Yeast nuclear extract contains two major forms of RNA polymerase II mediator complexes.
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 x 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 Rgrl 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 x Med complex in vitro.
Liu
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The Fred Hutchinson Cancer Research Center and the Howard Hughes Medical Institute, Seattle, WA 98109-1024, USA.
Ranish
J A
JA
Aebersold
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R
Hahn
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S
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D056892:Mediator Complex; D008954:Models, Biological; D011485:Protein Binding; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D056892:Mediator Complex; C095061:RGR1 protein, S cerevisiae; C103856:ROX3 protein, S cerevisiae; D012097:Repressor Proteins; C082149:SRB5 protein, S cerevisiae; C082150:SRB6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012319:RNA Polymerase II