Human Mediator enhances basal transcription by facilitating recruitment of transcription factor IIB during preinitiation complex assembly.
The multisubunit Mediator is a well established transcription coactivator for gene-specific activators. However, recent studies have shown that, although not essential for basal transcription by purified RNA polymerase II (pol II) and general initiation factors, Mediator is essential for basal transcription in nuclear extracts that contain a more physiological complement of factors (Mittler, G., Kremmer, E., Timmers, H. T., and Meisterernst, M. (2001) EMBO Rep. 2, 808-813; Baek, H. J., Malik, S., Qin, J., and Roeder, R. G. (2002) Mol. Cell. Biol. 22, 2842-2852). Here, mechanistic studies with immobilized DNA templates, purified factors, and factor-depleted HeLa extracts have shown (i) that Mediator enhancement of basal transcription correlates with Mediator-dependent recruitment of pol II and general initiation factors (transcription factor (TF) IIB and TFIIE) to the promoter; (ii) that Mediator and TFIIB, which both interact with pol II, are jointly required for pol II recruitment to the promoter and that TFIIB recruitment is Mediator-dependent, whereas Mediator recruitment is TFIIB-independent; (iii) that a high level of TFIIB can bypass the Mediator requirement for basal transcription and pol II recruitment in nuclear extract, thus indicating a conditional restriction of TFIIB function and a key role of Mediator in overcoming this restriction; and (iv) that an earlier rate-limiting step involves formation of a TFIID-Mediator-promoter complex. These results support a stepwise assembly model, rather than a preformed holoenzyme model, for Mediator-dependent assembly of a basal preinitiation complex and, more important, identify a step involving TFIIB as a key site of action of Mediator.
Baek
Hwa Jin
HJ
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021, USA.
Kang
Yun Kyoung
YK
Roeder
Robert G
RG
D001665:Binding Sites; D004257:DNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D056892:Mediator Complex; D046912:Multiprotein Complexes; D011401:Promoter Regions, Genetic; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D035581:Transcription Factor TFIIB / Q000737:chemistry / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
C460107:MED12 protein, human; D056892:Mediator Complex; D046912:Multiprotein Complexes; D011988:Receptors, Thyroid Hormone; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035581:Transcription Factor TFIIB; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D004257:DNA Polymerase II