The oscillating mechanism of a circadian clock

Circadian clocks provide a biochemical representation of local time inside cells and control the timing of gene expression in anticipation of sunrise and sunset. In cyanobacteria, a circadian oscillator comprised of three Kai proteins — KaiA, KaiB, and KaiC — relay temporal information downstream through two kinases, SasA and CikA, to regulate the transcription factor RpaA. Identifying the mechanisms by which circadian clocks exert temporal control over gene expression has been challenging in the complex milieu of cells. Thus, we reassembled an intact clock including oscillator and signal transduction components under defined conditions in vitro. Together with structural studies and biochemical analyses of partial clock reactions, we acquired new insights into mechanisms by which the cyanobacterial circadian clock functions to control gene expression. The in vitro oscillator is known to function under a relatively narrow set of Kai protein concentrations; we show here that a KaiABC-only mixture that fails to oscillate in a sustained manner due to limiting levels of KaiB can be rescued by SasA, which acts to recruit KaiB to the KaiC hexamer through heterotropic cooperativity. Cooperativity is based on structural mimicry between SasA and KaiB, and mutations that eliminate heterocooperativity in vitro profoundly affect circadian rhythms in vivo. CikA also rescues period defects under low levels of KaiA; together, our data help explain how the clock compensates in vivo for changes in concentrations of oscillator components that occur as part of the transcription-translation feedback loop and protein turnover. The coupling between oscillator and input-output components blurs their distinction. We developed the in vitro clock to establish causal links between clock biochemistry and in vivo phenotypes, and it provides a platform to explore how changes in factors such as temperature or ATP levels are propagated to regulate transcription.