SummaryCellular life emerged ~3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles due to the Earth’s rotation. The advantage conferred upon organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterising their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular time-keeping with redox homeostatic mechanisms following the Great Oxidation Event ~2.5 billion years ago.

A biochemical oscillator can be reconstituted in vitro with three purified proteins, that displays the salient properties of circadian (daily) rhythms, including self-sustained 24-h periodicity that is temperature compensated. We analyze the biochemical basis of this oscillator by quantifying the time-dependent interactions of the three proteins (KaiA, KaiB, and KaiC) by electron microscopy and native gel electrophoresis to elucidate the timing of the formation of complexes among the Kai proteins. The data are used to derive a dynamic model for the in vitro oscillator that accurately reproduces the rhythms of KaiABC complexes and of KaiC phosphorylation, and is consistent with biophysical observations of individual Kai protein interactions. We use fluorescence resonance energy transfer (FRET) to confirm that monomer exchange among KaiC hexamers occurs. The model demonstrates that the function of this monomer exchange may be to maintain synchrony among the KaiC hexamers in the reaction, thereby sustaining a high-amplitude oscillation. Finally, we apply the first perturbation analyses of an in vitro oscillator by using temperature pulses to reset the phase of the KaiABC oscillator, thereby testing the resetting characteristics of this unique circadian oscillator. This study analyzes a circadian clockwork to an unprecedented level of molecular detail.

Analysis of the cyanobacterial circadian biological clock reveals a complex interdependence between a transcription/translation feedback loop and a biochemical oscillator.

No abstract

Three proteins from cyanobacteria (KaiA, KaiB, and KaiC) can reconstitute circadian oscillations in vitro. At least three molecular properties oscillate during this reaction, namely rhythmic phosphorylation of KaiC, ATP hydrolytic activity of KaiC, and assembly/disassembly of intermolecular complexes among KaiA, KaiB, and KaiC. We found that the intermolecular associations determine key dynamic properties of this in vitro oscillator. For example, mutations within KaiB that alter the rates of binding of KaiB to KaiC also predictably modulate the period of the oscillator. Moreover, we show that KaiA can bind stably to complexes of KaiB and hyperphosphorylated KaiC. Modeling simulations indicate that the function of this binding of KaiA to the KaiB•KaiC complex is to inactivate KaiA's activity, thereby promoting the dephosphorylation phase of the reaction. Therefore, we report here dynamics of interaction of KaiA and KaiB with KaiC that determine the period and amplitude of this in vitro oscillator.