Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA
Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA
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DOI：
10.1073/pnas.2035253100
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发表时间：
2003-10-14
影响因子：
11.1
通讯作者：
Berk, AJ
中科院分区：
文献类型：
--
作者：
Cantin, GT;Stevens, JL;Berk, AJ
激活剂与介体的相互作用已被提出来刺激RNA聚合酶II（Pol II）preinitiation复合物的组装，但这个模型的测试很少。主要的腺病毒E1 A和促分裂原活化蛋白激酶磷酸化的Elk 1激活结构域与多细胞动物介体亚基中唯一的Sur 2结合，以及WT和Sur 2（-/-）胚胎干细胞的转录活性核提取物的发展，这一发现允许对模型进行直接测试。我们发现，而VP 16，E1 A，和磷酸化的Elk 1激活结构域的介质，Pol II，和一般的转录因子的启动子DNA的结合刺激从WT细胞提取物，只有VP 16刺激他们的结合从sur 2（-/-）细胞提取物。这种刺激介质，Pol II，和一般的转录因子结合到启动子DNA与转录激活这些激活因子在WT和突变体提取物。因为突变介体在与VP 16活化结构域的反应中是有活性的，所以响应于E1 A和Elk 1活化结构域的活性的缺乏不是由于广义介体功能的丧失，而是突变介体不能被EIIA和Elk 1结合。这些结果直接表明，激活结构域与介体的相互作用刺激启动子DNA上的预起始复合物组装。
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 EIIA and Elk1. These results directly demonstrate that the interaction of activation domains with mediator stimulates preinitiation complex assembly on promoter DNA.