Elucidating the Ticking of an In Vitro Circadian Clockwork

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.

Nonparametric entrainment of the in vitro circadian phosphorylation rhythm of cyanobacterial KaiC by temperature cycle

“…As the KaiC phosphorylation rhythms was already reported to satisfy self-sustainability of rhythm and temperature compensation of the period, the result here implies that the Kai oscillator satisfies all three criteria of circadian rhythm. That is, we confirmed entrainability of the in vitro rhythm in this study, whereas phase shifting of the rhythm by temperature pulse itself was previously reported (19).…”

“…S2). Other processes that may cause the phase shifting include changes of the KaiC intramolecular structure that can be associated with the timebase ATPase activity (4), changes in the state of the KaiC hexamer, or changes in complex formation between KaiC and KaiB or KaiA (19,20). At present, we have not yet found such changes, which may be too subtle to detect directly.…”

A Cyanobacterial Circadian Clock Based on the Kai Oscillator

“…In addition, note that temperature would be an effective factor to entrain the circadian oscillation of cyanobacteria. Mori et al (2007) reported phase shifting due to temperature shifts, whereas we found that in vitro KaiC phosphorylation rhythms can be entrained to a temperature cycle (T. Yoshida et al, unpubl.). On the other hand, changes in intracellular factors, such as the ATP level, the ratio of KaiA to KaiC, pH, and the concentrations of other metabolites produced by photosynthesis, can be used to test the parametric entrainment model; the effect of these parameters on the ATPase activity of KaiC would be the basis of this entrainment model.…”

The cyanobacterial circadian clock is based on the intrinsic ATPase activity of KaiC

“…Moreover, the in vitro reaction is not a homogeneous mixture of one complex that changes sequentially over time but rather is a mixture of complexes in which the proportion of each species oscillates. KaiC is present at all times in free hexamers as well as in hexamers bound to KaiA, and KaiB associates with both types of complexes during the dephosphorylation phase (15). During this phase, monomeric KaiC can also be detected.…”

“…The in vitro reconstitution of the circadian rhythm in KaiC phosphorylation has enabled the examination of associations among the Kai proteins both biochemically (14,15) and microscopically (15). KaiA associates with the KaiC hexamer to promote KaiC phosphorylation; KaiB then associates with the KaiC-KaiA complex and inactivates KaiA, initiating dephosphorylation.…”