Kondo T
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Cold Spring Harb Symp Quant Biol. 2007;72:47-55. doi: 10.1101/sqb.2007.72.029.
In the cyanobacterium Synechococcus elongatus PCC 7942, the products of three genes (kaiA, kaiB, and kaiC) have been identified as essential components of the circadian clock. Recently, we reconstituted the self-sustainable circadian oscillation of the KaiC phosphorylation state by incubating purified KaiC with KaiA, KaiB, and ATP. This in vitro oscillation persisted for at least three cycles and the period was compensated against temperature changes. Period lengths observed in vivo in various kaiC mutants were consistent with those measured using in vitro mixtures containing the respective mutant KaiC proteins. These results demonstrate that the oscillation of KaiC phosphorylation is the primary pacemaker of the cyanobacterial circadian clock and reveal a novel function of proteins as timing devices that govern cellular metabolism. We further analyzed four aspects of the KaiC phosphorylation cycle in vitro: the interactions among KaiA, KaiB, and KaiC; the functions of the two phosphorylation sites, the energetics that determine the circadian period, and the mechanisms that synchronize the components of the Kai oscillator. From these analyses, we have proposed a circadian program consisting of the three proteins that keeps biological time in a living cell.
在蓝藻聚球藻PCC 7942中，三个基因（kaiA、kaiB和kaiC）的产物已被确定为生物钟的基本组成部分。最近，我们通过将纯化的KaiC与KaiA、KaiB和ATP一起孵育，重建了KaiC磷酸化状态的自我维持昼夜节律振荡。这种体外振荡持续了至少三个周期，并且周期对温度变化具有补偿作用。在各种kaiC突变体中体内观察到的周期长度与使用含有相应突变体KaiC蛋白的体外混合物测量的结果一致。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，并揭示了蛋白质作为控制细胞代谢的计时装置的新功能。我们进一步在体外分析了KaiC磷酸化循环的四个方面：KaiA、KaiB和KaiC之间的相互作用；两个磷酸化位点的功能、决定昼夜节律周期的能量学以及使Kai振荡器各组分同步的机制。通过这些分析，我们提出了一个由这三种蛋白质组成的昼夜节律程序，该程序在活细胞中保持生物时间。
