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Molecular and Cellular Biology, September 2003, p. 6229-6242, Vol. 23, No. 17
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.17.6229-6242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

Shwu-Yuan Wu, Tianyuan Zhou,{dagger} and Cheng-Ming Chiang*

Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

Received 23 April 2003/ Accepted 29 May 2003

Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by enhancing activator-mediated recruitment of RNA polymerase II (pol II), whereas Mediator-P.85 works mainly by stimulating overall basal transcription. The coactivator function of Mediator-P.5 was not impaired when TATA-binding protein (TBP) was used in place of TFIID, but it was abolished when another general cofactor, PC4, was omitted from the reaction or when Mediator-P.5 was added after pol II entry into the preinitiation complex. Moreover, Mediator- P.5 is able to enhance TBP binding to the TATA box in an activator-dependent manner. Our data provides biochemical evidence that Mediator functions by facilitating activator-mediated recruitment of pol II and also promoter recognition by TBP, both of which can occur in the absence of TBP-associated factors in TFIID.

* Corresponding author. Mailing address: Department of Biochemistry, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4935. Phone: (216) 368-8550. Fax: (216) 368-3419. E-mail: c-chiang{at}biochemistry.cwru.edu.

{dagger} Present address: Howard Hughes Medical Institute, Department of Medicine, University of California—San Diego, La Jolla, CA 92093.

Molecular and Cellular Biology, September 2003, p. 6229-6242, Vol. 23, No. 17
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.17.6229-6242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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