Structural Studies of Bacterial Transcription Initiation

描述(申请人提供)：转录，遗传信息从DNA转移到RNA，是基因表达的第一步。RNA转录本是由DNA依赖的RNA聚合酶(RNAP)的催化核心产生的，RNAP在所有细胞生物中都是高度保守的。在细菌中，核心RNAP与Sigma因子相互作用形成全酶，该全酶特异性地结合位于转录起始点上游的启动子DNA序列。在大多数细菌启动子中，具有转录功能的RNAP全酶开放启动子复合体(RPO)由最初的闭合启动子复合体(RPC)自发形成，途径包括瞬时中间体(RPI)。从RPC到RPO的这条途径是许多转录调控因子的靶标，这些转录调控因子能够选择性地表达某些基因，以响应环境胁迫。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关于RPO形成机制或其调控的重要问题。最近的研究发现，CARD是一种重要的RNAP结合蛋白，在结核分枝杆菌中调节rRNA转录，但其功能的分子机制尚不清楚。这项建议旨在解决有关RPO形成的详细机制，以及CARD通过测定相关大分子络合物的X射线晶体结构来调控转录启动的问题。具体地说，我们建议：1)对RPO形成途径上的中间体(RPI)进行结构表征。使用新的DNA结构，我们已经确定了初步的晶体结构，显示了预期的中间体特征。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。所获得的信息将为深入了解转录起始的这一关键步骤提供2)使用结构和功能相结合的方法来阐明CARD调控转录起始的分子机制。将对嗜热嗜热菌RNAP转录起始复合体进行结构和详细的生化研究。这些研究的结果将与结核分枝杆菌CARD的体内研究(来自合作者)相结合，以提供CARD的体外机制和体内功能的综合图景。 与公共健康相关：转录是从DNA制造基因产品所需的基本细胞过程，对细胞生存至关重要。这项建议旨在了解转录的中心酶RNA聚合酶启动转录的机制，以及它在细菌中的调控，包括在重要的病原体结核分枝杆菌中。 所获得的见解将为开发选择性靶向细菌RNA聚合酶功能的抗菌剂提供基础。

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. PUBLIC HEALTH RELEVANCE: Transcription is a fundamental cellular process required to make gene products from DNA and is essential for cell viability. This proposal aims to understand the mechanism of transcription initiation by RNA polymerase, the central enzyme of transcription, and its regulation in bacteria, including in the important pathogen M. tuberculosis. The insights obtained will provide a basis for the development of antimicrobial agents that selectively target bacterial RNA polymerase function.