The Linchpin that Joins the Circadian Oscillator to Clock Output

项目摘要/摘要 好了！ 许多生命形式，包括细菌、真菌、植物和动物，都在不知不觉中调节着它们的 预测日出和日落的新陈代谢、生理和行为。这些生理节律 节律产生于内源性的分子计时系统，称为生物钟，它 产生时钟组件的组装和拆卸的浪潮。未知是暂时的 以及这些短暂事件之间的因果关系，以及它们对时钟的重要性 功能。利旺实验室的目标是阐明这些 波是由昼夜节律振荡器通过时钟输出产生和传输的 转录因子-DNA结合节律的途径。方法是进行实时 完整重组的蓝藻生物钟的体外测量(6 蛋白质和DNA)在活细胞的环境之外。尽管有一套近乎完整的设备 蓝藻时钟蛋白复合体的高分辨结构(手稿正在审查中 在《科学》杂志上，这一系统的动力机制仍然不明。 因此，利旺实验室正在对重组蓝藻进行实时实验 生物钟反应来解开这个系统的动态计时机制。在目标1中， 利旺实验室将阐明短暂相互作用之间的时间和因果关系 分子水平上的事件，使用实时核磁共振波谱(核磁共振)。 时间信号从KaiABC振荡器传播时将被跟踪，通过 由Sasa和Cika蛋白介导的产生昼夜节律的拮抗输出途径 RpaA-DNA结合的节律。在目标2中，时间调节的信号传输网络 涉及KaiC(时钟的心脏)的特定残基的地图将从 KaIA结合位点的磷酸化(白天)以及Kaib和Sasa结合位点的磷酸化(夜间)， 使用实时核磁共振。这两个目标的初步数据都证明了可行性。这项建议是 创新，因为这将是第一次可以观察到时间信号的涟漪 昼夜节律时钟蛋白的长度及其从一个时钟分量到 下一个，一个接一个的生理周期，实时的。这一提议意义重大，因为解决 时钟组件内部和之间的时间相互作用将揭示动态机制 它产生和传播时钟信号波，从而提供对 从静态结构中无法获得的生物计时。

PROJECT SUMMARY/ABSTRACT ! Numerous life forms including bacteria, fungi, plants, and animals involuntarily regulate their metabolism, physiology, and behavior in anticipation of sunrise and sunset. These circadian rhythms arise from endogenous molecular timekeeping systems, called circadian clocks, which generate waves of assembly and disassembly of clock components. Unknown are the temporal and causal relationships between these transitory events, and how they are important to clock function. The objective of the LiWang lab is to elucidate the dynamic mechanism by which these waves are produced by and transmitted from the circadian oscillator, through clock-output pathways, to transcription factor-DNA binding rhythms. The approach is to carry out real-time measurements in vitro on the cyanobacterial circadian clock, reconstituted in its entirety (six proteins and DNA) outside the milieu of live cells. Despite the availability of a near-complete set of high-resolution structures of cyanobacterial clock protein complexes (manuscript under review at Science, submitted on 06/04/16), the dynamic mechanism of this system remains obscure. Thus, the LiWang lab is conducting real-time experiments on reconstituted cyanobacterial circadian clock reactions to unravel the dynamic timekeeping mechanism of this system. In aim 1, the LiWang lab will elucidate the temporal and causal relationships between transitory interaction events at the molecular level, using real-time nuclear magnetic resonance spectroscopy (NMR). Time signals will be followed as they propagate from the KaiABC oscillator, through the antagonistic output pathways, mediated by SasA and CikA proteins, to generate circadian rhythms of RpaA-DNA binding. In aim 2, the temporally regulated signal transmission network involving specific residues across KaiC (the heart of the clock) will be mapped, from sites of phosphorylation to the KaiA-binding sites (daytime) and KaiB- and SasA-binding sites (nighttime), using real-time NMR. Preliminary data on both aims demonstrate feasibility. This proposal is innovative, because for the first time, it will be possible to observe the rippling of time signals across the lengths of circadian clock proteins and their transmission from one clock component to the next, cycle after circadian cycle, in real time. This proposal is significant, because resolving temporal interactions within and between clock components will reveal the dynamic machinery that generates and propagates waves of clock signals, and thereby provide insights into biological timekeeping that are unobtainable from static structures.