Macromolecular Crowding effects on DNA mechanics, topology and transcription

大分子拥挤对 DNA 力学、拓扑和转录的影响

• 批准号: 10623720
• 负责人: Laura Finzi
• 金额: $ 38.44万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623720
• 关键词: Affect; Affinity; Binding; Biophysics; Cell Nucleolus; Crowding; Cytoplasmic Granules; DNA; DNA Maintenance; DNA Structure; DNA-Directed RNA Polymerase; Escherichia coli; Genetic Transcription; Genome; Genomic Segment; Genomics; In Vitro; Learning; Liquid substance; Magnetism; Measurement; Mechanics; Mediating; Modeling; Molecular Biology; Motor; Oligonucleotides; Phase; Physical condensation; Probability; Process; Proteins; Regulation; Shapes; System; Techniques; Temperature; experimental study; in vivo; laser tweezer; melting; novel; optic tweezer; segregation; single molecule; synthetic biology; tool

Effects of macromolecular crowding on DNA mechanics, topology, transcription, and condensation ABSTRACT Macromolecular crowding (MMC) changes the concentration and affinities of intracellular biomolecules and promotes liquid phase separation. MMC has been shown to change the melting temperature of DNA oligos, but broad characterization of how it affects the mechanical stability of DNA is incomplete. Crowded DNA condensates may generate sub-piconewton retractile tension on DNA, which can be conveniently explored using magnetic/optical tweezers. While many experiments on DNA motors employ tensions opposing or assisting translocation of several to tens of pN, our group showed that sub-piconewton tension affects DNA topology, from supercoiling to protein-mediated looping, as well as the probability that an elongating E. coli RNA polymerase (RNAP) surpasses a protein roadblock. Surprisingly, the effect of MMC on topologies such as supercoiling and protein-mediated loops, and processes such as transcription, protein spreading, and condensation has not been well characterized. This proposal aims to assess the effects of MMC on DNA configurations including unwinding and looping, protein spreading, and liquid phase separation to integrate these features into our understanding of intracellular molecular biology. To do so, we integrate single-molecule, in vitro experiments with in vivo measurements and computational/theoretical approaches Over the next five years, we will analyze both model and/or novel systems with single-molecule techniques to learn how MMC changes DNA structure, affects protein-mediated looping, and alters transcription. We will also investigate how MMC influences ParB-mediated spreading along DNA and liquid-liquid phase separation (LLPS) which requires crowding agents in vitro. Then we propose to build artificial LLPS systems with which to learn what components are required to localize a liquid-liquid phase separated droplet on a DNA segment. P-granules, Cajal bodies, segrosome, and the nucleolus are some examples of LLPS that include specific genomic regions and demonstrate the ubiquity and importance of this phenomenon. Macromolecular crowding generates forces that affect fundamental DNA mechanics and topology and in the last decade MMC has emerged as a driver of LLPS. We will integrate in vitro experiments with computational and theoretical approaches and compare with appropriate in vivo measurements performed by a collaborator. Discovering the mechanisms by which crowding modifies DNA configurations, transactions, and segregation will advance our understanding of genome biophysics and regulation and provide new tools for synthetic biology.

大分子拥挤对DNA力学，拓扑，转录和冷凝的影响 抽象的 大分子拥挤（MMC）改变了细胞内生物分子和亲和力 促进液相分离。 MMC已显示会改变DNA寡聚的熔化温度，但 它如何影响DNA的机械稳定性的广泛表征是不完整的。拥挤的DNA 冷凝水可能会在DNA上产生亚皮孔沃顿伸缩张力，可以使用它方便地探索 磁性/光学镊子。虽然许多关于DNA电动机的实验采用相反或协助的紧张局势 我们的小组表明，几个到数十只PN的易位，亚皮孔张力会影响DNA拓扑。 超串联到蛋白质介导的环，以及拉长大肠杆菌RNA聚合酶的概率 （RNAP）超过蛋白质障碍。令人惊讶的是，MMC对诸如超螺旋和拓扑的影响 蛋白质介导的环，以及诸如转录，蛋白质扩散和冷凝等过程 特征很好。该建议旨在评估MMC对DNA配置的影响 并循环，蛋白质扩散和液相分离，以将这些特征整合到我们的理解中 细胞内分子生物学。为此，我们将单分子的体外实验与体内整合 测量和计算/理论方法 在接下来的五年中，我们将使用单分子技术分析模型和/或新型系统 了解MMC如何改变DNA结构，影响蛋白质介导的循环并改变转录。我们也会 研究MMC如何影响PARB介导的沿DNA和液体液相分离（LLP）的扩散 这需要体外拥挤的代理。然后，我们建议建立人工LLPS系统 需要哪些组件将液态相分离的液滴定位在DNA段中。 P颗粒， Cajal体，表层和核仁是包括特定基因组区域的LLP的一些例子 并证明这种现象的无处不在和重要性。大分子拥挤产生力 这会影响基本的DNA力学和拓扑，在过去的十年中，MMC已成为 llps。我们将与计算和理论方法进行体外实验，并与 合作者由合作者执行的合适体内测量。发现拥挤的机制 修改DNA构型，交易和隔离将提高我们对基因组的理解 生物物理学和调节，并为合成生物学提供新的工具。