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。为了使用这些工具来响应信号并执行不同的行为，细胞仔细地控制何时、何地和多少基因被打开。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足够好的作用，以预测和构建将在正确的地点、正确的时间和正确的水平启动的基因。这个项目探索了这样一种观点，即基因调控是一个多步骤的循环，在特定的时间点进行调控，比如开车。汽车包含许多小的相互作用的部件，这些部件必须按照特定的顺序动作才能产生力和移动，但要驱动它，一个人只需要操作点火、油门、方向盘和刹车。该项目的研究人员正在试图确定哪些步骤受到调控，以及哪些分子对它们进行调控。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，使治疗疾病的新药的设计成为可能，并将为提高作物产量提供新的方法，以及其他将受益于精确控制生物学和生物技术的能力的应用。几十年来，转录研究一直由一个概念上简单的模型驱动：蛋白质在平衡时与调节DNA结合，以调节RNA聚合酶(RNAP)的招募。这导致了转录因子(TF)、辅因子和基本转录机制之间的合作和竞争物理相互作用的关注，并为寻找仍然假设的TF结合位点的顺式调节代码奠定了基础。这个项目通过研究动力学过程如何组合调控转录来挑战这种占主导地位的转录控制范式。因为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来测量多个启动子的慢速率的数量，并测试该速率谱是否对细胞类型敏感(目标1)。TFS通过其不同的生化活动加速或延缓这一复杂、动态循环的不同部分。该项目将使用功能基因组学和基因敲除来表征内源基因和合成增强剂中的转铁蛋白功能(目标2)。长期目标是通过进行与生物物理学理论相联系的定量实验，预测和建立合成的基因调控系统，既能阐明基础生物学，又能发挥有用的功能。

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.