Nucleosomal Proofreading of Activator-Promoter Recognition

本研究计划研究随机分子行为在通过“转录激活因子”调节基因表达中的作用，这些分子通过结合DNA中的特定调节序列来控制基因的活性。该项目向来自不同背景的学生介绍分子遗传学和统计物理学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。在教学方面，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——即基因对特定信号的转录反应——被认为完全取决于激活子基因识别的特异性。然而，对于许多真核生物激活因子来说，正确和错误序列结合之间的能量差异非常小，小到无法解释观察到的调节特异性。特异性问题可以通过“动力学校对”来解决，即在激活子-启动子结合和转录之间插入一个自由的能量转导延迟步骤。该机制不是通过增加正确和错误联想之间的能量差异来增强特异性，而是通过在延迟步骤之前和之后两次利用相同的差异来增强特异性，当自由能量耗散有利于复杂解离而不是形成并使悬浮延迟机制返回其抑制状态时。激活子校对的可能延迟机制之一是atp依赖的染色质重塑，它以激活子依赖的方式解除核小体抑制的基因。这一命题的逻辑含义，包括预测染色质重塑的扰动会影响激活子区分正确和不正确基因的能力，将通过在酵母中使用活细胞多焦点荧光显微镜、单分子荧光原位杂交、纳米孔测序和反向遗传学的组合来测试。这些结果将为生物系统的接近确定性行为如何从其分子组分的随机行为中产生提供新的见解。该项目由生物科学理事会分子与细胞生物科学部的遗传机制项目和数学与物理科学理事会数学科学部的数学生物学项目共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.