Molecular and cellular mechanisms of circadian timekeeping in a prokaryote model

这个项目利用蓝藻模型系统来回答以下问题：什么是分子 在细胞中标记时间流逝的相互作用，它们在细胞中的哪里发生，它们如何调节时间 事件的调节，以及为什么生物时机对健康很重要？昼夜节律生物钟是一个 驱动生物活动24小时节律的振荡计时器。人类的生物钟功能障碍与 健康状况的范围，如心血管疾病、癌症、代谢综合征、精神疾病和 睡眠障碍。然而，生物钟在哺乳动物之外无处不在，促进不同物种的健康。 整个系统发育树上的有机体。蓝藻聚球藻的生物钟 细长线虫产生真正的遗传、生理和代谢活动的昼夜节律，以满足所有 定义真核生物生物钟的标准。在这种遗传上易驯化的模式生物中，有可能 系统地改变时钟蛋白的物理和生化特性，并追踪这些影响 从它们的近端效应，通过蛋白质相互作用网络，到表达的昼夜节律的变化 表型。一种新的体外制剂，包括振荡器蛋白KaIA，Kaib和KaiC，以及 激活Cika和Sasa以及转录因子RpaA，重构了RpaA结合的昼夜节律 与其目标启动子的实时读数。该项目将应用体外时钟和其他技术和 生物化学、细胞学、基因组学和生理学目标的概念性进展将回答 目标问题。体外时钟将揭示当时钟重置到 环境计时提示，识别调节计时电路的核苷酸的作用部位，并确定 RpaA和第二个受环境信号调控的转录因子RpaB是如何协同工作的 影响昼夜节律。Sasa和Cika这两种激动素赋予波动的耐受性的发现 振荡器成分浓度将克服在大肠杆菌中建立昼夜节律电路的过去障碍 Coli作为探索细胞生理学和生物技术时钟连接的幼稚模型系统 申请。高分辨率冷冻电子断层扫描和聚焦离子束球磨将用于可视化 时钟控制细胞内组织和时钟复合体本身的每日变化。分子基础和 自然变化的昼夜节律控制的适应度优势将被确定。条码转座子 首次用于鉴定光自养生长所需所有基因的文库将用于鉴定新的 在昼夜循环中为健身做出贡献。配合生理和新陈代谢测试，这些实验将 回答问题：为什么分子事件的时间很重要？总之，这些方法将阐明 时钟机制和时钟对昼夜生理学的价值，并将推动生物技术 光合作用和传统细菌生产系统中控制新陈代谢的机会。

This project leverages a cyanobacterial model system to answer the following questions: what are the molecular interactions that mark the passage of time in a cell, where do they occur in the cell, how do they mediate temporal regulation of events, and why does biological timing matter for fitness? The circadian biological clock is an oscillatory timer that drives 24-h rhythms of biological activities. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. However, the circadian clock is pervasive well beyond mammals, promoting fitness in diverse organisms throughout the phylogenetic tree. The circadian clock of the cyanobacterium Synechococcus elongatus generates bona fide circadian rhythms of genetic, physiological, and metabolic activities that fulfill all criteria that define circadian clocks in eukaryotes. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. A new in vitro preparation comprising the oscillator proteins KaiA, KaiB, and KaiC, along with the kinases CikA and SasA and the transcription factor RpaA, reconstitutes the circadian rhythm of binding of RpaA to its target promoter with a real-time readout. This project will apply the in vitro clock and other technical and conceptual advances towards biochemical, cytological, genomic, and physiological objectives that will answer the target questions. The in vitro clock will reveal the molecular events that occur when the clock resets to an environmental timing cue, identify the sites of action of nucleotides that modulate the timing circuit, and determine how RpaA and a second transcription factor that is regulated by environmental signals, RpaB, work together to influence circadian phasing. The discovery that the kinases SasA and CikA impart tolerance to fluctuating oscillator component concentrations will overcome past hurdles for establishing a circadian circuit in Escherichia coli as a naïve model system for exploring clock connections to cellular physiology and for biotechnology applications. High-resolution cryo-electron tomography and focused ion-beam milling will be used to visualize clock-controlled daily changes in intracellular organization and the clock complex itself. The molecular basis and fitness advantage of circadian control of natural transformation will be determined. A bar-coded transposon library first used to identify all genes required for photoautotrophic growth will be used to identify new loci that contribute to fitness in a day-night cycle. Paired with physiological and metabolic assays, these experiments will answer the question: why does the timing of molecular events matter? Together, these approaches will elucidate clock mechanisms and the value of the clock to diurnal physiology, and will advance biotechnological opportunities for controlling metabolism in both photosynthetic and traditional bacterial production systems.