Anti-MED7 polyclonal antibody

Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved "hinge" in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly.

Activation of gene transcription in metazoans is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA, These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus(1), One class of co-activator, the TAF(II) subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize core promoter sequences necessary for transcription initiation(2-5). Transcriptional activation by enhancer-binding factors such as Sp1 (ref, 6) requires TFIID, but the identity of other necessary cofactors has remained unknown. Here we describe a new human factor, CRSP, that is required together with the TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies a complex of approximate relative molecular mass 700,000 (M(r)similar to 700K) that contains nine subunits with M-r values ranging from 33K to 200K. Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of the yeast mediator subunit Med7 (ref. 7), whereas CRSP150 contains a domain conserved in yeast mediator subunit Rgr1 (ref. 8), CRSP p200 is identical to the nuclear hormone-receptor co-activator subunit TRIP2/PBP9,10. CRSPs 34, 77 and 130 are new proteins, but the amino terminus of CRSP70 is homologous to elongation factor TFIIS11. Immunodepletion studies confirm that these subunits have an essential cofactor function. The presence of common subunits in distinct cofactor complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional activation.