title: Requirement of TRAP/mediator for both activator-independent and activator-dependent transcription in conjunction with TFIID-associated TAF(II)s.
abstract: The multiprotein human TRAP/Mediator complex, which is phylogenetically related to the yeast SRB/Mediator coactivator, facilitates activation through a wide variety of transcriptional activators. However, it remains unclear how TRAP/Mediator functions in the context of other coactivators. Here we have identified a previously uncharacterized integral subunit (TRAP25) of the complex that is apparently metazoan specific. An antibody that is specific for TRAP25 allowed quantitative immunodepletion of essentially all TRAP/Mediator components from HeLa nuclear extract, without detectably affecting levels of RNA polymerase II and corresponding general transcription factors. Surprisingly, the TRAP/Mediator-depleted nuclear extract displayed severely reduced levels of both basal and activator-dependent transcription from DNA templates. Both activities were efficiently restored upon readdition of purified TRAP/Mediator. Moreover, restoration of basal and activator-dependent transcription to extracts that were simultaneously depleted of TRAP/Mediator and TFIID (TBP plus the major TAF(II)s) required addition of both TBP and associated TAF(II)s, as well as TRAP/Mediator. These observations indicate that TAF(II)s and Mediator are jointly required for both basal and activated transcription in the context of a more physiological complement of nuclear proteins. We propose a close mechanistic linkage between these components that most likely operates at the level of combined effects on the general transcription machinery and, in addition, a direct role for Mediator in relaying activation signals to this machinery.
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA.
authors.forename: Hwa Jin
authors.initials: HJ
authors.lastname: Baek
authors.forename: Sohail
authors.initials: S
authors.lastname: Malik
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
authors.forename: Robert G
authors.initials: RG
authors.lastname: Roeder
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D021122:Protein Subunits; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; C079092:MtrB protein, Bacteria; D021122:Protein Subunits; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII