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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Cells, Cultured
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Cyclin-Dependent Kinase 8
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism
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Deoxyribonuclease I / genetics
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Deoxyribonuclease I / metabolism
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Humans
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Immediate-Early Proteins
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Membrane Proteins
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Promoter Regions, Genetic / physiology*
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Protein Isoforms / isolation & purification
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RNA Polymerase II / genetics
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RNA Polymerase II / metabolism*
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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TATA-Binding Protein Associated Factors / genetics
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TATA-Binding Protein Associated Factors / metabolism*
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TATA-Box Binding Protein / genetics
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TATA-Box Binding Protein / metabolism*
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Trans-Activators / genetics
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Trans-Activators / metabolism
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Transcription Factor TFIID / genetics
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Transcription Factor TFIID / metabolism
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Transcription Factors / genetics
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Transcription Factors / isolation & purification
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Transcription Factors / metabolism*
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Transcription, Genetic
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Transcriptional Activation / physiology
Substances
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IFRD2 protein, human
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Immediate-Early Proteins
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Membrane Proteins
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Protein Isoforms
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Recombinant Proteins
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Repressor Proteins
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TATA-Binding Protein Associated Factors
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TATA-Box Binding Protein
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Trans-Activators
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Transcription Factor TFIID
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Transcription Factors
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CDK8 protein, human
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Cyclin-Dependent Kinase 8
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Cyclin-Dependent Kinases
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RNA Polymerase II
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Deoxyribonuclease I