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

基因以RNA和蛋白质的形式编码分子工具。为了使用这些工具对信号做出反应并执行不同的行为，细胞仔细控制何时、何地以及多少基因被激活。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充分发挥作用，以预测和构建将在正确的地点、正确的时间和正确的水平上被激活的基因。这个项目探索了基因调控是一个多步骤的循环，在特定的点上受到调控，比如开车。一辆汽车包含许多相互作用的小部件，它们必须按照特定的顺序行动，以产生力量和移动，但要驾驶它，只需要操作点火装置、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是受调节的，哪些分子调节它们。这项工作将有助于开发新工具，这些工具将推动生物制造，使设计治疗疾病的新药成为可能，并将提供提高作物产量的新方法，以及将受益于精确控制生物学和生物技术能力的其他应用。几十年来，转录研究一直受到一个概念简单的模型的驱动：蛋白质与调节DNA平衡结合，以调节RNA聚合酶（RNAP）的募集。这导致了对转录因子（TF）、辅因子和基础转录机制之间的合作和竞争物理相互作用的关注，并奠定了对TF结合位点的“顺式调控代码”的研究的基础。该项目通过解决动力学过程如何组合调节转录来挑战转录控制的主导范式。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的慢速率数量，并测试该速率谱是否对细胞类型敏感（Aim 1）。tf通过其独特的生化活动加速或延缓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除来表征内源性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，该系统将通过进行基于生物物理学理论的定量实验来阐明基础生物学并执行有用的功能。

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.