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Originally published In Press as doi:10.1074/jbc.M009586200 on December 11, 2000

J. Biol. Chem., Vol. 276, Issue 10, 7169-7175, March 9, 2001
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Yeast Nuclear Extract Contains Two Major Forms of RNA Polymerase II Mediator Complexes*

Ying LiuDagger , Jeffrey A. Ranish§, Ruedi Aebersold§, and Steven HahnDagger

From Dagger  The Fred Hutchinson Cancer Research Center and the Howard Hughes Medical Institute, Seattle, Washington 98109 and the § Institute for Systems Biology, Seattle, Washington 98105

The yeast Mediator complex is required for transcription by RNA polymerase II (pol II) in vivo and in vitro. This complex of over 20 polypeptides associates with pol II and is recruited to transcription complexes at promoters. Previous isolations of yeast Mediator-containing complexes in different laboratories have identified several distinct complexes. To identify the major forms of Mediator in yeast, Mediator was isolated from nuclear extracts using a two-step chromatographic procedure, avoiding ion exchange chromatography and high salt conditions to prevent dissociation of subunits during purification. Components of the Mediator complexes were identified by mass spectrometry and Western analysis. The major form of Mediator, termed pol II·Med, contained pol II and Mediator, including the Srb8-11 module. A second lower molecular size complex was also identified, termed Mediator core (Medc), which lacked pol II, Srb8-11, Rox3, Nut1, and the Rgr1 module. Both of these complexes were active in transcription in vitro, although the Medc complex had significantly lower activity and could compete with the activity of the pol II·Med complex in vitro.

* This work was supported by grants from the Natinal Institutes of Health (to S. H. and J. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

An associate investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Basic Sciences Division, The Fred Hutchinson Cancer Research Center and the Howard Hughes Medical Institute, 1100 Fairview Ave. N, Mailstop A1-162, P. O. Box 19024, Seattle, Washington 98109-1024. Tel.: 206-667-5261; Fax: 206-667-6497; E-mail: shahn@fhcrc.org.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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