Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854, USA.
Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19830-5. doi: 10.1073/pnas.0908782106. Epub 2009 Nov 10.
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-A 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 (alphaCTD), interactions of alphaCTD 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.
我们展示了一个完整的激活剂依赖转录起始复合物的实验确定的 3D 结构，该复合物由大肠杆菌代谢物激活蛋白（CAP）、RNA 聚合酶全酶（RNAP）和一个包含位置 -78 到 +20 的 DNA 片段组成，该片段包含一个 CAP 位点在位置 -61.5 和一个预熔化的转录泡的 I 类 CAP 依赖启动子。通过在碳砂负染色中可视化的单颗粒的迭代基于投影的匹配获得了 20-A 电子显微镜重建，并使用 CAP、RNAP 和 DNA 的原子坐标集进行拟合。该结构定义了 I 类 CAP-RNAP-启动子复合物的组织，并支持 CAP 与 RNAP alpha 亚基 C 末端结构域（alphaCTD）的先前提出的相互作用、alphaCTD 与 sigma(70)区域 4 的相互作用、CAP 和 RNAP 与启动子 DNA 的相互作用以及相移 DNA 弯曲依赖性的 DNA 围绕复合物的部分缠绕。该结构还揭示了 RNAP beta'、beta 和 sigma(70)亚基中特定于物种的结构域的位置和形状。
