[22] RNA polymerase III and class III transcription factors from Saccharomyces cerevisiae

RNA polymerase III is specifically recruited at the transcription start sites via a cascade of protein-protein-DNA interactions involving various transcription factors. Remarkably, complex assemblies are formed that involve more than 25 polypeptides and cover the entire transcription units. The transcriptional components and the process of transcription complex formation on various class III genes have been best analyzed in Saccharomyces cerevisiae. This chapter describes the purification of yeast RNA polymerase III and of yeast factors TFIIIA (transcription factor IIIA), TFIIIB, and TFIIIC. RNA polymerase III and TFIIIC can be purified to near homogeneity as stable multisubunit assemblies. In contrast, TFIIIB can be easily dissociated into three components (TBP, TFIIIB, and B"). TFIIIA, TFIIIB and TBP can be obtained in active form as recombinant proteins made in Escherichia coli. Twenty components of the yeast class III transcription machinery have already been cloned and mutagenized.