Structural Dynamics and Mechanochemical Coupling in Nucleoprotein Machines

摘要 核蛋白是DNA生物学和生物技术的核心功能。 机器。在这些动态的大分子组装中，DNA双链被结合并 在与蛋白质和有时与RNA的复合体中扭曲。生物物理测量和 需要模型来理解这些机器的机制，在这些机器中，协调 蛋白质和核酸成分的构象变化与化学物质相耦合 如骨架裂解或核苷酸水解等步骤。这是一份续签申请 这是我们之前开发的高分辨率和多模单分子 方法及其在阐明三磷酸腺苷依赖的机械力化学偶联中的应用 来自大肠杆菌的超卷曲电机DNA旋转酶。在这里，我们建议利用我们的方法和 剖析附加核蛋白机器的动力学和机制的见解， 聚焦于RNA引导的核酸酶Cas9和Cas12a并比较DNA旋转酶马达 跨物种。我们将描述DNA审问和DNA超级卷曲中的子步骤， 能量格局和动力学的分子决定因素，以及机械的影响 基因组中经历过的菌株。如果成功，该项目将确定物理 RNA引导的核酸酶在动态和机械中询问DNA的机制 详细信息，提供对符合以下条件的酶的目标搜索过程的定量描述 目前正被用于基因编辑和快速扩展的一系列其他应用 涉及针对基因组中的位置的特定靶向的活动。新的DNA旋转酶 测量将进一步阐明生物物理特化、结构特性和 作为抗菌药物重要靶点的酶的机械调节。最后， 在这些生物物理问题的推动下，单分子方法的发展将具有广阔的前景 在从转录到核小体重塑的各种系统中的应用。

SUMMARY Central functions in DNA biology and biotechnology are carried out by nucleoprotein machines. In these dynamic macromolecular assemblies, the DNA duplex is bound and distorted in complex with protein and sometimes RNA. Biophysical measurements and models are needed to understand the mechanisms of these machines, in which coordinated conformational changes in protein and nucleic acid components are coupled with chemical steps such as backbone cleavage or nucleotide hydrolysis. This is a renewal application for a grant in which we previously developed high-resolution and multimodal single-molecule approaches and applied them to elucidate mechanochemical coupling in the ATP-dependent supercoiling motor DNA gyrase from E. coli. Here, we propose to leverage our methods and insights to dissect the dynamics and mechanics of additional nucleoprotein machines, focusing on the RNA-guided nucleases Cas9 and Cas12a and comparing DNA gyrase motors across species. We will characterize substeps in DNA interrogation and DNA supercoiling, molecular determinants of energy landscapes and kinetics, and the effects of mechanical strains experienced in the genome. If successful, the project will determine the physical mechanisms of DNA interrogation by RNA-guided nucleases in dynamic and mechanical detail, providing a quantitative description of the target search process for enzymes that are currently being exploited for gene editing and for a rapidly expanding set of other applications involving specific targeting of activities to sites in the genome. New DNA gyrase measurements will further elucidate biophysical specializations, structural properties, and mechanical regulation of enzymes that are important targets for antibacterial drugs. Finally, single-molecule methods development driven by these biophysical questions will have broad applications in systems ranging from transcription to nucleosome remodeling.