The Pathway that Sets the Cyanobacterial Circadian Clock

描述（由申请人提供）：该项目将通过在蓝藻模型系统中整合遗传、生化、生物物理和细胞生物学方法，定义24小时（昼夜节律）生物钟内使内源性昼夜节律振荡器与自然昼夜周期同步的机制。模式生物长聚球菌（Synechococcus elongatus） 2.7 Mb基因组测序完成，全球功能基因组学项目正在进行中，先进的遗传工具可用，核心时钟组件的三维结构已知，基本振荡可以在体外重现。这些资源为全面了解生物计时机制提供了非凡的潜力。我们的研究结果预测了一个模型，在这个模型中，输入通路通过调节昼夜节律振荡器蛋白KaiA的C端结构域刺激的磷酸化状态来重置蓝藻时钟，以响应环境线索。KaiC磷酸化状态在昼夜周期中振荡，并且对于形成高阶复合物至关重要，这些复合物在每个昼夜周期中组装和拆卸一次。在之前的资助期内，我们确定了以下内容：环境信号的关键积分器（CikA），这些信息所影响的KaiA结构域，关键结构域的三维结构，信号转导途径中的其他相互作用组件，以及时钟组件感知细胞氧化还原状态的证据。拟议的项目将通过定义从环境感知到与振荡器交互的步骤来测试我们的夹带模型，从而使时钟与外部每日周期同步。具体目的是：(1)确定CikA组氨酸蛋白激酶下游的分子事件；(2)定义已知和候选输入通路成员的生化功能，以定义流经时钟的分子信号；(3)定义CikA与细胞内伙伴的物理相互作用，包括功能时钟复合物的细胞内定位。概要：该项目将为生物体的生物钟如何与环境同步提供新的见解，并将展示生物钟如何通过外部刺激进行调整，这与治疗干预的设计相关。这项研究将揭示在致病菌中也起作用的调节蛋白的新结构域的功能。

DESCRIPTION (provided by applicant): This project will define the mechanisms within a 24-h (circadian) biological clock that synchronize the endogenous circadian oscillator with natural day/night cycles by integrating genetic, biochemical, biophysical, and cell biological approaches in a cyanobacterial model system. The 2.7 Mb genome of the model organism Synechococcus elongatus is fully sequenced, a global functional genomics project is underway, sophisticated genetic tools are available, 3-dimensional structures are known for the core clock components, and the basic oscillation can be recapitulated in vitro. These resources offer exceptional potential for a comprehensive mechanistic understanding of biological timekeeping. Our findings predict a model in which input pathways reset the cyanobacterial clock in response to environmental cues by modulating the phosphorylation state of the circadian oscillator protein KaiC, which is stimulated by the C- terminal domain of the oscillator protein KaiA. The KaiC phosphorylation state oscillates during the circadian cycle, and is essential for formation of higher order complexes that assemble and disassemble once per circadian cycle. In the prior funding period we identified the following: a key integrator of environmental signals (CikA), the domain of KaiA on which this information impinges, 3-dimensional structures of key domains, other interacting components in the signal transduction pathway, and evidence of cellular redox state sensing by clock components. The proposed project will test our entrainment model by defining the steps from environmental sensing to interaction with the oscillator that enable synchronization of the clock with the external daily cycle. The Specific Aims are to: (1) identify the molecular events downstream of the CikA histidine protein kinase; (2) define the biochemical functions of known and candidate input pathway members to define the molecular signals that flow through the clock; and (3) define the physical interactions of CikA with partners in the cell, including intracellular localization of a functional clock complex. Lay summary: The project will provide new insights into how an organism's circadian clock becomes synchronized with the environment and will show how circadian clocks can be adjusted by external stimuli, which is of relevance for the design of therapeutic interventions. The research will reveal the functions of novel domains of regulatory proteins that also operate in pathogenic bacteria.