A cyanobacterial circadian clock based on the Kai oscillator.

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.