Nucleosomal Proofreading of Activator-Promoter Recognition

这项研究计划调查随机分子行为在通过“转录激活物”调节基因表达方面的作用，转录激活物是通过与DNA中特定的调控序列结合来控制基因活动的分子。该项目向来自不同背景的学生介绍了分子遗传学和统计物理之间的一个基本理论问题，以及解决这个问题的先进实验工具。在教学上，该项目旨在将数学、物理和定量推理整合到本科生和研究生的生物学课程中，并为高中生提供研究机会，以增加对STEM领域的参与。转录调控的特异性-哪些基因被转录为对特定信号的反应-被认为完全取决于激活基因识别的特异性。然而，对于许多真核细胞激活剂来说，正确和错误序列结合之间的能量差异非常小--太小了，无法解释观察到的调控特异性。特异性问题可以通过“动力学校对”来解决，即在激活-启动子结合和转录之间插入一个自由能量传递延迟步骤。该机制不是通过增加正确和不正确关联之间的能量差异来提高特异性，而是通过在延迟步骤之前和之后两次利用相同的差异，当自由能量耗散有利于复杂的解离而不是形成并使暂停的延迟机制返回其抑制状态时。激活剂校正的可能延迟机制中的一个主要候选机制是ATP依赖的染色质重塑，它以激活剂依赖的方式解除基因的核小体抑制。这一命题的逻辑含义，包括染色质重塑的扰动影响激活剂区分正确和不正确基因的能力的预测，将通过结合使用活细胞多焦点荧光显微镜、单分子荧光原位杂交、纳米孔测序和酵母中的反向遗传学来进行测试。这些结果将为生物系统如何从其分子成分的随机行为中产生接近确定性的行为提供新的见解。该项目由生物科学局分子和细胞生物科学部的遗传机制计划和数学和物理科学局数学科学部的数学生物学计划联合资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This research program investigates the role of random molecular behavior in regulating gene expression by "transcriptional activators", molecules that control the activity of genes by binding to specific regulatory sequences in DNA. The project introduces students from diverse backgrounds to a fundamental theoretical problem at the interface between molecular genetics and statistical physics as well as advanced experimental tools to address this problem. Pedagogically, the program aims to integrate mathematical, physical, and quantitative reasoning into the biology curriculum for undergraduate and graduate students and provide research opportunities to high school students to increase participation in STEM fields.The specificity of transcriptional regulation – which genes are transcribed in response to a particular signal – is thought to rest entirely with the specificity of activator-gene recognition. Yet, for many eukaryotic activators the energetic differences between correct and incorrect sequence binding are remarkably small – too small to explain observed regulatory specificities. The specificity problem may be solved by "kinetic proofreading", i.e., insertion of a free energy-transducing delay step between activator-promoter binding and transcription. The mechanism enhances specificity not by increasing the energetic difference between correct and incorrect associations but by exploiting the same difference twice, before the delay step and afterward, when free energy dissipation favors complex dissociation over formation and return of the suspended delay mechanism to its repressive state. A prime candidate among possible delay mechanisms for activator proofreading is ATP-dependent chromatin remodeling, which relieves genes from nucleosomal repression in an activator-dependent manner. Logical implications of this proposition, including the prediction that perturbation of chromatin remodeling affects the ability of activators to distinguish between correct and incorrect genes, will be tested by employing a combination of live-cell multifocus fluorescence microscopy, single molecule-fluorescence in situ hybridization, nanopore sequencing, and reverse genetics in yeast. The outcomes will provide new insights on how near deterministic behavior of biological systems emerges from the random behavior of their molecular components.This project is jointly funded by the Genetic Mechanisms program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate and the Mathematical Biology program of the Mathematical Sciences Division in the Mathematical and Physical Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.