Structural Biology of the S. elongatus Circadian Clock

S. elongatus 昼夜节律钟的结构生物学

• 批准号: 7591719
• 负责人: MARTIN EGLI
• 金额: $ 28.32万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7591719
• 关键词: Address; Affect; Behavioral; Binding; Biochemical; Biochemical Reaction; Biological; Biological Clocks; Biological Models; Cells; Characteristics; Chromosomes; Circadian Rhythms; Clock protein; Complement; Complex; Crystallization; Cyanobacterium; DNA; Deletion Mutation; Diagnostic; Dissection; Electrons; Event; Exhibits; Feedback; Financial compensation; Foundations; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Homeostasis; Homo; Hour; Individual; Length; Measures; Metabolic; Microscopic; Molecular; Molecular Structure; Mutation; Organism; Phase; Phosphorylation; Phosphorylation Site; Physiological; Play; Property; Protein S; Proteins; Rationalization; Regulation; Reporting; Research; Research Personnel; Resolution; Roentgen Rays; Role; Running; Sleep Wake Cycle; Structure; Synechococcus; System; Techniques; Temperature; Time; Translations; Work; X-Ray Crystallography; base; circadian pacemaker; dimer; in vivo; insight; mutant; overexpression; programs; promoter; protein function; protein protein interaction; research study; stoichiometry; structural biology; three dimensional structure

Circadian rhythms are endogenous biological programs that time metabolic and/or behavioral events to occur at optimal phases of the daily cycle. They have three diagnostic characteristics: (i) In constant conditions, the programs "free-run" with a period that is close to, but not exactly, 24 hours in duration; (ii)in an appropriate environmental cycle (usually a light/dark and/or temperature cycle), the rhythm will take on the period of the environmental cycle, i.e., they will entrain; (iii) the period of the free-running rhythm is nearly the same at different constant ambient temperatures within the physiological range, i.e.they are temperature-compensated. One of the fascinations of circadian rhythms is to explain how a biochemical mechanism can keep time so precisely over such a long time constant (~24h) at different ambient temperatures. Cyanobacteria are the simplest organisms that display circadian rhythms and provide a model system for the circadian clock. The long-term goal of this proposal is a structural characterization of the circadian clockwork of Synechococcus elongatus. Close to 80% of the genes of 5. elongatus are regulated with a circadian rhythm. Three relevant loci have been identified by genetic screens: kaiA, kaiB and kaiC. The corresponding proteins physically associate and autoregulate gene expression to produce circadian molecular cycling. Thus, cycling gene expression and autoregulation appear to always provide the molecular foundation for circadian rhythms. In S. elongatus, inactivation of any single kai gene abolished the circadian rhythms and reduced kaiBC-promoter activity.Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Therefore, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, KaiA sustains the oscillation by enhancing kaiC expression and KaiB is an anagonist of KaiA. Thus, KaiC plays a role as a 'state variable' of the circadian oscillator and emerges as a key component of the circadian clockwork. Remarkably and of importance for the specific aims proposed here, it was shown very recently that the KaiABC clock keeps time independent of de novo transcription and translation. As part of the dissection of the fundamental mechanism of the cyanobacterial clock, we have determined the three-dimensional structure of the KaiC protein by X-ray crystallography. The specific aims of this proposal are: (1) A structure-based mutational analysis of KaiC; (2) The determination of X-ray crystal structures of selected KaiC mutants; (3) The crystal structure determination of the complex between KaiC and KaiA; and (4) The crystal structure determination of the complex between KaiC and KaiB. Because circadian rhythms are evolutionarily convergent, insights gained from the structural analyses of Kai proteins may provide clues as to the general mechanism of controlling sleep-wake cycles.

昼夜节律是内源性生物程序，可以安排代谢和/或行为事件在最佳状态下发生 每日周期的各个阶段。它们具有三个诊断特征： (i) 在恒定条件下，程序“自由运行” 持续时间接近但不完全是 24 小时； (ii) 在适当的环境循环中（通常是 光/暗和/或温度循环），节奏将呈现环境循环的周期，即，它们将 夹带； (iii) 在不同的恒定环境温度下，自由运行节律的周期几乎相同 生理范围，即它们是温度补偿的。昼夜节律的魅力之一是 解释生化机制如何在不同的条件下在如此长的时间常数（~24小时）内如此精确地保持时间 环境温度。蓝细菌是显示昼夜节律并提供模型的最简单的生物体 生物钟系统。该提案的长期目标是昼夜节律的结构特征 细长聚球藻的发条装置。 5. elongatus 近 80% 的基因受昼夜节律调节 韵律。通过遗传筛选已鉴定出三个相关基因座：kaiA、kaiB 和 kaiC。相应的蛋白质 物理关联并自动调节基因表达以产生昼夜节律分子循环。因此，循环基因 表达和自动调节似乎总是为昼夜节律提供分子基础。在 S. elongatus 中， 任何单个 kai 基因的失活都会消除昼夜节律并降低 kaiBC 启动子活性。连续 kaiC 过表达抑制 kaiBC 启动子，而 kaiA 过表达则增强它。颞钙 过度表达会重置节奏的相位。因此，KaiC 对 kaiC 表达的负反馈控制 在蓝藻中产生昼夜节律振荡，KaiA 通过增强 kaiC 表达和 KaiB 来维持振荡 是 KaiA 的拮抗剂。因此，KaiC 发挥着昼夜节律振荡器“状态变量”的作用，并成为关键 昼夜节律的组成部分。对于此处提出的具体目标而言，值得注意且重要的是 最近的研究表明，KaiABC 时钟保持时间独立于从头转录和翻译。作为一部分 通过对蓝藻生物钟基本机制的剖析，我们确定了三维 通过 X 射线晶体学分析 KaiC 蛋白的结构。本提案的具体目标是： (1) 基于结构的 KaiC突变分析； (2)选定KaiC突变体的X射线晶体结构测定； (3)晶体 KaiC和KaiA复合物的结构测定； (4)晶体结构测定 KaiC 和 KaiB 之间的复杂关系。由于昼夜节律在进化上是趋同的，因此从 Kai 蛋白的结构分析可能为控制睡眠-觉醒周期的一般机制提供线索。