Wu Shwu-Yuan, Zhou Tianyuan, Chiang Cheng-Ming
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Mol Cell Biol. 2003 Sep;23(17):6229-42. doi: 10.1128/MCB.23.17.6229-6242.2003.
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.
中介体是一种与许多转录激活因子功能相关的通用辅因子。尽管已分离出具有不同蛋白质组成的中介体，但尚不清楚中介体如何促进依赖激活因子的转录，而不依赖于其对基础转录的一般刺激。为了确定中介体功能的机制，我们分离出两种形式的人中介体复合物（中介体-P.5和中介体-P.85），并证明中介体-P.5通过增强激活因子介导的RNA聚合酶II（pol II）募集来发挥作用，而中介体-P.85主要通过刺激整体基础转录来发挥作用。当使用TATA结合蛋白（TBP）代替TFIID时，中介体-P.5的共激活因子功能未受损，但当反应中省略另一种通用辅因子PC4或在pol II进入起始前复合物后添加中介体-P.5时，其功能被消除。此外，中介体-P.5能够以依赖激活因子的方式增强TBP与TATA框的结合。我们的数据提供了生化证据，表明中介体通过促进激活因子介导的pol II募集以及TBP对启动子的识别来发挥作用，这两者都可以在不存在TFIID中TBP相关因子的情况下发生。
