Structural Biology of the S. elongatus Circadian Clock

描述(由申请人提供)：昼夜节律是一种内源性生物程序，它将代谢和/或行为事件定在每日周期的最佳阶段。它们具有三个诊断特征：(I)在恒定条件下，程序的“自由运行”周期接近但不准确地持续24小时；(Ii)在适当的环境周期(通常是明/暗和/或温度周期)中，节奏将呈现环境周期的周期，即它们将缠绕；(Iii)在生理范围内的不同恒定环境温度下，自由运行节奏的周期几乎相同，即它们被温度补偿。昼夜节律的魅力之一是解释生物化学机制如何在不同的环境温度下，在如此长的时间常数(~24小时)内如此精确地保持时间。蓝藻是最简单的生物，它显示出昼夜节律，并为生物钟提供了一个模型系统。这项提案的长期目标是对细长聚球藻昼夜节律的结构特征进行表征。长链霉菌近80%的基因受昼夜节律调控。基因筛查已鉴定出三个相关的基因座：KaIa、Kaib和KaiC。相应的蛋白质在物理上结合并自动调节基因表达，以产生昼夜分子循环。因此，循环基因的表达和自我调节似乎总是为昼夜节律提供分子基础。在细长链霉菌中，任何一个KAI基因的失活都会消除昼夜节律，并降低KaiBC启动子的活性。持续的KaiC过表达抑制了KaiBC启动子的表达，而KaiA的过表达则增强了KaiBC启动子。短暂性kaiC的过度表达重置了节律的相位。因此，KaiC对kaiC表达的负反馈控制在蓝藻中产生昼夜振荡，KaiA通过促进kaiC表达来维持这种振荡，Kaib是KaiA的拮抗剂。因此，KaiC扮演着昼夜节律振荡器的“状态变量”的角色，并作为昼夜节律时钟的关键组件出现。值得注意的是，对于这里提出的特定目标来说，最近的研究表明，KaiABC时钟保持时间与从头转录和翻译无关。作为对蓝藻生物钟基本机制的剖析的一部分，我们通过X射线结晶学确定了KaiC蛋白的三维结构。这项建议的具体目的是：(1)基于结构的突变分析；(2)选定的KaiC突变体的X射线晶体结构的测定；(3)KaiC与KaiA之间的络合物的晶体结构测定；(4)KaiC与Kaib之间的络合物的晶体结构测定。由于昼夜节律在进化上是收敛的，从KAI蛋白的结构分析中获得的见解可能为控制睡眠-觉醒周期的一般机制提供线索。

DESCRIPTION (provided by applicant): 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 (~24 h) 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 S. 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.