Complexes of CAP in Transcription Activation

描述(申请人提供)：我们目前的重点是两个简单的三组分分子复合体的结构分析，这些复合体在转录激活过程中由大肠杆菌分解代谢激活蛋白(CAP)、RNA聚合酶(RNAP)和启动子DNA形成。CAP和RNAP及其各自与启动子DNA元件的复合体的结构知识非常先进，在过去几年中取得了特别快的进展。相比之下，关于CAP和RNAP在两个简单的转录激活类别中对启动子DNA的相互作用的知识仍然主要基于生化和遗传数据。这项拟议工作的目标是推进这些相互作用的结构知识，以产生完整、准确的完整转录复合体的分子图像。目前的目标是使用X射线结晶学获得I类和II类CAP-RNAP-启动子DNA复合体的高分辨结构，以及使用冷冻电子显微镜获得完整的I类和II类CAP-RNAP-启动子DNA复合体的低分辨结构。这项工作将为两类主要的CAP依赖的启动子提供完整、准确的完整转录起始复合体的分子描述，包括对蛋白质-蛋白质相互作用、蛋白质-DNA相互作用、启动子DNA结构和溶剂作用的描述。CAP的转录激活比细菌和真核生物中的大多数转录激活例子要简单得多，后者可能涉及大分子成分和/或DNA结合位点的复杂排列。对CAP系统的拟议研究为我们提供了一种快速推进我们对转录控制基本结构原理的理解的手段。

DESCRIPTION (provided by applicant): Our current focus is on structural analysis of two simple three-component molecular complexes formed in transcription activation by the E. coil catabolite activator protein (CAP), RNA polymerase (RNAP), and promoter DNA. Structural knowledge of both CAP and RNAP and their individual complexes with promoter DNA elements is quite advanced, with particularly rapid progress made in the past few years. In contrast, knowledge about about the interactions between CAP and RNAP on promoter DNA in the two simple classes of transcription activation is still based largely on biochemical and genetic data. The goal of the proposed work is to advance structural knowledge of these interactions to yield complete, accurate molecular pictures of intact transcription complexes. The immediate aims are to obtain high-resolution structures of sub-assemblies of Class I and Class II CAP-RNAP-promoter DNA complexes using X-ray crystallography, and low resolution structures of intact Class I and Class II CAP-RNAP-promoter DNA complexes using cryo-electron microscopy. The work will yield complete, accurate molecular descriptions of intact transcription initiation complexes for the two major simple classes of CAP-dependent promoters, including descriptions of protein-protein interactions, protein-DNA interactions, promoter DNA structure, and role of solvent. Transcription activation by CAP is substantially simpler than most examples of transcription activation in bacteria and eukaryotes, which can involve complex arrangements of macromolecular components and/or DNA binding sites. The proposed studies of the CAP system provide a means to rapidly advance our understanding of fundamental structural principles underlying transcriptional control.