Formation of transcribing mononucleosome-eukaryotic RNA polymerase II complexes in vitro as a simple model of active chromatin.
Mononucleosomes obtained from cultured mouse hepatoma cells were incubated with RNA polymerase II from wheat germ. No free DNA was liberated as available templates under the experimental condition employed. Size analysis of the transcripts showed that the polymerase initiated transcription from either terminus and read through the DNA template of mononucleosomes. Sucrose density gradient centrifugation of the reaction mixture resolved mononucleosome-polymerase complexes from free materials. The complexes were characterized by the enrichment of DNA fragments containing the nucleosome linker region, the presence of H1 histone, and the increased susceptibility to DNase I. Both the complexes formed in the presence and absence of precursor nucleotides were susceptible. These suggest that RNA polymerase II prefers to bind to the linker region, and the polymerase-bound nucleosomes are structurally altered. The data were discussed in context with possible mechanisms of transcription of the nucleosome structure.
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000201:enzymology*; D004247:DNA / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D051379:Mice; D009707:Nucleosomes / Q000201:enzymology*; D012319:RNA Polymerase II / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D002843:Chromatin; D009707:Nucleosomes; D004247:DNA; D012319:RNA Polymerase II; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I