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Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; approximately 140,000 unengaged polymerases (mainly of form IIA) were released. Only approximately 4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (approximately 110,000 molecules of form IIO) and 10,000 to 30,000 molecules of each of TFIIB, TFIIEalpha, TFIIEbeta, TFIIF-RAP74, TFIIF-RAP30, and TFIIH-MAT1 remained tightly associated with the nuclear substructure. Most proteins and run-on activity were retained when approximately 50% of the chromatin was detached with a nuclease, but approximately 45,000 molecules of bound TATA binding protein (TBP) were detached. Similar results were obtained after cross-linking living cells with formaldehyde. The results provide little support for the existence of a large pool of soluble holoenzyme; they are consistent with TBP-promoter complexes in nuclease-sensitive chromatin being assembled into preinitiation complexes attached to the underlying structure.

Type

Journal article

Journal

Mol Cell Biol

Publication Date

08/1999

Volume

19

Pages

5383 - 5392

Keywords

Cell Nucleus, Centrifugation, Density Gradient, Chromatin, Cross-Linking Reagents, Formaldehyde, HeLa Cells, Holoenzymes, Humans, Immunoblotting, Macromolecular Substances, Micrococcal Nuclease, Neoplasm Proteins, Nucleoproteins, RNA Polymerase II, RNA, Messenger, RNA, Neoplasm, Transcription Factors, Transcription, Genetic