Requirement of TRAP/mediator for both activator-independent and activator-dependent transcription in conjunction with TFIID-associated TAFIIs
Requirement of TRAP/mediator for both activator-independent and activator-dependent transcription in conjunction with TFIID-associated TAFIIs
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DOI：
10.1128/mcb.22.8.2842-2852.2002
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发表时间：
2002-04-01
影响因子：
5.3
通讯作者：
Roeder, RG
中科院分区：
文献类型：
--
作者：
Baek, HJ;Malik, S;Roeder, RG
多蛋白人类TRAP/介体复合物，其与酵母SRB/介体共激活因子在遗传学上相关，通过多种转录激活因子促进激活。然而，目前尚不清楚TRAP/Mediator如何在其他辅激活因子的背景下发挥作用。在这里，我们已经确定了一个以前未知的完整的亚基（TRAP 25）的复杂的，显然是后生动物的具体。特异于TRAP 25的抗体允许从HeLa核提取物中定量免疫耗竭基本上所有TRAP/介体组分，而不会可检测地影响RNA聚合酶II和相应的一般转录因子的水平。令人惊讶的是，TRAP/介体耗尽的核提取物显示来自DNA模板的基础和激活物依赖性转录水平严重降低。在再加入纯化的TRAP/介体后，两种活性都有效地恢复。此外，恢复同时耗尽TRAP/介体和TFIID（TBP加上主要TAF（II））的提取物的基础和激活子依赖性转录需要添加TBP和相关TAF（II）以及TRAP/介体。这些观察结果表明，TAF（II）和介体共同需要的基础和激活的转录的背景下，一个更生理的补充核蛋白。我们提出了一个密切的机制联系，这些组件，最有可能在一般的转录机器的组合效应的水平上运作，此外，中介在中继激活信号到这个机器的直接作用。
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.