Reconstitution of an intact clock that generates circadian DNA binding in vitro

Abstract

Circadian clocks control gene expression to provide an internal representation of time. Here, we reconstituted in vitro under defined conditions the cyanobacterial circadian clock system which includes an oscillator, signal-transduction pathways, transcription factor, and promoter DNA. The system oscillates autonomously, remains phase coherent for many days, and allows real-time observation of each component simultaneously without user intervention. We show that the circadian sensor histidine kinase SasA engages directly with KaiB and KaiC proteins to regulate the period and enhance robustness of the reconstituted circadian oscillator in vitro, particularly under limiting concentrations of KaiB, by using structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex. The other circadian sensor histidine kinase, CikA, also contributes directly to robustness in vitro by enhancing rhythms under limiting concentrations of KaiA.