The Biochemistry of Clock Function in Fluctuating Environments

项目摘要 昼夜节律是行为的每日振荡，具有近24小时的周期，由 一个内在的生物钟。在许多生物体中，当昼夜节律发生变化时，健康和体能会受到损害 时钟与外部环境中的日常周期不适当地同步。它是这样的 对于了解时钟如何响应具有挑战性的波动环境至关重要 不规律的输入是现代生活的典型。这个问题在概念上具有挑战性，因为 生物钟是一个复杂的系统。一般来说，有一个由生化组成的核心振荡器 一种产生有节奏的日常信号的电路，这种节奏的时序可以通过输入进行调整 向振荡器传递有关环境信息的信号。然而，这个振荡器 也嵌入了细胞生理学的其余部分，因此它对不断变化的环境的反应是 可能取决于新陈代谢和其他信号通路的状态。我们用的是细菌 模式生物长聚球藻破解时钟与环境相互作用的问题 因为这种生物具有核心振荡器可以在体外重组的显著特征 使用纯化的蛋白质。因此，我们将使用简化论的方法来建立一个完整的 受环境波动影响时，完整细胞中时钟函数的数学模型。在……里面 目标1，我们将研究纯化的试管振荡器，收集大量的动力学测量数据集 在不同温度、代谢物浓度和蛋白质下的核心时钟蛋白 化学计量学。使用高级统计方法，然后我们将约束一个基本的 揭示温度补偿、新陈代谢感知和夹带作用的反应 核心振荡器。在目标2中，我们将研究时钟如何随着环境波动而改变。 在活着的细胞里。在这里，我们将使用一种新的方法来分离时钟灵敏度的历史依赖性 这在核心振荡器中是不存在的。然后我们将使用基因分析来找到关键的途径 用于调节体内的时钟灵敏度。然后，这些数据将被合并到扩展的 描述生物钟在环境条件下的作用的数学模型 波动。在目标3中，我们将开发一种深度诱变扫描方法，以寻找时钟表型 同时存在10000个点突变的时钟基因的竞争性生长缺陷。这 不仅可以让我们发现时钟蛋白上的关键相互作用位点，而且还可以获得 破坏温度补偿的周期突变和突变的综合清单。因为 这种分析是基于生物钟中普遍存在的转录反馈环，它可以 一般也适用于其他时钟系统。

Project Summary Circadian rhythms are daily oscillations in behavior with a nearly 24-hour period that are generated by an internal biological clock. Across many organisms, health and fitness are impaired when the circadian clock does not appropriately synchronize with the daily cycles in the external environment. It is thus critical to understand how clocks respond to challenging fluctuating environments with intermittent or irregular inputs that are typical of modern life. This problem is conceptually challenging because circadian clocks are complex systems. In general, there is a core oscillator consisting of biochemical circuitry that generates a rhythmic daily signal, and the timing of this rhythm can be adjusted by input signals that communicate information about the environment to the oscillator. However, this oscillator is also embedded in the rest of cellular physiology, and so its response to a changing environment is likely contingent on the status of metabolism and other signaling pathways. We are using the bacterial model organism Synechococcus elongatus to crack the problem of clock-environment interaction because this organism has the remarkable feature that the core oscillator can be reconstituted in vitro using purified proteins. We will thus use a reductionistic approach to build up to an integrated mathematical model of clock function in the intact cell when subject to environmental fluctuations. In Aim 1, we will study the purified test tube oscillator, collecting a large data set of kinetic measurements on the core clock proteins at various temperatures, metabolite concentrations, and protein stoichiometries. Using advanced statistical approaches, we will then constrain a model of elementary reactions to uncover how temperature compensation, metabolic sensing, and entrainment function in the core oscillator. In Aim 2, we will study how the clock shifts in response to environmental fluctuations in the living cell. Here we will use a novel assay to isolate the history-dependence of clock sensitivity that is absent from the core oscillator. We will then use a genetic analysis to find the key pathways used to modulate clock sensitivity in vivo. These data will then be incorporated into a expanded mathematical model that describes the function of the clock in vivo when environmental conditions fluctuate. In Aim 3, we will develop a deep mutagenic scanning approach, to find the clock phenotype and competitive growth defects of 10,000s of point mutations in the clock genes simultaneously. This will not only allow us to discover critical interaction sites on the clock proteins, but also to obtain a comprehensive list of period mutants and mutants that disrupt temperature compensation. Because this assay is based on the transcriptional feedback loops ubiquitous in circadian clocks, it can be generally applied to other clock systems as well.