Molecular and cellular mechanisms of circadian timekeeping in a prokaryote model

原核生物模型中昼夜节律的分子和细胞机制

• 批准号: 10592430
• 负责人: SUSAN S GOLDEN
• 金额: $ 67.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-04 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592430
• 关键词: Address; Anacystisnidulans; Animals; Bar Codes; Binding; Biochemical; Biological; Biological Assay; Biological Clocks; Biological Models; Biological Rhythm; Biotechnology; Cardiovascular Diseases; Cell physiology; Cells; Circadian Dysregulation; Circadian Rhythms; Clock protein; Complex; Cryo-electron tomography; Cues; Cyanobacterium; Cytology; Disease; Environment; Escherichia coli; Eukaryota; Event; Functional disorder; Genes; Genetic; Genomics; Growth; Health; Human; In Vitro; Ions; Libraries; Malignant Neoplasms; Mammals; Mediating; Mental disorders; Metabolic; Metabolic syndrome; Metabolism; Modeling; Molecular; Nucleotides; Organism; Personal Satisfaction; Phase; Phenotype; Phosphotransferases; Phylogenetic Analysis; Physiological; Physiology; Preparation; Production; Prokaryotic Cells; Property; Protein Biochemistry; Proteins; Regulation; Resolution; Sasa; Signal Transduction; Site; Sleep Disorders; System; Time; Trees; Visualization; Work; circadian; circadian pacemaker; experimental study; fitness; model organism; pathogen; promoter; reconstitution; transcription factor

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.

这个项目利用蓝藻模型系统来回答以下问题： 这些相互作用标志着细胞中时间的流逝，它们在细胞中发生在哪里，它们如何介导时间 以及为什么生物计时对健康很重要？昼夜节律生物钟是一种 振荡计时器驱动24小时生物活动的节奏。人类生物钟功能障碍与 一系列健康状况，如心血管疾病、癌症、代谢综合征、精神疾病和 睡眠障碍然而，生物钟的普遍性远远超出了哺乳动物， 在整个系统发育树中。蓝细菌聚球藻的生物钟 细长体产生真正的遗传、生理和代谢活动的昼夜节律， 定义真核生物生物钟的标准。在这种遗传上易于处理的模式生物中， 系统地改变生物钟蛋白的物理和生化特性，并追踪这些生物钟蛋白的影响。 通过蛋白质相互作用网络，从其近端效应到表达的昼夜节律的变化 表型一种新的体外制剂，其包含振荡蛋白KaiA、KaiB和KaiC，沿着 激酶CikA和SasA以及转录因子RpaA，重建RpaA结合的昼夜节律 与它的目标启动子实时连接。本项目将应用体外时钟等技术， 生物化学，细胞学，基因组学和生理学目标的概念进展，将回答 目标问题。体外生物钟将揭示当生物钟重置到一个特定的时间点时发生的分子事件。 环境时间线索，识别调节时间电路的核苷酸的作用位点，并确定 RpaA和第二个受环境信号调节的转录因子RpaB如何共同作用， 影响昼夜节律定相。发现激酶SasA和CikA赋予对波动的耐受性， 振荡器成分浓度将克服过去在埃希氏菌中建立昼夜节律回路的障碍 大肠杆菌作为探索生物钟与细胞生理学和生物技术联系的初始模型系统 应用.高分辨率冷冻电子断层扫描和聚焦离子束铣削将用于可视化 生物钟控制细胞内组织和生物钟复合体本身的日常变化。分子基础和 将确定自然转化的昼夜节律控制的适应性优势。一个条形码转座子 首先用于鉴定光自养生长所需的所有基因的文库将用于鉴定新的基因座， 有助于健康的昼夜循环。与生理和代谢测定配对，这些实验将 回答这个问题：为什么分子事件的时间很重要？总之，这些方法将阐明 生物钟的机制和昼夜生理时钟的价值，并将推动生物技术 在光合和传统细菌生产系统中控制代谢的机会。