Reconstitution of an intact clock reveals mechanisms of circadian timekeeping.
Reconstitution of an intact clock reveals mechanisms of circadian timekeeping.
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DOI：
10.1126/science.abd4453
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发表时间：
2021-10-08
期刊：
影响因子：
--
通讯作者：
LiWang A
中科院分区：
文献类型：
--
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
昼夜节律时钟提供了细胞内本地时间的内部表示，并控制基因表达的时间以预测日出和日落。在蓝细菌中，计时是通过包含三个 Kai（日语中的“循环”）蛋白 KaiA、KaiB 和 KaiC 的振荡器来实现的，它们通过两个传感器组氨酸激酶 SasA 和 CikA 将时间信息向下游传递，以调节转录因子 RpaA。事实证明，在复杂的细胞环境中，识别生物钟对基因表达进行时间控制的具体机制具有挑战性。因此，我们在体外规定的条件下重新组装了一个完整的时钟，包括核心振荡器和信号传导组件。结合部分时钟反应的结构研究和生化分析，我们深入了解了蓝藻生物钟控制基因表达的机制。尽管核心振荡器可以仅用 Kai 蛋白在体外重建，但通过 SDS-聚丙烯酰胺凝胶电泳检测的这些反应仅报告 KaiC 的磷酸化状态，因此无法提供时钟蛋白相互作用或信号转导的机制见解。因此，我们开发了一种基于荧光偏振的体外全时钟系统，包括 KaiABC 振荡器、SasA 和/或 CikA、RpaA 以及带有时钟启动子的 DNA 片段。这种体外时钟 (IVC) 可以自主振荡数天，并可以实时单独监测每个组件，揭示从计时器到 RpaA 与 DNA 结合的大分子组装体的形成和阶段。 IVC 用于检查 SasA 或 CikA 在恒定条件下维持时钟输出的充足性，并确定各自如何对 RpaA 的 DNA 结合阶段做出贡献；它甚至可以剖析 RpaA 心律失常突变体的信号传导崩溃。我们挑战了一个长期存在的范例，即振荡器的计时仅取决于 Kai 蛋白，而 SasA 和 CikA 仅提供输入-输出信号。已知体外振荡器在相对较窄的 Kai 蛋白浓度范围内发挥作用；我们在这里表明，由于 KaiB 水平有限而无法以持续方式振荡的仅 KaiABC 混合物可以通过 SasA 来挽救，SasA 可以通过异方协同作用将 KaiB 募集到 KaiC 六聚体中。协同性基于 SasA 和 KaiB 之间的结构模仿，消除体外异协同性的突变会深刻影响体内的昼夜节律。 CikA 还可挽救低水平 KaiA 下的月经缺陷。总之，我们的数据有助于解释时钟如何在体内补偿作为转录翻译反馈环和蛋白质周转的一部分而发生的振荡器成分浓度的变化。振荡器和输入输出组件之间的紧密耦合模糊了它们的区别，尽管这种耦合在多大程度上可以推广到真核生物钟仍然是一个悬而未决的问题。我们开发了重组的 IVC，以建立时钟生物化学和体内表型之间的因果关系。我们的系统提供了一个强大的平台来探索，例如，如何补偿温度或三磷酸腺苷水平等因素的变化，以产生可靠的转录昼夜节律。事实上，这次IVC只是一个起点，一个有很多可能性的起点。例如，这里提供的实时数据将使蓝藻时钟的数学模型扩展到 KaiABC 振荡器之外，以解决影响生理和新陈代谢时间控制的事件。下一步，将这种 IVC 移植到人造细胞中可以在更接近生理的条件下对这种简化的时钟进行单细胞显微镜实验。在人工细胞系统中，可以包含转录和翻译网络，从而允许设计更复杂的定制频率和输出，从而为合成生物学应用打开了大门。 ■ 在体外重建完整的时钟。将蓝藻昼夜节律蛋白 KaiA、KaiB、KaiC、SasA、CikA、RpaA 和带有启动子的 DNA 片段重构为体外时钟。结合结构研究和生化分析，该系统可以对生物钟系统进行解剖，以证明核心振荡器的概念应该扩展到包括输出蛋白 SasA 和 CikA。虚线将主观白天和主观夜晚分开，指的是恒定条件下昼夜节律的两半，对应于明暗循环中的白天和黑夜。 S，丝氨酸； T，苏氨酸； p，磷酸化。昼夜节律时钟控制基因表达以提供当地时间的内部表示。我们报告了完整的蓝藻生物钟的体外重建，包括中央振荡器、信号转导途径、下游转录因子和启动子DNA。整个系统自主振荡，并通过基于荧光的读数保持相位相干多日，无需用户干预即可同时实时观察每个组件。我们确定了心律失常突变体循环丧失的分子基础，并探索了蓝藻时钟计时的基本机制。我们发现 SasA（一种与时钟输出相关的昼夜节律传感器组氨酸激酶）直接与 KaiC 六聚体上的 KaiB 结合，调节中央振荡器的周期和振幅。 SasA 使用结构拟态将 KaiB 的罕见折叠转换构象协同招募到 KaiC 六聚体，形成夜间抑制复合物并增强振荡器的节律性，特别是在 KaiB 浓度有限的情况下。因此，扩展的体外时钟揭示了蓝藻昼夜节律系统在不同蛋白质浓度下维持节奏和节律的先前未知的机制。
Circadian clocks provide an internal representation of local time inside cells and control the timing of gene expression in anticipation of sunrise and sunset. In cyanobacteria, timekeeping is achieved by way of an oscillator comprising three Kai (meaning “cycle” in Japanese) proteins, KaiA, KaiB, and KaiC, which relay temporal information downstream through two sensor histidine kinases, SasA and CikA, to regulate the transcription factor RpaA. Identifying the specific mechanisms by which circadian clocks exert temporal control over gene expression has proven challenging in the complex milieu of cells. Thus, we reassembled an intact clock, including the core oscillator and signal transduction components, under defined conditions in vitro. Together with structural studies and biochemical analyses of partial clock reactions, we acquired insights into mechanisms by which the cyanobacterial circadian clock functions to control gene expression. Although the core oscillator can be reconstituted in vitro with just the Kai proteins, these reactions assayed by SDS–polyacrylamide gel electrophoresis report only on the phosphorylation status of KaiC and have therefore not offered mechanistic insight into clock protein interactions or signal transduction. Thus, we developed a fluorescence polarization–based in vitro whole-clock system that includes the KaiABC oscillator, SasA and/or CikA, RpaA, and a clock promoter–bearing DNA fragment. This in vitro clock (IVC) oscillates autonomously for days and allows monitoring of each component individually in real time, revealing the formation and phase of macromolecular assemblies from the timekeeper to DNA binding by RpaA. The IVC was used to examine the sufficiency of SasA or CikA to maintain clock output under constant conditions and identify how each contributes to the phase of DNA binding by RpaA; it even allowed the dissection of the breakdown in signaling of an arrhythmic mutant of RpaA. We challenged a long-standing paradigm that timekeeping by the oscillator depends solely on the Kai proteins, with SasA and CikA providing only input-output signaling. The in vitro oscillator is known to function under a relatively narrow set of Kai protein concentrations; we show here that a KaiABC-only mixture that fails to oscillate in a sustained manner owing to limiting levels of KaiB can be rescued by SasA, which acts to recruit KaiB to the KaiC hexamer through heterotropic cooperativity. Cooperativity is based on structural mimicry between SasA and KaiB, and mutations that eliminate heterocooperativity in vitro profoundly affect circadian rhythms in vivo. CikA also rescues period defects under low levels of KaiA. Together, our data help to explain how the clock compensates in vivo for changes in concentrations of oscillator components that occur as part of the transcription-translation feedback loop and protein turnover. The intimate coupling between oscillator and input-output components blurs their distinction, although the extent to which this coupling can be generalized to eukaryotic clocks remains an open question. We developed the reconstituted IVC to establish causal links between clock biochemistry and in vivo phenotypes. Our system provides a powerful platform to explore, for example, how changes in factors such as temperature or adenosine triphosphate levels are compensated to allow generation of reliable circadian rhythms of transcription. In fact, this IVC is just a starting point, one with many possibilities. For example, the real-time data presented here will allow mathematical models of the cyanobacterial clock to extend beyond the KaiABC oscillator to address the events that effect temporal control of physiology and metabolism. As a next step, transplanting this IVC to artificial cells could enable single-cell microscopy experiments of this streamlined clock under conditions that are much closer to physiological. In an artificial cellular system, transcriptional and translational networks could be included, allowing higher-complexity customized frequencies and outputs to be engineered, opening the door to synthetic biology applications. ■ Reconstituting an intact clock in vitro. Cyanobacterial circadian proteins KaiA, KaiB, KaiC, SasA, CikA, RpaA, and a promoter-bearing DNA fragment were reconstituted into an in vitro clock. Together with structural studies and biochemical assays, this system allowed dissection of a circadian clock system to demonstrate that the concept of the core oscillator should be extended to include output proteins SasA and CikA. The dashed line separates subjective day and subjective night, which refer to the halves of a circadian period under constant conditions that correspond to day and night in a light-dark cycle. S, serine; T, threonine; p, phosphorylated. Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.
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影响因子：
14.8
作者：
通讯作者：
--
DOI：
10.1073/pnas.1104221108
发表时间：
2011-08-30
影响因子：
11.1
作者：
Chang, Yong-Gang;Kuo, Nai-Wei;LiWang, Andy
通讯作者：
LiWang, Andy
影响因子：
2.9
作者：
Chow, Gary K.;Chavan, Archana G.;Britt, R. David
通讯作者：
Britt, R. David
DOI：
10.1126/science.1260031
发表时间：
2015-07-17
期刊：
Science (New York, N.Y.)
影响因子：
--
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
影响因子：
2.4
作者：
Heisler, Joel;Chavan, Archana;LiWang, Andy
通讯作者：
LiWang, Andy