Assays and affinity purification of biotinylated and nonbiotinylated forms of double-tagged core RNA polymerase II from Saccharomyces cerevisiae.

Publisher Summary This chapter reviews the Pol II core enzyme which has been isolated, using a combination of ion-exchange chromatography and immunoaffinity chromatography with an antibody against the Rpb1 subunit. The alternative to the immunoaffinity approach is introduction of an affinity tag. This strategy was applied successfully to mammalian Pol II, where a FLAG epitope was inserted in the Rpb9 subunit, which allowed achieving a 1000-fold purification in one step. The chapter introduces hexahistidine and biotin acceptor peptide tags in the amino terminus of the Rpb3 subunit of yeast Pol II. According to the Pol II core enzyme and elongation complex crystal structures, tags are located on a surface of the enzyme opposite from the active center and sites of interaction with the DNA and RNA. The tagged enzyme is, indeed, indistinguishable from the wild-type Pol II in the in vitro activity test, and tagged Rpb3 rescues the rpb3 temperature-sensitive phenotype as efficiently as wild-type Rpb3. The chapter also highlights that an alternative protocol, which includes three chromatographic steps, was developed for the purification of nonbiotinylated histidine-tagged Pol II.