Chapter 1. Isolation and Assay of Eukaryotic DNA-Dependent RNA Polymerases

Abstract

This chapter describes the most recent and general procedures used in the purification of eukaryotic nuclear RNA polymerases,with special emphasis on those enzymes that have been extensively purified. Detailed methods are presented for the purification of yeast RNA polymerases that have been selected because of the convenience of their source, the authors' interest and experience, and the possible general applicability of this procedure for the purification of polymerases from other sources. Eukaryotic cells contain multiple RNA polymerases. The polymerase content in higher cells is quite low and unlike the bacterial polymerase the enzymes are difficult to extract as they occur tightly bound to chromosomal components, such as DNA, RNA, histones, and acidic proteins. Eukaryotic RNA polymerases are complex structures composed of multiple subunits, and losses of activity are usually encountered owing to the apparently delicate nature of these molecules.