Chapter 5 DNA-Dependent RNA Polymerases from Yeasts

Abstract

This chapter focuses on DNA-dependent RNA polymerases from yeasts. Eukaryotic cells contain multiple forms of DNA-dependent RNA polymerases. These enzymes are localized in different compartments. One enzyme is found in the nucleolus and it synthesizes rRNA. Another RNA polymerase can be isolated from the nucleoplasm that synthesizes the bulk of nucleoplasmic RNA, including mRNA. These different RNA polymerases can be clearly distinguished by their chromatographic behavior, their response to the action of α-amanitin, their subunit structure, and their sedimentation value. Other DNA-dependent RNA polymerases are localized in the mitochondria and in the chloroplasts of eukaryotic cells. Mitochondria1 and chloroplast enzymes differ from nuclear enzymes, as they are smaller and are solubilized only by methods different from those used for nuclear enzymes. In the chapter, methods for the purification of DNA-dependent RNA polymerases from yeasts are described. The chapter discusses the separation of RNA polymerases A and B, isolation and purification of RNA polymerase C, and purification of mitochondrial RNA polymerase. Characterization of the RNA polymerases of yeasts is also discussed in the chapter.