Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York, USA.
Nat Struct Mol Biol. 2014 Dec;21(12):1028-34. doi: 10.1038/nsmb.2914. Epub 2014 Nov 10.
The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.
进化保守的中介复合物（Mediator complex）是 RNA 聚合酶 II（Pol II）介导的转录的关键共激活因子。在这里，我们报告了一个功能性的 15 亚基人类核心中介复合物的重建及其通过功能测定和化学交联与 MS（CX-MS）的表征。虽然重建的头部和中部模块可以稳定地结合，但只有在将 MED14 纳入双模块复合物后，才能获得基础和共激活功能。这是由于包含 MED14 的复合物与 Pol II 结合的能力大大增强。总的来说，我们的分析将 MED14 鉴定为中介复合物的结构和功能骨干。我们还进一步确定了后生动物特异性 MED26 的条件性要求，在存在异源核因子时，这种要求变得明显。这种通用方法为系统剖析与中介复合物相关的多个功能层铺平了道路。
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