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Molecular and Cellular Biology, July 2001, p. 4604-4613, Vol. 21, No. 14
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.14.4604-4613.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

Gang Wang, Greg T. Cantin, Jennitte L. Stevens, and Arnold J. Berk*

Molecular Biology Institute, University of California, Los Angeles, California 90095-1570

Received 11 January 2001/Returned for modification 24 February 2001/Accepted 18 April 2001

A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (~500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (~2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at ~500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 × 105 to 6 × 105 molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the ~2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The ~2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.

* Corresponding author. Mailing address: University of California Los Angeles, Molecular Biology Institute, 611 Charles Young Dr. E, Box 951570, Los Angeles, CA 90095-1570. Phone: (310) 206-6298. Fax: (310) 206-7286. E-mail: berk{at}mbi.ucla.edu.

Molecular and Cellular Biology, July 2001, p. 4604-4613, Vol. 21, No. 14
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.14.4604-4613.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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