The Linchpin that Joins the Circadian Oscillator to Clock Output

连接昼夜节律振荡器和时钟输出的关键

• 批准号: 10152597
• 负责人: Andy LiWang
• 金额: $ 32.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152597
• 关键词: Animals; Applications Grants; Bacteria; Basic Science; Behavior; Binding; Binding Proteins; Binding Sites; Biological; Biological Rhythm; Biology; Cells; Circadian Rhythms; Clock protein; Complex; Cyanobacterium; DNA; DNA Binding; Data; Event; Face; Gene Expression; Heart; In Vitro; Knowledge; Length; Life; Manuscripts; Maps; Mechanics; Mediating; Metabolism; Mission; Molecular; Motion; NMR Spectroscopy; Output; Pathway interactions; Phase; Phosphorylation Site; Phosphotransferases; Physiology; Planet Earth; Plants; Proteins; Reaction; Regulation; Research; Resolution; Rotation; Sasa; Science; Signal Transduction; Spectrum Analysis; Structure; System; Testing; Time; United States National Institutes of Health; Work; activating transcription factor; circadian; circadian pacemaker; experimental study; fungus; innovation; insight; protein complex; protein protein interaction; reconstitution; temporal measurement; transcription factor; transmission process

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.

项目总结/摘要 ! 包括细菌、真菌、植物和动物在内的许多生命形式都不自觉地调节它们的 新陈代谢，生理和行为的预期日出和日落。这些昼夜节律 生物节律起源于内源性分子计时系统，称为生物钟， 产生组装和拆卸时钟部件的波。未知的是时间 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LiWang实验室的目标是阐明这些 波由昼夜节律振荡器产生并通过时钟输出从昼夜节律振荡器传输 途径，转录因子-DNA结合的节奏。该方法是进行实时 在体外对蓝藻生物钟的测量，在其整体上重建（六 蛋白质和DNA）。尽管有一套近乎完整的 蓝藻生物钟蛋白复合物的高分辨率结构（正在审查的手稿 在科学，提交于06/04/16），这个系统的动力机制仍然模糊。 因此，LiWang实验室正在对重组蓝藻进行实时实验 生物钟反应，以解开这个系统的动态计时机制。在目标1中， 李旺实验室将阐明瞬时相互作用之间的时间和因果关系， 在分子水平上的事件，使用实时核磁共振光谱（NMR）。 时间信号将随着它们从KaiABC振荡器传播通过 拮抗输出途径，由SasA和CikA蛋白介导，以产生昼夜节律 RpaA-DNA结合的节律。在目标2中，时间调节信号传输网络 涉及跨KaiC（时钟的心脏）的特定残基将被映射，从 KaiA结合位点（白天）和KaiB和SasA结合位点（夜间）的磷酸化， 使用实时核磁共振。关于这两个目标的初步数据证明了可行性。这项建议是 创新，因为这是第一次，它将有可能观察到时间信号的涟漪 跨越生物钟蛋白质的长度以及它们从一个时钟组件到另一个时钟组件的传输， 下一个，在真实的时间里，一个又一个昼夜循环。这一建议意义重大，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 它产生并传播时钟信号波，从而提供对 静态结构无法实现的生物计时。

项目成果

Reconstitution of an intact clock reveals mechanisms of circadian timekeeping.

• DOI: 10.1126/science.abd4453
• 发表时间: 2021-10-08
• 期刊: Science (New York, N.Y.)
• 作者: Chavan AG;Swan JA;Heisler J;Sancar C;Ernst DC;Fang M;Palacios JG;Spangler RK;Bagshaw CR;Tripathi S;Crosby P;Golden SS;Partch CL;LiWang A
• 通讯作者: LiWang A

Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria.

• DOI: 10.1016/j.jmb.2013.09.040
• 发表时间: 2014-01-23
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Tseng R;Chang YG;Bravo I;Latham R;Chaudhary A;Kuo NW;Liwang A
• 通讯作者: Liwang A

Nuclear magnetic resonance spectroscopy of the circadian clock of cyanobacteria.

蓝藻生物钟的核磁共振波谱。

• DOI: 10.1093/icb/ict054
• 发表时间: 2013
• 期刊: Integrative and comparative biology
• 影响因子: 2.6
• 作者: Chang,Yong-Gang;Tseng,Roger;Kuo,Nai-Wei;LiWang,Andy
• 通讯作者: LiWang,Andy

Circadian rhythms. A protein fold switch joins the circadian oscillator to clock output in cyanobacteria.

• DOI: 10.1126/science.1260031
• 发表时间: 2015-07-17
• 期刊: Science (New York, N.Y.)
• 作者: Chang YG;Cohen SE;Phong C;Myers WK;Kim YI;Tseng R;Lin J;Zhang L;Boyd JS;Lee Y;Kang S;Lee D;Li S;Britt RD;Rust MJ;Golden SS;LiWang A
• 通讯作者: LiWang A

Structural basis of the day-night transition in a bacterial circadian clock.

• DOI: 10.1126/science.aag2516
• 发表时间: 2017-03-17
• 期刊: Science (New York, N.Y.)
• 作者: Tseng R;Goularte NF;Chavan A;Luu J;Cohen SE;Chang YG;Heisler J;Li S;Michael AK;Tripathi S;Golden SS;LiWang A;Partch CL
• 通讯作者: Partch CL