Requirement of TRAP/mediator for both activator-independent and activator-dependent transcription in conjunction with TFIID-associated TAF(II)s.
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.
Baek
Hwa Jin
HJ
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA.
Malik
Sohail
S
Qin
Jun
J
Roeder
Robert G
RG
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
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