Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

Abstract

Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by enhancing activator-mediated recruitment of RNA polymerase II (pol II), whereas Mediator-P.85 works mainly by stimulating overall basal transcription. The coactivator function of Mediator-P.5 was not impaired when TATA-binding protein (TBP) was used in place of TFIID, but it was abolished when another general cofactor, PC4, was omitted from the reaction or when Mediator-P.5 was added after pol II entry into the preinitiation complex. Moreover, Mediator-P.5 is able to enhance TBP binding to the TATA box in an activator-dependent manner. Our data provides biochemical evidence that Mediator functions by facilitating activator-mediated recruitment of pol II and also promoter recognition by TBP, both of which can occur in the absence of TBP-associated factors in TFIID.Initiation of transcription in eukaryotes requires many proteins, including gene-specific transcription factors, protein cofactors, and the general transcription machinery (52, 91). Gene-specific transcription factors (or activators) are necessary to turn on specific gene expression by recruiting components of the general transcription machinery, which includes TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), as well as chromatin cofactors, such as ATP-dependent chromatin remodeling complexes and histone acetyltransferases. This communication between activators and the general transcription machinery also requires at least one of the three general cofactors: Mediator, TATA-binding protein (TBP)-associated factors (TAFs) in TFIID, and upstream stimulatory activity (USA)-derived protein components, such as positive cofactor 4 (PC4).TAFs in TFIID act collectively as transcriptional coactivator to enhance activator-dependent transcription from chromatin (68, 97), to contact the activation domain (26, 33) or the DNAbinding domain (14) of activator, to facilitate the recruitment of the initiation form of pol II to the promoter region (94), or to induce DNA wrapping on the TFIID-bound promoter region (72). TAFs also enhance promoter recognition through multiple protein-DNA interactions with Initiator (87) and downstream core promoter elements (9). In addition, the enzymatic activities of TFIID, due to the presence of TAF II 250, also lead to acetylation of histones H3 and H4 (63), phosphorylation of PC4 (45) and the RAP74 component of TFIIF (18), as well as ubiquitin activation and conjugation of histone H1 (76). However, TAFs are not universally required for activator function (11,22,47,60,64,73,88,93,96), nor are they needed for transcriptional repressio...

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