Abstract
A defining feature of circadian clocks that enables adaptation to Earth's rotation is their ability to sustain an approximately 24 h period with precision, regardless of environmental factors such as temperature. This remarkable reliability of circadian timing can be reconstituted in vitro using only three cyanobacterial clock proteins: KaiA, KaiB, and KaiC. In vivo circadian rhythms, however, are governed not only by this Kai protein-based oscillator but also by transcription-translation feedback loops and additional clock components. The contribution of the Kai protein-based circadian oscillator to the overall reliability of the in vivo circadian rhythm remains unclear. In this study, we compared over 20 KaiC period mutants with periods ranging from 15 to 60 h under in vitro and in vivo conditions. In both cases, the period was insensitive to environmental conditions, suggesting a compensatory mechanism independent of metabolic state or rhythm amplitude. The ATPase activity of KaiC, the pacemaker of the cyanobacterial circadian clock, exhibited a stronger correlation with in vitro circadian frequency than in vivo circadian frequency. These results indicate that the KaiC ATPase-driven protein-based circadian oscillator inherently encodes a reliable circadian period independent of rhythm amplitude or environmental conditions. This intrinsic property likely plays a critical role in preserving the precision and stability of circadian timing in vivo while being influenced by the intracellular environment.