Baek Hwa Jin, Malik Sohail, Qin Jun, Roeder Robert G
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA.
Mol Cell Biol. 2002 Apr;22(8):2842-52. doi: 10.1128/MCB.22.8.2842-2852.2002.
The multiprotein human TRAP/Mediator complex, which is phylogenetically related to the yeast SRB/Mediator coactivator, facilitates activation through a wide variety of transcriptional activators. However, it remains unclear how TRAP/Mediator functions in the context of other coactivators. Here we have identified a previously uncharacterized integral subunit (TRAP25) of the complex that is apparently metazoan specific. An antibody that is specific for TRAP25 allowed quantitative immunodepletion of essentially all TRAP/Mediator components from HeLa nuclear extract, without detectably affecting levels of RNA polymerase II and corresponding general transcription factors. Surprisingly, the TRAP/Mediator-depleted nuclear extract displayed severely reduced levels of both basal and activator-dependent transcription from DNA templates. Both activities were efficiently restored upon readdition of purified TRAP/Mediator. Moreover, restoration of basal and activator-dependent transcription to extracts that were simultaneously depleted of TRAP/Mediator and TFIID (TBP plus the major TAF(II)s) required addition of both TBP and associated TAF(II)s, as well as TRAP/Mediator. These observations indicate that TAF(II)s and Mediator are jointly required for both basal and activated transcription in the context of a more physiological complement of nuclear proteins. We propose a close mechanistic linkage between these components that most likely operates at the level of combined effects on the general transcription machinery and, in addition, a direct role for Mediator in relaying activation signals to this machinery.
多蛋白人TRAP/中介体复合物与酵母SRB/中介体共激活因子在系统发育上相关，它通过多种转录激活因子促进转录激活。然而，TRAP/中介体在其他共激活因子的背景下如何发挥作用仍不清楚。在这里，我们鉴定出该复合物中一个以前未被表征的整合亚基（TRAP25），它显然是后生动物特有的。一种对TRAP25特异的抗体能够从HeLa细胞核提取物中定量免疫去除几乎所有的TRAP/中介体成分，而不会显著影响RNA聚合酶II和相应的通用转录因子的水平。令人惊讶的是，去除TRAP/中介体的细胞核提取物中，DNA模板的基础转录和激活因子依赖的转录水平都严重降低。重新添加纯化的TRAP/中介体后，这两种活性都得到了有效恢复。此外，对于同时去除了TRAP/中介体和TFIID（TBP加上主要的TAF(II)s）的提取物，要恢复基础转录和激活因子依赖的转录，需要同时添加TBP和相关的TAF(II)s以及TRAP/中介体。这些观察结果表明，在更接近生理状态的核蛋白补充情况下，TAF(II)s和中介体对于基础转录和激活转录都是共同必需的。我们提出这些成分之间存在紧密的机制联系，很可能在对通用转录机制的综合作用水平上发挥作用，此外，中介体在将激活信号传递给该机制方面具有直接作用。
