Hrs1/Med3 is a Cyc8-Tup1 corepressor target in the RNA polymerase II holoenzyme

Manolis Papamichos-Chronakis, R. Steven Conlan, Niki Gounalaki, Tjana Copf, Dimitris Tzamarias

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73 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8397-8403
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number12
DOIs
StatePublished - Mar 24 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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