Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in Vitro

TLDR

Kai proteins globally regulate circadian gene expression in cyanobacteria, and the KaiC phosphorylation cycle persists without transcription or translation, serving as a basic timing mechanism of the clock. This study enables in‑vitro investigation of the circadian clock by examining interactions among the three Kai proteins. The authors reconstituted a self‑sustaining KaiC phosphorylation oscillation in vitro by incubating KaiC with KaiA, KaiB, and ATP. The in‑vitro oscillation exhibited temperature‑compensated, stable periods that matched the circadian periods measured in vivo in KaiC mutant strains.

Abstract

Kai proteins globally regulate circadian gene expression of cyanobacteria. The KaiC phosphorylation cycle, which persists even without transcription or translation, is assumed to be a basic timing process of the circadian clock. We have reconstituted the self-sustainable oscillation of KaiC phosphorylation in vitro by incubating KaiC with KaiA, KaiB, and adenosine triphosphate. The period of the in vitro oscillation was stable despite temperature change (temperature compensation), and the circadian periods observed in vivo in KaiC mutant strains were consistent with those measured in vitro. The enigma of the circadian clock can now be studied in vitro by examining the interactions between three Kai proteins.