Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo‐EM
Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo‐EM
复制标题
DOI：
10.1038/emboj.2012.205
复制
发表时间：
2012-08
期刊：
影响因子：
--
通讯作者：
Yi-Min Wu;Jen-wei Chang;Chun-Hsiung Wang;Yen-chen Lin;Pei-lun Wu;Shih-Hsin Huang;Chia-Chi Chang;Xiaopeng Hu;A. Gnatt;Wei-Hau Chang
中科院分区：
文献类型：
--
作者：
Yi-Min Wu;Jen-wei Chang;Chun-Hsiung Wang;Yen-chen Lin;Pei-lun Wu;Shih-Hsin Huang;Chia-Chi Chang;Xiaopeng Hu;A. Gnatt;Wei-Hau Chang
在哺乳动物中，一种独特的RNA聚合酶II形式，RNAPII(G)含有一个新的亚基Gdown 1(由POLR2M编码)，它抑制基因的激活，但被多亚单位介体共激活物逆转。在这里，我们利用单粒子冷冻电子显微镜(CRYO-EM)揭示了RNAPII(G)、RNAPII和RNAPII与转录起始因子TFIIF的复合体的结构，均为∼19 ä。差异分析将Gdown 1主要定位于RNAPII Rpb5支架-Rpb1颌骨，并得到抗体标记实验的支持。这些结构特征与RNAP II(G)对RNA链延长效率的适度提高有关。此外，我们更新的RNAPII-TFIIF图谱显示，TFIIF连接了DNA结合裂隙周围的多个区域，与交联和生化图谱一致。Gdown 1‘S结合位点与TFIIF有广泛的重叠，Gdown 1立体地将TFIIF排除在RNAPII之外，本文通过大小排除层析竞争分析证明了这一点。综上所述，我们的工作为Gdown 1通过阻断TFIIF而阻碍启动子的启动奠定了结构基础，说明了中介在激活转录中的额外依赖作用。
In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded byPOLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co‐activator. Here, we employed single‐particle cryo‐electron microscopy (cryo‐EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ∼19 Å. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf‐Rpb1 jaw, supported by antibody labelling experiments. These structural features correlate with the moderate increase in the efficiency of RNA chain elongation by RNAP II(G). In addition, our updated RNAPII–TFIIF map showed that TFIIF tethers multiple regions surrounding the DNA‐binding cleft, in agreement with cross‐linking and biochemical mapping. Gdown1's binding sites overlap extensively with those of TFIIF, with Gdown1 sterically excluding TFIIF from RNAPII, herein demonstrated by competition assays using size exclusion chromatography. In summary, our work establishes a structural basis for Gdown1 impeding initiation at promoters, by obstruction of TFIIF, accounting for an additional dependent role of Mediator in activated transcription.