X-Ray Crystallographic Studies of Multi-Subunit Nucleic Acid Polymerases

多亚基核酸聚合酶的 X 射线晶体学研究

基本信息

批准号：
9203058
负责人：
Katsuhiko Murakami
金额：
$ 33.79万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-15 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9203058
关键词：
Address Antibiotics Bacteria Bacterial RNA Biochemical Biochemistry Cells Complex Complex Analysis Consensus Crystallization DNA DNA-Directed RNA Polymerase Development Disease Elongation Factor Enzymes Escherichia coli Evolution Freezing Gene Expression Gene Expression Regulation Genetic Code Genetic Transcription Genome Goals Holoenzymes Human Laboratories Modeling Molecular Motion Mycobacterium tuberculosis Nucleic Acids Organism Phase Play Polymerase Positioning Attribute Process RNA RNA analysis Regulation Reporting Research Resolution Roentgen Rays Staphylococcus aureus Structure System Time Transcript Transcription Elongation Transcriptional Regulation X-Ray Crystallography antitermination base design experimental study genetic approach in vivo inhibitor/antagonist insight interest mutant novel overexpression pathogen public health relevance scaffold screening time use transcription factor transcriptome

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand transcription mechanisms of cellular RNA polymerase (RNAP) and its regulation. During the next project period, we will study the transcription machinery in bacteria to provide a fundamental mechanism of transcription, which is conserved from bacteria to human. Recently, we reported the first X-ray structure of the Escherichia coli RNAP s70 holoenzyme. This enzyme is the most studied RNAP and has been used as a model RNAP for understanding the mechanism of transcription. E. coli RNAP is conveniently prepared using an overexpression system, which allows exploring new directions in RNAP structural studies including the transcription elongation complex, paused transcription complex, RNAP in complex with a variety of transcription factors and inhibitors, as well as RNAP mutants. Here, we propose structural and biochemical studies of E. coli RNAP transcription to address three specific aims. Aim1. Structural basis for productive-phase transcription: We crystallized an E. coli RNAP elongation complex and determined its X-ray structure at 6 Å resolution, which provides a framework for the structure- based study of the transcription mechanism. Further experiments are proposed: (1) to determine the atomic resolution structure of the elongation complex for analyzing interactions between RNAP and nucleic acids; (2) to determine structures of the elongation complex with elongation factors NusG or RfaH for determining the positions of the non-template DNA in the transcription bubble and the upstream DNA for the first time in the context of an intact elongation complex; and (3) to determine the structure of the elongation complex with an RNAP mutant prone to transcription slippage for understanding transcriptional fidelity. Our E. coli RNAP elongation complex crystal can extend multiple RNA bases in crystal form. Therefore, we will carry out in crystallo transcription and record motions of the bridge helix and trigger loo as well as translocation of nucleic acids during transcription elongation using time-resolved soak-trigger-freeze X-ray crystallography. Aim 2. Elucidate the molecular mechanism of transcription pausing by the "pause-trigger" sequence: Nascent transcript sequencing of the E. coli transcriptome identified a consensus pause sequence in the E. coli genome. We will determine the crystal structure of the elongation complex containing the consensus pause sequence to reveal the interplay between RNAP and nucleic acids during transcription pausing and to provide novel insight into gene regulation. Aim 3. Structural basis for RNAP modulation by NusA: Most transcription elongation complexes in vivo associate with NusA, which stimulates the effect of RNA hairpins for pausing and termination. We will determine the crystal structures of NusA in complex with RNAP and also with the elongation complex to elucidate the structural basis for NusA-dependent pausing and termination.

描述（通过应用程序提供）：我们研究的长期目标是了解细胞RNA聚合酶（RNAP）的转录机制及其调节。在下一个项目时期，我们将研究细菌中的转录机制，以提供转录的基本机制，该机制是从细菌到人类的保守的。最近，我们报道了大肠杆菌RNAP S70 Holoenzyme的第一个X射线结构。该酶是研究最多的RNAP，已用作理解转录机理的模型RNAP。大肠杆菌RNAP是使用过表达系统方便地制备的，该系统允许在RNAP结构研究中探索新方向，包括转录复合物，暂停的转录复合物，与多种转录因子和抑制剂以及RNAP突变体的复合物中的RNAP。在这里，我们提出了大肠杆菌转录的结构和生化研究，以解决三个特定目标。 AIM1。生产阶段转录的结构基础：我们结晶了大肠杆菌RNAP伸长率复合物，并以6Å分辨率确定了其X射线结构，这为基于结构的转录机制提供了框架。提出了进一步的实验：（1）确定伸长复合物的原子分辨率结构，以分析RNAP和核酸之间的相互作用；（2）确定用NUSG或RFAH确定伸长率复合物的结构，以确定在完整的伸长复合物的背景下，首次在转录气泡和上游DNA中确定了非转换DNA的位置；（3）用RNAP突变体的伸长率复合物的结构容易转录，以理解转录保真度。我们的大肠杆菌RNAP伸长复合物晶体可以以晶体形式延伸多个RNA碱基。因此，我们将使用时间分辨的浸泡 - 触发冻结X射线晶体图进行转录伸长期间的桥螺旋和触发液体的核酸螺旋和核酸的易位。目标2。阐明了通过“暂停触发”序列暂停转录的分子机制：大肠杆菌转录组的新生转录测序确定了大肠杆菌基因组中的共有暂停序列。我们将确定包含共有暂停序列的伸长络合物的晶体结构，以揭示转录过程中RNAP和核酸之间的相互作用，并提供对基因调节的新颖洞察力。 AIM 3。由NUSA调制RNAP的结构基础：体内大多数转录伸长复合物与NUSA辅助，这刺激了RNA发夹对暂停和终止的影响。我们将确定NUSA与RNAP的晶体结构以及伸长复合物的复合物，以阐明NUSA依赖性暂停和终止的结构基础。