Purification of RNA polymerase IIO from calf thymus.

Abstract

Three subspecies of RNA polymerase II, designated IIO, IIA, and IIB, have been described in calf thymus and shown to differ in the apparent molecular weight of their largest subunits, designated IIo, IIa, and IIb, respectively. The objective of this study was to develop a procedure for the purification of RNA polymerase IIO. This form of the enzyme predominates in vivo and is responsible for the transcription of most cellular genes. RNA polymerase II is solubilized from isolated calf thymus nuclei in the presence of high concentrations of chelators, precipitated with polyethyleneimine, extracted with salt, and precipitated with (NH4)2SO4. The solubilized enzyme is resolved from factors that destabilize RNA polymerase IIO by chromatography on heparin-Sepharose CL-4B and DE52. RNA polymerase IIO is then partially resolved from RNA polymerases IIA and IIB by chromatography on DEAE-5PW and further purified by chromatography on Phenyl-Superose and Mono Q. RNA polymerase IIO was purified 1000-fold from the polyethyleneimine eluate resulting in about 130 micrograms of RNA polymerase IIO from 300 g of calf thymus. The specific activity of RNA polymerase IIO, in nonselective assays using calf thymus DNA as template, is 440 units/mg and not significantly different from that of RNA polymerases IIA and IIB. The similar transcriptional activities in nonselective assays suggest that the C-terminal domain of the largest RNA polymerase II subunit does not play a major role in the elongation phase of the reaction when deproteinized DNA serves as template. The small subunits of RNA polymerase IIO are indistinguishable from those of RNA polymerases IIA and IIB.