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Papers In Press, published online ahead of print December 11, 2000
J. Biol. Chem, 10.1074/jbc.M009586200
Submitted on October 19, 2000
Revised on November 20, 2000
Accepted on December 11, 2000

Yeast nuclear extract contains two major forms of RNA polymerase II mediator complexes

Ying Liu, Jeffrey A Ranish, Ruedi Aebersold, and Steven Hahn

Basic sciences division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024

Corresponding Author: shahn{at}fhcrc.org

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.


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