Life in every kingdom has evolved an endogenous molecular mechanism, termed circadian (~24-h) clock, to synchronize their physiology and behavior with the daily environmental changes such as light and temperature, due to Earth’s rotation. Circadian clocks have been demonstrated to confer fitness advantage, and in humans, they have been implicated in cancer, aging, and immunity. The circadian clock found in cyanobacteria consists of only three core protein components, KaiA, KaiB, and KaiC, and three output proteins SasA, CikA, and RpaA, and represents a simple model system for elucidating circadian clock mechanisms and signal transduction. In 2005 Nakajima and coworkers showed that these three core proteins could generate an approximately 24-hour cycle of phosphorylation rhythm in the presence of only adenosine triphosphate (ATP). Despite significant research efforts, elucidating the phase relationships between the core oscillator and output proteins of the cyanobacterial clock remains unknown. Utilizing fluorescence anisotropy of the extended oscillator in real-time is an innovative leap over the method used presently, gel electrophoresis, which only monitors phosphorylation of KaiC. Here by applying the new method, we show a 4-hour temporal window for SasA-KaiC binding before KaiB competes for the same binding site, revealing real-time clock signal propagation.

Real-time ticking of a biological clock, assembled in a test tube

255th National Meeting (2018)

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