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: 10201243
• 负责人: SUSAN S GOLDEN
• 金额: $ 67.66万
• 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/10201243
• 关键词: Address; Anacystisnidulans; Animal Model; 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; Work; circadian; circadian pacemaker; experimental study; fitness; human 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.

该项目利用蓝藻模型系统来回答以下问题：分子是什么？ 标记细胞中时间流逝的相互作用，它们发生在细胞中的何处，它们如何介导时间 事件的调节，以及为什么生物时间对于健身很重要？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。人类的时钟功能障碍与 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，生物钟的存在远远超出了哺乳动物的范围，促进了多种动物的健康。 整个系统发育树中的生物体。蓝藻聚球藻的生物钟 elongatus 产生真正的遗传、生理和代谢活动的昼夜节律，以满足所有需要 定义真核生物生物钟的标准。在这种遗传上易于处理的模型生物体中，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，包含振荡器蛋白 KaiA、KaiB 和 KaiC，以及 激酶 CikA 和 SasA 以及转录因子 RpaA，重建 RpaA 结合的昼夜节律 通过实时读数发送到其目标启动子。本项目将应用体外时钟等技术和 生化、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。体外时钟将揭示当时钟重置为某个值时发生的分子事件。 环境计时线索，识别调节计时电路的核苷酸的作用位点，并确定 RpaA 和受环境信号调节的第二种转录因子 RpaB 如何共同作用 影响昼夜节律。发现激酶 SasA 和 CikA 赋予波动耐受性 振荡器成分浓度将克服过去在大肠杆菌中建立昼夜节律回路的障碍 大肠杆菌作为一个简单的模型系统，用于探索时钟与细胞生理学和生物技术的联系 应用程序。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 细胞内组织和时钟复合体本身受时钟控制的日常变化。分子基础和 健身的优势将由昼夜节律控制的自然转化决定。条形码转座子 首先用于识别光自养生长所需的所有基因的文库将用于识别新的基因座 有助于昼夜循环的健身。与生理和代谢测定相结合，这些实验将 回答这个问题：为什么分子事件的时间很重要？这些方法将共同阐明 时钟机制和时钟对昼夜生理学的价值，并将推进生物技术 控制光合作用和传统细菌生产系统中新陈代谢的机会。