Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in Vitro

  title={Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in Vitro},
  author={Masato Nakajima and Keiko Imai and Hiroshi Ito and Taeko Nishiwaki and Yoriko Murayama and Hideo Iwasaki and Tokitaka Oyama and Takao Kondo},
  pages={414 - 415}
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… Expand
In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB
KaiA and KaiB were found to be “parameter‐tuning” and “state‐switching” regulators of KaiC phosphorylation rhythm, respectively, suggesting a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins. Expand
Autonomous synchronization of the circadian KaiC phosphorylation rhythm
The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three purified clock proteins, KaiA, KaiB and KaiC, with ATP and it is demonstrated that synchronization is tightly linked with KaiC dephosphorylation and is mediated by monomer exchange between KaiC hexamers during the early deph phosphorylation phase. Expand
Circadian Rhythmicity by Autocatalysis
This model, based upon autocatalysis instead of transcription-translation negative feedback, shows temperature-compensated circadian limit-cycle oscillations with KaiC phosphorylation profiles and has period lengths and rate constant values that are consistent with experimental observations. Expand
Evolution Analysis of the Circadian Clock Protein KaiB
Here, the evolution of the KaiB protein is studied to help understand the development of the circadian clock system in cyanobacteria. Expand
Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo.
Here, a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro is provided and a protocol that facilitates analysis of phosphorylation states of KaiC and other phosphorylated proteins from in vivo samples is presented. Expand
A Molecular Dynamics Study of the Cyanobacterial Clock Protein KaiA
Regulation of daily physiological functions with a ~24-hour periodicity, or circadian rhythms, exists in both eukaryotes and prokaryotes. So far, cyanobacteria are only known prokaryotes proved toExpand
Synchronization of circadian oscillation of phosphorylation level of KaiC in vitro.
This study extends the previous model of oscillation by explicitly taking two phosphorylation sites of KaiC into account and applies the extended model to the problem of synchrony of two oscillatory samples mixed at different phases, suggesting that the combined mechanism of the allosteric transition of Kai C hexamers and the monomer shuffling between them plays a key role in synchronization among KaiC hexamer and hence underlies the population-level oscillation of the ensemble of Kai proteins. Expand
Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity.
Circadian clocks of cyanobacteria are the simplest and one of the best studied models and can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol. Expand
Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro.
The molecular model proposed here provides mechanisms for circadian timing systems and confirmed that a similar model can be applied in cyanobacterial cells. Expand
A model for circadian rhythm of cyanobacteria , which maintains oscillation without gene expression
An intriguing property of the cyanobacterial circadian clock is that endogenous rhythm persists when protein abundances are kept constant either in the presence of translation and transcriptionExpand


KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria
It is shown that activation of kaiBC expression by kaiA requires KaiC, suggesting a positive feedback control in the cyanobacterial clockwork, and proposed that KaiA-stimulated circadian KaiC phosphorylation is important for circadian timing. Expand
KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system
A novel role for KaiB is proposed in a regulatory link among subcellular localization, protein–protein interactions and post‐translational modification of Kai proteins in the cyanobacterial clock system. Expand
No Transcription-Translation Feedback in Circadian Rhythm of KaiC Phosphorylation
Temperature-compensated, robust circadian cycling of KaiC phosphorylation even without kaiBC messenger RNA accumulation under continuous dark conditions is demonstrated, and the cyanobacterial clock can keep time independent of de novo transcription and translation processes. Expand
Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria.
A negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiA expression. Expand
Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC
It is concluded that the circadian period in cyanobacteria is determined by the phosphorylation status of KaiC and also by the degradation rate of Kai C, which is integrated into a model proposing rhythmic changes in chromosomal status. Expand
Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.
  • T. Nishiwaki, Y. Satomi, +11 authors T. Kondo
  • Biology, Medicine
  • Proceedings of the National Academy of Sciences of the United States of America
  • 2004
The results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins. Expand
Global gene repression by KaiC as a master process of prokaryotic circadian system.
It is confirmed that the Kai-based oscillator is the dominant circadian oscillator functioning in cyanobacteria and the nature of this regulation is probed, finding that excess KaiC represses not only kaiBC but also the rhythmic components of all genes in the genome. Expand
Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.
Daily modulations of KaiC activity by phosphorylation at T432 and S431/T426 seem to be key components of the circadian clockwork in cyanobacteria. Expand
The Neurospora Circadian System
Overall, the FRQ/white collar complex feedback loop appears to coordinate the circadian system through its activity to regulate downstream-target clock-controlled genes, either directly or via regulation of driven FLOs. Expand
ATP‐induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC
Background: KaiA, KaiB and KaiC are cyanobacterial circadian clock proteins. KaiC contains two ATP/GTP‐binding Walker's motif As, and mutations in these regions affect the clock oscillations.