Human mediator enhances activator-facilitated recruitment of RNA polymerase II and promoter recognition by TATA-binding protein (TBP) independently of TBP-associated factors.
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.
Wu
Shwu-Yuan
SY
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Zhou
Tianyuan
T
Chiang
Cheng-Ming
CM
D002478:Cells, Cultured; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D011401:Promoter Regions, Genetic / Q000502:physiology*; D020033:Protein Isoforms / Q000302:isolation & purification; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D035381:TATA-Binding Protein Associated Factors / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D035362:Transcription Factor TFIID / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000502:physiology
C490043:IFRD2 protein, human; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012097:Repressor Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D014157:Transcription Factors; C095686:CDK8 protein, human; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D003850:Deoxyribonuclease I