The Structural and Biochemical Bases of Circadian Oscillator Rhythmicity

从细菌到人类的生物体昼夜节律的中心振荡器 使生物活动与日照和温度的每日变化同步，以及 已被证明对健康和生存很重要。中央振荡器可以是 被认为是生物钟的心脏。自我维持的基础是什么？ 节律性，为什么周期是~24小时，为什么昼夜节律的频率 时钟比大多数酶对温度变化的抵抗力强得多吗？ 这些问题是所有昼夜节律中心振荡器的基础。我们的长期目标 是为一个中央振荡器提供这些问题的全面答案 生化和结构分辨率的模型系统。 蓝藻聚球藻生物钟的中枢振荡器 拉长蛋白只由三种蛋白质组成，即KaIA、Kaib和KaiC。一种简单的混合物 由这些时钟蛋白和ATP的重组形式组成的试管中可以 重建KaiC的自我维持和温度补偿的昼夜节律 几个循环中的磷酸化。一个周期有两个部分：大约12个小时 自动磷酸化阶段和~12小时的自动去磷酸化阶段。交替 KaiC分别与Kaia和Kaib的相互作用，在KaiC之间缓慢摆动 每天都会出现过度和低磷酸化。 这种管中式中央振荡器提供了一个绝佳的机会来回答 提出了上述问题，并开发了一种生化和结构详细的 对生物计时器的理解。我们的具体目标如下： 1.确定Kaia如何设置时钟的~12小时自动磷酸化阶段 句号。 2.确定Kaib如何调节~12小时的自去磷酸化阶段 时钟周期。 3.确定KaiC的自身磷酸化和自身去磷酸化是否 合作进程。 在这里，我们从生物化学和结构上解决了长达24小时的 昼夜节律的周期是确定的，如何缓冲该周期 温度的变化，以及振荡器在12小时后如何逆转方向。

The central oscillators of circadian clocks of organisms ranging from bacteria to humans synchronize biological activities with daily changes in sunlight and temperature, and have been shown to be important to health and survival. The central oscillator can be thought of as the heart of the circadian clock. What is the basis of the self -sustained rhythmicity, why are the periods ~24 hours, and why are the frequencies of circadian clocks much more resistant than the rates of most enzymes to changes in temperature? These questions are fundamental to all circadian central oscillators. Our long-term goal is to provide comprehensive answers to these questions for a central oscillator of a model system at biochemical and structural resolution. The central oscillator of the circadian clock in the cyanobacterium Synechococcus elongatus is comprised of only three proteins, KaiA, KaiB, and KaiC. A simple mixture consisting of the recombinant forms of these clock proteins and ATP in a test tube can reconstitute a self-sustained and temperature-compensated circadian rhythm of KaiC phosphorylation over several cycles. A single cycle has two halves: an ~12 hour autophosphorylation phase and an ~12 hour autodephosphorylation phase. Alternating interactions of KaiC with KaiA and KaiB, respectively, slowly swing KaiC between hyper- and hypophosphorylation over each day. This central oscillator-in-a-tube provides an outstanding opportunity to answer the questions posed above and to develop a biochemically and structurally detailed understanding of a biological timekeeper. Our specific aims are the following: 1. Determine how KaiA sets the ~12-hour autophosphorylation phase of the clock period. 2. Determine how KaiB mediates the ~12 hour autodephosphorylation phase of the clock period. 3. Determine whether autophosphorylation and autodephosphorylation of KaiC are cooperative processes. Here, we address biochemically and structurally the problems of how the long ~24 hour period of the circadian rhythm is established, how that period is buffered against changes in temperature, and how an oscillator reverses direction after 12 hours.