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Temporal integration of cellular system by circadian clock in cyanobacteria

蓝藻生物钟对细胞系统的时间整合

基本信息

批准号：
15GS0308
负责人：
KONDO Takao
金额：
$ 264.33万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Creative Scientific Research
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2007
项目状态：
已结题

项目摘要

In the cyanobacterium, Synechococcus elongatus PCC 7942, three genes (kaiA, kaiB and kaiC) code essential components of the circadian clock, and negative feedback regulation of kaiBC expression by Kai proteins was confirmed and proposed as a core loop of prokaryotic circadian oscillator. However, it would be difficult to explain the key feature of circadian oscillation, that is, about 24-hour periodicity and its stability, by the feedback model of clock gene expression. To address this question, we developed DNA-chips and bioluminescence reporter strain for all transcripts and elucidated basic feature of global regulation of transcription by Kai proteins.Recently, by studying kaiC phosphorylation in continuous darkm conditions, we found robust circadian cycling of kaiC phosphorylation even without kaiBC mRNA accumulation. Moreover, by incubating kaiC with kaiA, kaiB, and ATP, we found the self-sustainable circadian oscillation of kaiC phosphorylation. The in vitro oscillation of kaiC phosphorylation persisted for at least three cycles and the period was compensated against temperature change. These results demonstrate that the oscillation of kaiC phosphorylation is the primary pacemaker of the cyanobacterial circadian clock. We further analyzed the in vitro kaiC phosphorylation cycle and found that extraordinarily weak (16 ATP/day/kaiC) but temperature-compensated ATPase activity of kaiC is a key reaction in defining the circadian period.

在蓝细菌Synechococcus elongatus PCC 7942中，三个基因（kaiA，kaiB和kaiC）编码生物钟的重要组成部分，Kai蛋白对kaiBC表达的负反馈调节被证实并被认为是原核生物昼夜节律振荡器的核心环。然而，生物钟基因表达的反馈模型难以解释昼夜节律振荡的关键特征，即大约24小时的周期性及其稳定性。为了解决这个问题，我们开发了所有转录本的DNA芯片和生物发光报告菌株，并阐明了Kai蛋白对转录的全局调节的基本特征。最近，通过研究连续黑暗条件下的kaiC磷酸化，我们发现即使没有kaiBC mRNA积累，kaiC磷酸化也有强大的昼夜循环。此外，通过将kaiC与kaiA、kaiB和ATP一起孵育，我们发现了kaiC磷酸化的自我持续的昼夜节律振荡。kaiC磷酸化的体外振荡持续至少三个周期，并且该周期针对温度变化进行补偿。这些结果表明，kaiC磷酸化的振荡是蓝藻生物钟的主要起搏器。我们进一步分析了体外kaiC磷酸化周期，发现kaiC的非常弱（16 ATP/天/kaiC）但温度补偿的ATP酶活性是定义昼夜节律周期的关键反应。