Protein-Mediated DNA Looping under Intrinsic and External Constraints

在这个项目中，PI将利用单分子技术，在一个基于大肠杆菌乳糖抑制物的模型系统中，研究机械约束对转录调控的影响。这个系统是蛋白质介导的DNA环的范例，是转录和复制调控的主要主题，在生化和结构上都有很好的特征。PI将研究细胞环境可能施加的三个机械约束，以及它们在蛋白质介导的DNA环路中的作用。首先，PI将使用带有特殊设计的弯曲的DNA结构和单分子竞争分析来研究限制DNA上操纵点相对于蛋白质的角度方向的影响。这些实验将阐明环路寿命中三个数量级差异的起源，通过整体竞争分析和单分子拴系粒子显微镜进行测量，并深入了解超螺旋如何增强环路的形成。其次，PI将研究来自波动的蜂窝环境的噪声如何影响环路形成过程的稳健性，这将有助于理解环路形成基因开关如何可靠地工作，尽管它对微小的力非常敏感。第三，PI将研究模拟细胞环境的凝胶中环的形成和分解，特别强调理解为什么DNA-蛋白质复合体在凝胶中显著更稳定，即使在孔径如此之大的情况下，凝胶应该几乎不会成为蛋白质扩散的障碍。PI将使用高度跨学科的方法，培训生物物理方面的研究生和本科生。将特别重视招收代表性不足的少数族裔学生，国际学生联合会还将开展广泛的外联工作，包括就生物物理普遍感兴趣的主题进行公开讲座，以及参加高中的单分子路演。

In this project the PI will study the effects of mechanical constraints on transcription regulation in a model system based on the lactose repressor in E. Coli, using single-molecule techniques. This system is a paradigm for protein-mediated DNA looping a major theme in the regulation of transcription and replication and is biochemically and structurally well characterized. The PI will look into three mechanical constraints that a cellular environment might impose, and their role in protein-mediated DNA looping. First, the PI will study the effect of constraining the angular orientation of the operator sites on the DNA with respect to the protein using DNA constructs with specially designed bends and single-molecule competition assays. These experiments will shed light on the origin of a three-order of magnitude discrepancy in loop lifetimes measure by bulk competition assays and single-molecule tethered-particle microscopy and give insight into how supercoiling enhanced loop formation. Second, the PI will study how noise from a fluctuating cellular environment affects the robustness of the loop formation process, which will help to understand how a loop-forming genetic switch can work reliably despite its extreme sensitivity to small forces. Third, the PI will look at loop formation and breakdown in gels that mimic cellular environments with particular emphasis on understanding why DNA-protein complexes are significantly more stable in gels even when the pore size is so large that the gel should hardly be an obstacle for protein diffusion. The PI will train graduate and undergraduate students in biological physics, using a highly interdisciplinary approach. Special emphasis will be placed on the recruitment of underrepresented minority students and the PI will also engage in a broad range of outreach efforts that include public lectures on topics of general interest in biological physics, and participation in a Single-Molecule Roadshow for high schools.