Characterization of a protein that interacts with the rat ribosomal gene promoter and modulates RNA polymerase I transcription

Abstract

An RNA polymerase I core promoter binding factor (CPBF) was purified to apparent homogeneity from rat adenocarcinoma ascites cells by chromatographic fractionation on a series of columns including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides with molecular masses of 44,000 and 39,000 daltons. The binding of the factor to the promoter was demonstrated by Southwestern blotting, UV cross-linking and electrophoretic mobility shift assay. The specificity of its binding to the core promoter was confirmed by competitive electrophoretic mobility shift assay using several unlabeled oligo probes in the assay. The addition of increasing amounts of purified CPBF to the in vitro transcription reaction that contains a limiting quantity of the factor resulted in dramatic stimulation of RNA polymerase I (pol I) transcription of rat ribosomal RNA gene. The transcription stimulatory activity associated with the purified CPBF fractions co-purified with the core promoter binding activity in an electrophoretic mobility shift assay. Finally, in a reconstitution transcription system which is devoid of the factor and is incapable of ribosomal gene transcription, purified CPBF could trans-activate the pol I promoter. These data indicate that CPBF is a novel pol I promoter binding factor required for ribosomal gene transcription.