Structural Studies of Bacterial Transcription Initiation

描述（申请人提供）：转录，即遗传信息从DNA到RNA的转移，是基因表达的第一步。 RNA转录物由DNA依赖性RNA聚合酶（RNAP）的催化核心产生，其在所有细胞生物体中高度保守。在细菌中，核心RNAP与σ因子相互作用以形成全酶，其特异性结合位于转录起始位点上游的启动子DNA序列。在大多数细菌启动子中，有转录能力的RNAP全酶开放启动子复合物（RPo）通过包括瞬时中间体（RPi）的途径从初始封闭启动子复合物（RPc）自发形成。从RPc到RPo的这一途径是许多转录调节因子的靶点，这些转录调节因子使某些基因能够响应环境胁迫而选择性表达。虽然RPo的详细结构模型与许多生化和生物物理实验的结果相一致，但这些模型并没有解决有关RPo形成机制及其调控的重要问题。最近的研究表明，CardD是一种重要的RNAP结合蛋白，在病原体结核分枝杆菌中调节rRNA转录，但CardD功能的分子机制尚不清楚。该提案旨在解决有关RPo形成的详细机制以及通过相关大分子复合物的X射线晶体学结构测定由CardD调节转录起始的问题。具体而言，我们提出：1）结构表征RPo形成途径上的中间体（RPi）。使用新的DNA结构，我们已经确定了初步的晶体结构，显示预期的功能的中间体。将进行进一步的结构和生化分析，以确定RPi模型并验证其相关性。所获得的信息将提供对转录起始的这一关键步骤的深入了解，以及2）使用结构和功能方法的组合来阐明由CardD调节转录起始的分子机制。对T.嗜热菌RNAP转录起始复合物与T.将进行嗜热菌CardD。这些研究的结果将与M.结核病CardD（来自合作者），以提供CardD体外机制和体内功能的全面图片。

DESCRIPTION (provided by applicant): Transcription, the transfer of genetic information from DNA to RNA, is the first step of gene expression. RNA transcripts are generated by the catalytic core of the DNA dependent RNA polymerase (RNAP), which is highly conserved among all cellular organisms. In bacteria, the core RNAP interacts with the sigma factor to form the holoenzyme that specifically binds to promoter DNA sequences located upstream of transcription start sites. At most bacterial promoters, the transcription-competent RNAP holoenzyme open promoter complex (RPo) forms spontaneously from the initial closed promoter complex (RPc) through a pathway that includes transient intermediates (RPi). This pathway from RPc to RPo is the target of many transcriptional regulators that enable selective expression of certain genes in response to environmental stress. While detailed structural models of RPo reconcile the results of many biochemical and biophysical experiments, these have not addressed important questions regarding the mechanism of RPo formation nor its regulation. Recent studies identified CarD as an essential RNAP binding protein that regulates rRNA transcription in the pathogen Mycobacterium tuberculosis, but the molecular mechanism for CarD function is unknown. This proposal aims to address questions regarding the detailed mechanism of RPo formation, and the regulation of transcription initiation by CarD through X-ray crystallographic structure determination of relevant macromolecular complexes. Specifically, we propose to: 1) structurally characterize intermediates (RPi) on the pathway of RPo formation. Using novel DNA constructs, we have determined preliminary crystal structures that show expected features of intermediates. Further structural and biochemical analyses will be conducted to determine RPi model and validate its relevance. The information obtained will provide insight into this key step of transcription initiation~ and 2) Use a combination of structural and functional approaches to elucidate the molecular mechanism for the regulation of transcription initiation by CarD. Structural and detailed biochemical studies of T. thermophilus RNAP transcription initiation complexes with T. thermophilus CarD will be conducted. The results of these studies will be combined with in vivo studies of M. tuberculosis CarD (from collaborators) to provide a comprehensive picture of CarD in vitro mechanism and in vivo function.