Abstract
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.
Highlights
Circadian clocks are self-sustained biochemical oscillators that underlie daily rhythms of sleep/waking, metabolic activity, gene expression, and many other biological processes
One of the proteins undergoes phase-dependent exchange of its monomers, and the model demonstrates that this monomer exchange allows the maintenance of robust oscillations
We show in this study that the rhythmicity of KaiABC complexes can be visualized by electron microscopic (EM) and quantified by two-dimensional blue-native/sodium dodecyl sulfate gel electrophoresis (2-D BN/SDS-PAGE)
Summary
Circadian clocks are self-sustained biochemical oscillators that underlie daily rhythms of sleep/waking, metabolic activity, gene expression, and many other biological processes. Their properties include temperature compensation, a time constant of approximately 24 h, and high precision. The atomic structures of these proteins have been determined, and a recent study reported the amazing observation that these three proteins can reconstitute a circadian oscillation in vitro, including its temperature-compensation property [1]. This in vitro circadian oscillator is the best available system for structural and biophysical analyses of a circadian clockwork
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