RNA polymerase from eukaryotic cells: isolation and purification of enzymes and factors from chromatin of coconut nuclei

Abstract

This paper describes the isolation and purification of two RNA polymerases (CI and CII) and several protein fractions (A, B and C) from the chromosomal acidic proteins of coconut endosperm nuclei. The method involves disruption of isolated nuclei in dilute salt medium, centrifugation to sediment the crude chromatin, solubilization of chromatin in concentrated salt, dialysis of soluble chromatin to dilute salt to precipitate DNA-histone complex and centrifugation to get the acidic proteins in the supernatant. Further purification involves ammonium sulphate fractionation and chromatography on DEAE-cellulose and QAE-Sephadex. RNA polymerase CI purified through the QAE-Sephadex step gives a single band on polyacrylamide gel electrophoresis. In gels containing dodecylsulfate, this enzyme shows multiple bands indicating its subunit nature. The pH optima for both RNA polymerases is 8.0. RNA polymerase CI is maximally activated by Mn²⁺; while CII, by Mg²⁺. The activities of both the enzymes are stimulated by fraction B. All the fractions except A are substantially free from nucleases. Both RNA polymerases require the addition of DNA for activity. Fraction B is ineffective either with denatured coconut DNA or native λ DNA.