RNA polymerase is usually assayed as an "aggregate enzyme" preparation consisting of the polymerase tightly bound to its deoxyribonucleoprotein template. In consequence changes in enzyme activity cannot be distinguished from alterations in available template in the aggregate. To overcome this difficulty, attempts have been made to release polymerase free of DNA from chicken embryo (Furth and Loh, 1963), bovine lymphosarcoma (Furth and Ho, 1965), rat testes (Ballard and Williams-Ashman, 1966) and from isolated rat liver nuclei (Ramuz et al., 1965; Cunningham and Steiner, 1967; Seifert and Sekeris, 1967) followed by measurement of the activity of the isolated enzyme using purified DNA as template. None of these authors obtained total release of the enzyme and in most cases it is evident that only a small proportion of the activity was solubilized. Since polymerase activity occurs both in Mg²⁺- and Mn²⁺-dependent forms in whole nuclei (Widnell and Tata, 1964) and in nucleoli (Jacob et al., 1968), extraction of only a fraction of the total activity does not allow meaningful conclusions to be drawn about the quantities of each type of activity present in intact nuclei. In the present communication, we report a procedure for release of more than 80% of the total polymerase activity from rat liver nuclei.