Structural Biology of the S. elongatus Circadian Clock

描述（由申请人提供）：昼夜节律是内源性的生物程序，它使代谢和/或行为事件发生在每日周期的最佳阶段。它们有三个诊断特征：(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（通常是光/暗及/或温度循环）中，节奏会与环境循环的周期一致，即它们会夹带；(3)在生理范围内不同的恒定环境温度下，自由奔跑节律的周期几乎相同，即它们是温度补偿的。昼夜节律的迷人之处在于，它解释了生物化学机制如何在不同环境温度下如此精确地保持时间在如此长的时间内恒定（~24小时）。蓝藻是显示昼夜节律的最简单的生物，并为昼夜节律钟提供了一个模型系统。这项建议的长期目标是长聚球菌生物钟的结构特征。近80%的长形丝瓜基因受昼夜节律调控。通过基因筛选鉴定出三个相关位点：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，循环基因表达和自我调节似乎总是为昼夜节律提供分子基础。在长形海参中，任何单个kai基因的失活都会破坏昼夜节律并降低kaibc启动子的活性。持续的kaiC过表达抑制了kaiBC启动子，而kaiA过表达则增强了kaiBC启动子。时间kaiC的过度表达重置了节律的阶段。因此，kaiC对kaiC表达的负反馈控制在蓝藻中产生昼夜振荡，KaiA通过增强kaiC表达维持振荡，KaiB是KaiA的拮抗剂。因此，KaiC作为昼夜节律振荡器的“状态变量”发挥作用，并作为昼夜节律时钟的关键组成部分出现。值得注意的是，对于本文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。作为蓝藻时钟基本机制解剖的一部分，我们通过x射线晶体学确定了KaiC蛋白的三维结构。本文的具体目标是：(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) KaiC与KaiA配合物的晶体结构测定；(4) KaiC与KaiB配合物的晶体结构测定。由于昼夜节律在进化上是趋同的，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.