Protein-Mediated DNA Looping under Intrinsic and External Constraints

内在和外在约束下蛋白质介导的 DNA 循环

• 批准号: 0957293
• 负责人: Jens Meiners
• 金额: $ 36万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0957293&HistoricalAwards=false
• 关键词: Protein; Mediated; DNA; Looping; under

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.

在这个项目中，PI将研究机械约束对基于大肠杆菌乳糖阻遏物的模型系统中转录调控的影响。大肠杆菌，使用单分子技术。该系统是蛋白质介导的DNA成环的范例，这是转录和复制调节的主要主题，并且在生物化学和结构上具有良好的特征。PI将研究细胞环境可能施加的三种机械约束，以及它们在蛋白质介导的DNA循环中的作用。首先，PI将使用具有专门设计的弯曲的DNA构建体和单分子竞争测定来研究限制DNA上的操纵位点相对于蛋白质的角取向的影响。这些实验将揭示了一个三个数量级的差异，在环的寿命测量散装竞争测定和单分子拴系粒子显微镜的起源，并深入了解如何超螺旋增强环的形成。其次，PI将研究来自波动的细胞环境的噪声如何影响环形成过程的鲁棒性，这将有助于理解环形成基因开关如何可靠地工作，尽管它对小力非常敏感。第三，PI将着眼于模拟细胞环境的凝胶中的环形成和分解，特别强调理解为什么DNA-蛋白质复合物在凝胶中显着更稳定，即使当孔径如此之大，凝胶几乎不应该成为蛋白质扩散的障碍。PI将使用高度跨学科的方法培养生物物理学的研究生和本科生。将特别强调招聘代表性不足的少数民族学生，PI还将参与广泛的外联工作，包括关于生物物理学普遍感兴趣的主题的公开讲座，以及参加高中单分子路演。