Structural Biology of the S. elongatus Circadian Clock

昼夜节律是一种内源性生物程序，它将代谢和/或行为事件定在最佳时间发生 每日周期的不同阶段。它们有三个诊断特征：(1)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；(2)处于适当的环境周期(通常是 光/暗和/或温度周期)，节奏将呈现环境周期的周期，即它们将 自由奔跑节律的周期在不同恒定的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 解释生物化学机制如何在如此长的时间恒定(~24小时)内如此精确地保持时间不变 环境温度。蓝藻是最简单的生物，它显示出昼夜节律，并提供了一个模型 生物钟系统。这项提议的长期目标是对昼夜节律的结构特征进行描述 细长聚球藻的发条。近80%的基因受昼夜节律调控 节奏。基因筛查已鉴定出三个相关的基因座：KaIa、Kaib和KaiC。相应的蛋白质 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 表达和自我调节似乎总是为昼夜节律提供分子基础。在长穗沙门氏菌中， 任何一个KAI基因的失活都会破坏昼夜节律，降低KaiBC启动子的活性。 KaiC的过表达抑制了KaiBC启动子的表达，而KaiA的过表达则增强了KaiBC的启动子。临时kaic 过度表达重置了节律的相位。因此，KaiC对KaiC表达的负反馈调控 在蓝藻中产生昼夜振荡，KaIA通过增强KaiC和Kaib的表达来维持这种振荡 是卡娅的镇静剂。因此，KaiC扮演着昼夜节律振荡器的‘状态变量’的角色，并作为一个关键出现 昼夜节律发条的组成部分。值得注意的是，对于这里提出的具体目标来说，它是 最近的研究表明，KaiABC时钟保持时间独立于从头转录和翻译。作为以下内容的一部分 剖析了蓝藻生物钟的基本机制，我们已经确定了三维 X射线结晶学研究KaiC蛋白的结构。这项建议的具体目标是：(1)以结构为基础 KaiC的突变分析；(2)选定的KaiC突变体的X射线晶体结构的测定；(3)晶体 KaiC-KaIA络合物的结构测定；(4)KaiC与KaIA的晶体结构测定 Kaic和Kaib之间的复合体。由于昼夜节律在进化上是收敛的，从 对KAI蛋白的结构分析可能会为控制睡眠-觉醒周期的一般机制提供线索。

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.