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、转录和缩合的影响 抽象的 大分子拥挤（MMC）改变细胞内生物分子的浓度和亲和力， 促进液相分离。 MMC 已被证明可以改变 DNA 寡核苷酸的熔解温度，但是 关于它如何影响 DNA 机械稳定性的广泛表征尚不完整。拥挤的DNA 冷凝物可能会对 DNA 产生亚皮牛顿的回缩张力，可以使用以下方法方便地进行探索 磁/光镊子。虽然许多关于 DNA 马达的实验都采用了反对或协助的张力 几个到几十 pN 的易位，我们的小组表明亚皮牛顿张力会影响 DNA 拓扑结构，从 超螺旋到蛋白质介导的环，以及延伸的大肠杆菌 RNA 聚合酶的可能性 (RNAP) 克服了蛋白质障碍。令人惊讶的是，MMC 对超螺旋和超螺旋等拓扑的影响 蛋白质介导的循环以及转录、蛋白质扩散和缩合等过程尚未被研究。 特征良好。该提案旨在评估 MMC 对 DNA 构型（包括解旋）的影响 以及循环、蛋白质扩散和液相分离，将这些特征融入我们的理解中 细胞内分子生物学。为此，我们将单分子体外实验与体内实验相结合 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/或新颖系统，以 了解 MMC 如何改变 DNA 结构、影响蛋白质介导的循环以及改变转录。我们还将 研究 MMC 如何影响 ParB 介导的 DNA 扩散和液-液相分离 (LLPS) 这需要体外拥挤剂。然后我们建议构建人工 LLPS 系统来学习 将液-液相分离的液滴定位在 DNA 片段上需要哪些成分。 P-颗粒， Cajal 小体、segrosome 和核仁是包含特定基因组区域的 LLPS 的一些例子 并证明这种现象的普遍性和重要性。大分子拥挤产生力 影响基本 DNA 力学和拓扑结构，并且在过去十年中 MMC 已成为 LLPS。我们将体外实验与计算和理论方法相结合，并与 由合作者进行适当的体内测量。发现拥挤的机制 修改 DNA 配置、交易和分离将增进我们对基因组的理解 生物物理学和调控，并为合成生物学提供新工具。