Structural Biology of the S. elongatus Circadian Clock

昼夜节律是内源性生物程序，其将代谢和/或行为事件的时间安排在最佳时间， 每日循环的各个阶段。它们具有三个诊断特征：（i）在恒定条件下，程序“自由运行” 时间接近但不完全是24小时;（ii）在适当的环境周期（通常是 光/暗和/或温度循环），节奏将呈现环境循环的周期，即，他们将 （iii）自由运行节奏的周期在不同的恒定环境温度下几乎相同， 生理范围，即它们是温度补偿的。昼夜节律的魅力之一是 解释一种生化机制如何在不同的温度下，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。蓝细菌是最简单的生物体，它显示昼夜节律，并提供了一个模型， 生物钟系统。这项提案的长期目标是对昼夜节律的结构特征进行研究， 细长聚球藻的发条近80%的基因5。细长体受昼夜节律的调节 节奏通过遗传筛选已经鉴定了三个相关基因座：kaiA、kaiB和kaiC。相应的蛋白 物理关联和自动调节基因表达以产生昼夜分子循环。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。In S.细长体， 任何单个kai基因的失活消除了昼夜节律并降低了kaiBC启动子活性。 kaiC过表达抑制了kaiBC启动子，而kaiA过表达增强了它。 过度表达会重置节律的相位因此，通过KaiC对KaiC表达的负反馈控制， 在蓝藻中产生昼夜振荡，KaiA通过增强KaiC表达维持振荡，KaiB通过增强KaiC表达维持振荡。 是KaiA的激动剂因此，KaiC作为昼夜节律振荡器的“状态变量”发挥作用，并作为关键因素出现。 生物钟的组成部分。值得注意的是，对于这里提出的具体目标来说， 最近的研究表明，KaiABC时钟保持时间独立于从头转录和翻译。的一部分 解剖的蓝藻时钟的基本机制，我们已经确定了三维 通过X射线晶体学分析KaiC蛋白的结构。本建议的具体目标是：（1）基于结构的 KaiC的突变分析;（2）选择的KaiC突变体的X射线晶体结构测定;（3）晶体 KaiC与KaiA配合物的结构测定;（4）KaiC与KaiA配合物的晶体结构测定 KaiC和KaiB之间的复杂性。由于昼夜节律在进化上是趋同的，因此从生物钟中获得的见解 Kai蛋白的结构分析可以提供关于控制睡眠-觉醒周期的一般机制的线索。

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.