Abstract
The yeast Mediator is composed of two subcomplexes, Rgr1 and Srb4, known to be required for diverse aspects of transcriptional regulation; however, their structural and functional organizations have not yet been deciphered in detail. Biochemical analyses designed to determine the subunit composition of the Rgr1 subcomplex revealed that the regulator-interacting subcomplex has a modular structure and is composed of the Gal11, Med9/Cse2, and Med10/Nut2 modules. Genome-wide gene expression and Northern analyses performed in the presence or absence of the various Mediator modules revealed a distinct requirement for the Gal11, Med9/Cse2, and Med10/Nut2 modules in transcriptional repression as well as activation. GST pull-down analysis revealed that the transcriptional repressor Tup1 binds to distinct but overlapping regions of the Gal11 module that were shown previously to be transcriptional activator binding sites. These data suggest that competition between transcriptional activators and repressors for a common binding site in the Mediator and distinct conformational changes in the Mediator induced by repressor binding may underlie the mechanism of transcriptional repression in eukaryotes.
Highlights
The Mediator complex was identified first as a coactivator essential for transcriptional activation in the yeast Saccharomyces cerevisiae [1, 2]
We demonstrated that a distinct Mediator module is required for transcriptional activator binding to h-polymerase II (Pol II) [8]
The 1 M urea wash removed mainly the polymerase subunits associated with the Mediator complex, whereas a 2 M urea wash removed the Srb4 subcomplex from the beads without affecting the association of the Rgr1 subcomplex with the beads (Fig. 1B)
Summary
Pol II, RNA polymerase II; h-Pol II, Pol II holoenzyme; GST, glutathione S-transferase; HA, hemagglutinin; Ab, antibody; YPD, yeast extract-peptone-dextrose medium; ts, temperature sensitive; HDA, high density oligonucleotide array. Yeast cells lacking the MED1 gene, which encodes a subunit of the Rgr subcomplex, are viable but show a complex phenotype that includes partial defects in both repression and induction of GAL gene transcription [21]. Deletion of the HRS1 gene, which encodes a subunit of the Gal module, causes derepression of the GAL1, PHO5, and HSP26 genes [22], and Hrs protein was shown to interact with Tup protein in vitro [23]. Taken together, these results suggest that several proteins in the Rgr subcomplex are required for transcriptional repression. To decipher the modular structure of Mediator and identify the functions associated with each module, we used a ureainduced disruption assay and reconstitution experiment with recombinant proteins to analyze the protein-protein interactions among the Mediator subunits and examined the role of each subunit in transcriptional regulation with genome-wide expression analysis
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