21] Purification, assay, and properties of RNA polymerase I and class I-specific transcription factors in mouse

Abstract

This chapter introduces the factors involved in initiation and termination of mouse ribosomal gene (rDNA) transcription and describes the protocols used for the purification of the individual activities. In eukaryotes, ribosomal gene (rDNA) transcription is governed by a dedicated set of transcription factors and a specialized RNA polymerase, the DNA-dependent RNA polymerase I (Pol I). To characterize the biochemical properties of these factors, it is necessary to purify them to homogeneity. The purified factors are required to analyze their function, to raise antibodies, and to isolate the genes encoding the proteins. It has been difficult, however, to isolate Pol I and the respective initiation and termination factors because they typically constitute less than 0.01% of the total cellular protein. The chapter outlines chromatographic procedures that allow reconstituting a Pol I transcription system with highly purified factors that mimicks all steps in basal transcription, that is, initiation, elongation, and termination.