Collaborative Research: NSF/MCB: Kinetic Control of the Transcription Cycle Revealed by Synthetic Enhancers

合作研究：NSF/MCB：合成增强剂揭示的转录周期动力学控制

• 批准号: 1715184
• 负责人: Angela DePace
• 金额: $ 70万
• 依托单位: Harvard University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1715184&HistoricalAwards=false
• 关键词: Collaborative; Research; NSF; MCB; Kinetic

Genes encode molecular tools in the form of RNA and protein. To use these tools to respond to signals and execute different behaviors, cells carefully control when, where and how much genes are turned on. This is called 'gene regulation'. A major goal of synthetic biology is to engineer cells to perform specific useful tasks. To accomplish this goal, researchers must learn how gene regulation works well enough to predict and build genes that will be turned on in exactly the right place, at the right time and to the right level. This project explores the idea that gene regulation is a multi-step cycle that is regulated at specific points, like driving a car. A car contains many small interacting parts that have to act in a specific order to produce force and move, but to drive it, one only need to operate the ignition, the gas, the steering wheel and the brakes. The investigators in the project are trying to identify which steps are regulated, and which molecules regulate them. The work will contribute to development of new tools that will advance biomanufacturing, enable the design of new drugs to treat disease, and will provide new methods for enhancing crop yield, and other applications that will benefit from the ability to precisely control biology and biotechnology.For decades, transcription research has been driven by a model that is conceptually simple: proteins bind to regulatory DNA at equilibrium to regulate recruitment of RNA polymerase (RNAP). This has led to a focus on cooperative and competitive physical interactions between transcription factors (TFs), cofactors and the basal transcriptional machinery, and underlies the search for the still hypothetical 'cis-regulatory code' of TF binding sites. This project challenges this dominant paradigm of transcriptional control by addressing how kinetic processes could combinatorially regulate transcription. Because promoters dictate rate limiting steps for transcription, we will measure the number of slow rates at multiple promoters using high resolution imaging, and test whether this rate spectrum is sensitive to cell type (Aim 1). TFs accelerate or retard different parts of this complex, dynamic cycle through their distinct biochemical activities. This project will use functional genomics and knock-down to characterize TF function at endogenous genes and at synthetic enhancers (Aim 2). The long-term goal is to predict and build synthetic gene regulatory systems that will both illuminate fundamental biology and perform useful functions, by performing quantitative experiments that are contextualized with theory based in biophysics.

基因以RNA和蛋白质的形式编码分子工具。为了使用这些工具来响应信号并执行不同的行为，细胞仔细控制基因何时，何地以及多少被打开。这被称为“基因调控”。合成生物学的一个主要目标是设计细胞来执行特定的有用任务。为了实现这一目标，研究人员必须了解基因调控如何发挥作用，以预测和构建基因，这些基因将在正确的地方，正确的时间和正确的水平被打开。 这个项目探讨了基因调控是一个多步骤循环的想法，在特定的点进行调节，就像驾驶汽车一样。 汽车包含许多相互作用的小部件，它们必须以特定的顺序产生力并移动，但要驾驶它，人们只需要操作点火，汽油，方向盘和刹车。 该项目的研究人员正试图确定哪些步骤受到调节，哪些分子调节它们。 这项工作将有助于开发新的工具，这些工具将促进生物制造，使新药的设计能够治疗疾病，并将提供提高作物产量的新方法，以及其他将受益于精确控制生物学和生物技术能力的应用。蛋白质在平衡状态下与调节DNA结合以调节RNA聚合酶（RNAP）的募集。这导致了一个焦点的合作和竞争性的物理相互作用之间的转录因子（TF），辅因子和基础的转录机制，并强调搜索仍然假设的“顺式调节代码”的TF结合位点。这个项目通过解决动力学过程如何组合调节转录来挑战这种主导的转录控制范式。由于启动子决定转录的速率限制步骤，我们将使用高分辨率成像测量多个启动子处的慢速率的数量，并测试该速率谱是否对细胞类型敏感（目标1）。TF通过其独特的生物化学活动加速或延迟这个复杂的动态循环的不同部分。该项目将使用功能基因组学和敲低来表征内源基因和合成增强子的TF功能（目标2）。长期目标是预测和构建合成基因调控系统，通过执行基于生物物理学理论的定量实验，阐明基础生物学并执行有用的功能。