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

• 批准号: 9236743
• 负责人: Katsuhiko Murakami
• 金额: $ 3.86万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 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/9236743
• 关键词: Address; Antibiotics; Bacteria; Bacterial RNA; Biochemical; Biochemistry; Cells; Complex; Consensus; 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; Health; Holoenzymes; Human; Laboratories; Modeling; Molecular; Motion; Mycobacterium tuberculosis; Nucleic Acids; Organism; Phase; Play; Polymerase; Positioning Attribute; Process; RNA; Regulation; Reporting; Research; Resolution; Roentgen Rays; Staphylococcus aureus; Structure; System; Time; Transcript; Transcription Elongation; X-Ray Crystallography; base; design; genetic approach; in vivo; inhibitor/antagonist; insight; interest; mutant; novel; overexpression; pathogen; research study; scaffold; screening; time use; transcription factor; transcriptome

 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（由申请人提供）：我们研究的长期目标是了解细胞RNA聚合酶（RNAP）的转录机制及其调控。在下一个项目期间，我们将研究细菌中的转录机制，以提供基本机制。最近，我们报道了大肠杆菌 RNAP s70 全酶的第一个 X 射线结构，这种酶是研究最多的 RNAP，并已被用作 RNAP 的模型。使用过表达系统可以方便地制备大肠杆菌 RNAP，从而探索 RNAP 结构研究的新方向，包括转录延伸复合物、暂停转录复合物、RNAP 与多种转录因子和抑制剂的复合物，如在这里，我们提出了大肠杆菌 RNAP 转录的结构和生化研究，以解决生产阶段转录的三个具体目标：我们结晶了大肠杆菌 RNAP。延伸复合物并在 6 Å 分辨率下确定了其 X 射线结构，这为基于结构的转录机制研究提供了框架：（1）确定延伸复合物结构的原子分辨率以分析相互作用。 RNAP和核酸之间；（2）用延伸因子NusG或RfaH确定延伸复合物的结构，以首次在转录泡中确定非模板DNA和上游DNA的位置。完整的延伸复合物；和（3）确定具有容易发生转录滑移的RNAP突变体的延伸复合物的结构，以了解转录保真度。因此，我们将以晶体形式延伸多个RNA碱基。使用时间分辨浸泡-触发-冷冻 X 射线晶体学进行晶体转录并记录桥螺旋和触发环的运动以及转录延伸过程中核酸的易位。 2.通过“暂停触发”序列阐明转录暂停的分子机制：大肠杆菌转录组的新生转录本测序鉴定出大肠杆菌基因组中的共有暂停序列我们将确定包含延伸复合物的晶体结构。目的 3. NusA 调节 RNAP 的结构基础共识：大多数转录延伸。我们将确定 NusA 与 RNAP 复合物以及延伸复合物的晶体结构，以阐明 NusA 依赖性暂停和终止的结构基础。