Macromolecular Crowding effects on DNA mechanics, topology and transcription

大分子拥挤对DNA力学、拓扑学、转录和凝聚的影响 摘要 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已显示改变DNA寡核苷酸的解链温度，但 对它如何影响DNA的机械稳定性的广泛表征是不完整的。拥挤的DNA 凝聚物可能会对DNA产生亚微微牛顿的伸缩张力，可以使用 磁性/光学镊子。虽然许多关于DNA马达的实验使用了张力， 几个到几十个pN的易位，我们的小组表明，亚皮牛顿张力影响DNA拓扑结构，从 蛋白质介导的成环的超螺旋，以及延长E.大肠杆菌RNA聚合酶 （RNAP）超越了蛋白质路障。令人惊讶的是，MMC对拓扑结构，如超螺旋和 蛋白质介导的环，以及转录、蛋白质扩散和缩合等过程尚未被发现。 很好的描述。该建议旨在评估MMC对DNA构型（包括解旋）的影响 以及成环、蛋白质扩散和液相分离，将这些特征整合到我们的理解中， 细胞内分子生物学为了做到这一点，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， 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。我们还将 研究MMC如何影响ParB介导的沿着DNA的扩散和液-液相分离（LLPS） 这需要体外的拥挤剂。然后，我们建议建立人工LLPS系统， 将液-液相分离的液滴定位在DNA片段上需要什么组分。P颗粒， Cajal小体、分裂体和核仁是包含特定基因组区域的LLPS的一些实例 并证明这种现象的普遍性和重要性。大分子拥挤产生力 影响基本的DNA力学和拓扑结构，在过去的十年中，MMC已经成为 LLPS。我们将把体外实验与计算和理论方法结合起来， 由合作者进行适当的体内测量。发现拥挤的机制 改变DNA的构型、交换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.