Macromolecular Crowding effects on DNA mechanics, topology and transcription

大分子拥挤对DNA力学、拓扑、转录和缩合的影响 摘要 大分子拥挤(MMC)改变了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寡聚的熔化温度，但 关于它如何影响DNA的机械稳定性的广泛描述是不完整的。拥挤的DNA 凝结物可能会在DNA上产生亚皮牛顿的伸缩张力，这可以方便地使用 磁性/光学镊子。虽然许多关于DNA马达的实验都使用了张力来反对或帮助 几个到几十个Pn的移位，我们的团队表明亚微微牛顿的张力影响DNA拓扑，从 超螺旋到蛋白质介导的环，以及延长的大肠杆菌RNA聚合酶 (RNAP)超越了蛋白质障碍。令人惊讶的是，MMC对超级卷曲和 蛋白质介导的环，以及转录、蛋白质扩散和缩合等过程还没有 很好的特点。该提案旨在评估MMC对DNA构型的影响，包括解离 以及循环、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 在接下来的五年里，我们将用单分子技术分析模型和/或新系统，以 了解MMC如何改变DNA结构、影响蛋白质介导的环路和改变转录。我们还将 研究MMC如何影响PARB介导的DNA扩散和液-液相分离(LLP) 这需要体外的拥挤剂。然后，我们建议建立人工LLP系统，用于学习 在DNA片段上定位液-液相分离的液滴需要哪些成分。P-颗粒， Cajal小体、segroome和核仁是包括特定基因组区域的LLP的一些例子 并证明了这一现象的普遍性和重要性。大分子拥挤产生力量 影响基本的DNA力学和拓扑结构，在过去的十年中，MMC已经成为 有限责任公司。我们将把体外实验与计算和理论方法相结合，并与 合作者进行适当的活体测量。发现拥挤的机制 修改DNA配置、交易和分离将促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的工具。

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.