Two species of chromatin-RNA polymerase II complex released from rat liver nuclei by nuclease digestion

Abstract

To investigate the structural organization of transcribing chromatin, rat liver nuclei were washed thoroughly to remove the free RNA polymerase and then digested with micrococcal nuclease or DNAase I. After extensive digestion with the nuclease in the presence of 0.1 to 0.25 m m-divalent cation, two species of the chromatin-RNA polymerase II complexes were separated by subsequent chromatography on a column of Bio-Gel A-15m (peak 1 and peak 2). The peak 1 complexes, recovered at the oligonucleosomal region, were RNA polymerase II-nucleosome monomer or dimer complexes. These complexes were further characterized by the possession of factors that enhanced chromatin transcription, and seemed to associate with ribonucleoprotein particles. The peak 2 complexes, eluted at the position of purified RNA polymerase II molecules, contained endogenous template, but were apparently not organized in the nucleosomal structure. The RNA polymerase II molecules in both complexes were found to be associated with nascent RNA synthesized in vivo. When the digestion with micrococcal nuclease was performed in the presence of 3 m m-MgCl 2, peak 2 was recovered as a relatively large chromatin (S 2-chromatin), while peak 1 was not extracted and remained in the residual chromatin (P 2-chromatin). Analysis of RNA transcripts synthesized from these chromatins indicated that the two forms of the enzyme transcribed different DNA sequences, and both of the products were found in the polysomal poly(A) + messenger RNA. These lines of evidence suggest that there are separate RNA polymerase II transcriptional systems, which transcribe different DNA sequences of the genome.