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

 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，以提供从细菌到人类的基本转录机制。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，并已被用作理解转录机制的模型RNAP。E.利用过表达系统可以方便地制备大肠杆菌RNAP，这使得RNAP结构研究的新方向得以探索，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以及RNAP突变体。在这里，我们提出了结构和生化研究的E。coli RNAP转录，以解决三个特定的目标。目标1.生产期转录的结构基础：我们结晶了一个E。coliRNAP延伸复合物，并在6 μ m分辨率下测定了其X射线结构，为基于结构的转录机制研究提供了框架。提出了进一步的实验：（2）确定具有延伸因子NusG或RfaH的延伸复合物的结构，用于在完整延伸复合物的情况下首次确定转录泡中的非模板DNA和上游DNA的位置;和（3）确定具有易于转录滑移的RNAP突变体的延伸复合物的结构，以理解转录保真度。我们的E。coli RNAP延伸复合物晶体可以以晶体形式延伸多个RNA碱基。因此，我们将在晶体中进行转录，并使用时间分辨浸泡-触发-冻结X射线晶体学记录桥螺旋和触发loo的运动以及转录延伸期间核酸的易位。目标二。通过“暂停-触发”序列阐明转录暂停的分子机制：E.在大肠杆菌转录组中发现了一个共有的暂停序列。coli基因组。我们将确定含有一致暂停序列的延伸复合物的晶体结构，以揭示RNAP和核酸在转录暂停过程中的相互作用，并为基因调控提供新的见解。目标3：NusA调节RNAP的结构基础：体内大多数转录延伸复合物与NusA相关，NusA刺激RNA发夹的暂停和终止作用。我们将确定与RNAP复合的NusA的晶体结构，并与延伸复合物一起阐明NusA依赖性暂停和终止的结构基础。

 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.