Hrs1/Med3 is a Cyc8-Tup1 corepressor target in the RNA polymerase II holoenzyme.
The Srb/Mediator, a multisubunit subcomplex of the RNA polymerase II (RNA pol II) holoenzyme has been proposed to function as a control panel regulating transcription in response to gene-specific activator proteins. In this report, we identify the Mediator subunit Hrs1/Med3 as a physical target for Cyc8-Tup1, a yeast transcriptional corepressor. Two-hybrid and glutathione S-transferase interaction assays show that Hrs1 can associate directly with Cyc8-Tup1. Moreover, affinity chromatography experiments, using yeast protein extracts, reveal that Cyc8-Tup1 co-purifies with Hrs1 and with additional Mediator subunits in a Hrs1-dependent manner. These observations suggest that Cyc8-Tup1 contacts the Mediator complex via its interaction with the Hrs1 subunit. Further on, genetic analysis indicates that increased Hrs1 dosage can alleviate Cyc8-Tup1-mediated repression, suggesting that Hrs1/Mediator's function is inhibited upon its interaction with Cyc8-Tup1. Finally, artificial holoenzyme recruitment assays support a model by which the contact between the corepressor and the Hrs1/Mediator may prevent pol II holoenzyme recruitment to the core promoter. These data, together with previous genetic evidence, establish a functional and physical interaction between the Cyc8-Tup1 corepressor and the RNA pol II holoenzyme and support a central role of the Mediator complex in transcriptional repression.
Papamichos-Chronakis
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M
Institute of Molecular Biology and Biotechnology Foundation of Research and Technology, University of Crete, Vassilika Vouton, P. O. Box 1527, GR-711 10 Heraklion, Crete, Greece.
Conlan
R S
RS
Gounalaki
N
N
Copf
T
T
Tzamarias
D
D
D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D020035:Holoenzymes / Q000378:metabolism; D056892:Mediator Complex; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
C065455:CYC8 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020035:Holoenzymes; D056892:Mediator Complex; D009687:Nuclear Proteins; C110281:PGD1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C066502:TUP1 protein, S cerevisiae; D014157:Transcription Factors; D012319:RNA Polymerase II