Isolation and Characterization of DNA-Dependent RNA Polymerase I of Tetrahymena pyriformis

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Abstract
A procedure for the separation and purification of DNA-dependent RNA polymerases [EC 2.7.7.6] from macronuclei of Tetrahymena pyriformis is described. We have used it to isolate and characterize the class I enzyme. RNA polymerase I was identified by its resistance against α-amanitin and its location in nucleoli. The purified enzyme consists of at least 12 major subunits with approximate molecular weights of 180,000, 118,000, 37,500, 36,000, 29,000, 27,500, 20,000, 18,500, 15,600, 14,500, 13,500, and 12,600. Chromatography on DEAE-Sephadex separated two forms of RNA polymerase I which differed in the presence of an additional polypeptide of 25 kDa. Independently of this polypeptide, the enzyme was found to segregate on DNA cellulose into a binding and a non-binding fraction. This type of heterogeneity was found to be unrelated to differences in molar ratios or molecular weights of the enzyme subunits. The catalytic properties of all enzyme subfractions were very similar and complied with the general characteristics of RNA polymerase I [cf. Roeder, R.G. (1976) in RNA Polymerase (Losick, R. & Chamberlin, M., eds.) pp. 285–329, Cold Spring Harbor Pub1. Co., New York].