Transcription of in vitro assembled chromatin templates in a highly purified RNA polymerase II system.
In mammalian cells RNA polymerase II efficiently transcribes nucleosome-packaged DNA. In this regard, a fundamental question concerns the nature and mechanism of action of the accessory factors that are necessary and sufficient for, or enhance, transcription through nucleosomal arrays by RNA polymerase II. Here we describe a highly purified system that allows for efficient activator-dependent transcription by RNA polymerase II from the promoter through several contiguous nucleosomes on defined chromatin templates. The system contains natural or recombinant histones, chromatin assembly factors, the histone-acetyltransferase p300, all components of the general transcription machinery, general coactivators and the elongation factor SII (TFIIS). As examples of the applicability of this system for mechanistic analyses of these and other factors, representative experiments show (i) that activated transcription from chromatin templates is concomitantly dependent on the activator, p300-mediated histone acetylation and elongation factor SII/TFIIS. (ii) that SII/TFIIS acts in a highly synergistic manner with p300 (and histone acetylation) at a step subsequent to preinitiation complex (PIC) formation and (iii) that SII/TFIIS works directly at the elongation step of chromatin transcription. Here we describe purification methods for the different factors employed and the specific transcriptional assays that led to the above-mentioned conclusions. This purified system will be very useful as an assay system for the discovery of new factors or the mechanistic analysis of known or candidate factors involved in transcription initiation or elongation on chromatin templates, including factors that effect specific histone modifications or nucleosomal remodeling.
Guermah
Mohamed
M
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. guermam@mail.rockefeller.edu
Kim
Jaehoon
J
Roeder
Robert G
RG
D002843:Chromatin / Q000737:chemistry / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006367:HeLa Cells; D051548:Histone Acetyltransferases / Q000502:physiology; D006657:Histones / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D008967:Molecular Biology / Q000379:methods; D010445:Peptide Elongation Factors / Q000502:physiology; D012319:RNA Polymerase II / Q000502:physiology*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000502:physiology; D050880:p300-CBP Transcription Factors / Q000502:physiology; D000099224:p300-CBP-Associated Factor
D002843:Chromatin; D051548:Histone Acetyltransferases; D006657:Histones; D055767:Immobilized Proteins; D010445:Peptide Elongation Factors; D012319:RNA Polymerase II; D035602:Transcriptional Elongation Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C018648:transcription factor S-II