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Molecular and Cellular Biology, August 1999, p. 5383-5392, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Quantitation of RNA Polymerase II and Its Transcription Factors in an HeLa Cell: Little Soluble Holoenzyme but Significant Amounts of Polymerases Attached to the Nuclear Substructure

Hiroshi Kimura,1 Yong Tao,2 Robert G. Roeder,2 and Peter R. Cook1,*

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom,1 and Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 100212

Received 10 March 1999/Returned for modification 24 April 1999/Accepted 30 April 1999

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; ~140,000 unengaged polymerases (mainly of form IIA) were released. Only ~4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (~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 ~50% of the chromatin was detached with a nuclease, but ~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.

* Corresponding author. Mailing address: Sir William Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford OX1 3RE, United Kingdom. Phone: (44/0) 1865 275528. Fax: (44/0) 1865 275515. E-mail: Peter.Cook{at}Path.OX.AC.UK.

Molecular and Cellular Biology, August 1999, p. 5383-5392, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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