Isolation of multiple protein factors involved in ribosomal DNA transcription

Abstract

Studies were made of the molecular mechanisms which regulate ribosomal gene transcription in response to changes in the growth rate of cells. Extracts prepared from exponentially growing Ehrlich ascites cells faithfully and efficiently transcribe cloned mouse rDNA, whereas extracts from growth-arrested cells are virtually inactive. In an attempt to identify and characterize functionally the proteins that mediate the accuracy and the control of transcription initiation, a fractionation procedure was developed which allows the purification of RNA polymerase I and four accessory factors that are required for transcription initiation at the ribosomal gene promoter. Starting from about 300 ml of cell extract, each of the individual factors and the polymerase was purified on at least four different chromatographic columns, including ion-exchange chromatography on DEAE-Sepharose, heparin-Ultrogel, Mono Q and Mono S, gel filtration and specific affinity chromatography. The resulting protein fractions are functionally active, as shown by reconstitution of specific rDNA transcription in the presence of purified polymerase and the additional factors.