Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA.
The interaction of activators with mediator has been proposed to stimulate the assembly of RNA polymerase II (Pol II) preinitiation complexes, but there have been few tests of this model. The finding that the major adenovirus E1A and mitogen-activated protein kinase-phosphorylated Elk1 activation domains bind to Sur2 uniquely among the metazoan mediator subunits and the development of transcriptionally active nuclear extracts from WT and sur2-/- embryonic stem cells, reported here, allowed a direct test of the model. We found that whereas VP16, E1A, and phosphorylated Elk1 activation domains each stimulate binding of mediator, Pol II, and general transcription factors to promoter DNA in extracts from WT cells, only VP16 stimulated their binding in extracts from sur2-/- cells. This stimulation of mediator, Pol II, and general transcription factor binding to promoter DNA correlated with transcriptional activation by these activators in WT and mutant extracts. Because the mutant mediator was active in reactions with the VP16 activation domain, the lack of activity in response to the E1A and Elk1 activation domains was not due to loss of a generalized mediator function, but rather the inability of the mutant mediator to be bound by E1A and Elk1. These results directly demonstrate that the interaction of activation domains with mediator stimulates preinitiation complex assembly on promoter DNA.
D018528:ATP-Binding Cassette Transporters; D017500:Adenovirus E1A Proteins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D051379:Mice; D018345:Mice, Knockout; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D024661:Potassium Channels, Inwardly Rectifying; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1
D018528:ATP-Binding Cassette Transporters; D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; C494113:Elk1 protein, mouse; D024661:Potassium Channels, Inwardly Rectifying; D011518:Proto-Oncogene Proteins; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D015534:Trans-Activators; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D004247:DNA; D006899:Mixed Function Oxygenases; D012319:RNA Polymerase II