Three-dimensional EM structure of an intact activator-dependent transcription initiation complex
Three-dimensional EM structure of an intact activator-dependent transcription initiation complex
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DOI：
10.1073/pnas.0908782106
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发表时间：
2009-11-24
影响因子：
11.1
通讯作者：
Lawson, Catherine L.
中科院分区：
文献类型：
--
作者：
Hudson, Brian P.;Quispe, Joel;Lawson, Catherine L.
我们提出了一个完整的激活子依赖的转录起始复合物的实验确定的三维结构，包括大肠杆菌分解代谢物激活蛋白（CAP），RNA聚合酶全酶（RNAP），和一个DNA片段，含有位置-78至+20的I类CAP依赖的启动子与CAP位点在位置-61.5和预熔化的转录泡。一个20埃的电子显微镜重建，通过迭代投影为基础的匹配的单粒子可视化碳三明治负染色，并拟合使用原子坐标集CAP，RNAP，和DNA。该结构定义了I类CAP-RNAP-启动子复合物的组织，并支持先前提出的CAP与RNAP α亚基C-末端结构域（α CTD）的相互作用，α CTD与σ（70）区4的相互作用，CAP和RNAP与启动子DNA的相互作用，以及复合物周围DNA的相DNA弯曲依赖性部分包裹。该结构还揭示了RNAP β '，β和sigma（70）亚基内物种特异性结构域的位置和形状。
We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-angstrom electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alpha CTD), interactions of alpha CTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.