Temporal integration of cellular system by circadian clock in cyanobacteria

在蓝藻长聚球菌（Synechococcus elongatus） PCC 7942中，三个基因（kaiA、kaiB和kaiC）编码了生物钟的重要组成部分，Kai蛋白对kaiBC表达的负反馈调控被证实为原核生物昼夜振荡器的核心环。然而，生物钟基因表达的反馈模型很难解释昼夜节律振荡的关键特征，即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蛋白全局调控转录的基本特征。最近，通过在持续黑暗条件下研究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一起培养，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了至少三个周期，并且这个周期被温度变化所补偿。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异常弱（16 ATP/天/kaiC），但温度补偿的ATP酶活性是确定昼夜节律周期的关键反应。

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.