CIRCADIAN RHYTHMS - Cyanobacteria - page 316

and temperature compensated also. Due to the replication of these parameters in some of the
KaiC mutants in vivo , Nakajima et al . (2005) proposed that the primary circadian oscillator KaiC in
cyanobacteria is autonomous and not dependent on transcription and translation feedback loop of
KaiBC. This chemical oscillator could reproduce temperature compensated rhythms and altered
phenotypes in mutants (Naef, 2005).
Emberly and Wingreen (2006) observed a perfect synchronization between the release of
monomers from KaiC hexamers during the day and formation of clusters of KaiC hexamers in the
night. They also highlighted the importance of collective assembly and disassembly of proteins in
biochemical networks in designing novel protein-based oscillators. The in vitro KaiC phosphorylation
cycle and the changes in Kai protein complexes were also confi rmed by Kageyama et al . (2006).
Each phosphorylation cycle seems to be promoted by active and repeated association of KaiA
with KaiC. High phosphorylation rates of KaiC induced the association of the KaiC hexamer with
KaiB and inactivated KaiA leading to a dephosphorylation cycle, coinciding with formation of
monomeric KaiC subunits from KaiC hexamers. Reduced phosphorylation led to the dissociation
of KaiB from KaiC reactivating KaiA in the process. They are of the view that similar association
and dissociation kinetics may be occurring in vivo . Mori et al . (2007) elucidated the ticking of an
in vitro circadian clock by visualizing the rhythmicity of the KaiABC complex formation and its
dissociation though electron microscopy and also quantifying by two-dimensional blue-native/
SDS-PAGE electrophoresis. Fluorescence resonance energy transfer with two populations of
fl uorescently labelled KaiC hexamers confi rmed that monomer exchange among hexamers occurs
(Fig. 3). According to them, this monomer exchange might be to maintain synchrony among KaiC
hexamers in the reaction that helps in maintaining a high amplitude oscillation for several days
(Fig. 4). In vitro oscillator has been subjected to entrainment by temperature pulses and could be
reset. Based on computer modelling of interactions, Yoda et al . (2007) confi rmed the experimental
fi ndings of Kageyama et al . (2006) for the existence of allosteric transition of KaiC hexamers as well
as monomer shuffl ing. Furthermore, Miyoshi et al . (2007) presented a mathematical model for the in
vitro functionality of KaiC phosphorylation cycle as well as its coupling to in vivo transcriptional/
translational feedback under LL conditions. According to van Zon et al . (2007) the synchronization
of KaiC phosphorylation and dephosphorylation cycles very much depends on the existence of two
types of hexamers of KaiC molecules, one the slowest and the other fastest. Due to a differential
affi nity, the former outcompetes the latter for KaiA molecules and in this process the fastest ones
are slowed down making the entire population of KaiC molecules to oscillate in phase. Eguchi et al .
(2008) explained the robustness of circadian oscillation of KaiC phosphorylation in vitro by putting
forward a mathematical model and suggested that it is resilient to changes in the concentration of
Kai proteins as well as a change in the system size.
Most of the mathematical models so far proposed took into consideration the equivalence of the
three phosphorylation sites at Ser431, Thr432 and Thr 426 on KaiC molecule (Emberly and Wingreen,
2006; Kurosawa et al ., 2006; Mehra et al ., 2006; Takigawa-Imamura and Mochizuki, 2006; Clodong
et al ., 2007; Miyoshi et al ., 2007; Mori et al ., 2007; van Zon et al ., 2007; Yoda et al ., 2007). However, the
work of Rust et al . (2007) brought to light that there exist four forms of KaiC molecules in vivo that
undergo phosphorylation differentially and each of these can be separated by means of SDS-PAGE
and could be quantifi ed by mass spectrometry. According to them, there are four forms of KaiC,
i.e. unphosphorylated KaiC, KaiC phosphorylated at Ser431 (S-KaiC), Thr432 (T-KaiC) and KaiC
phosphorylated both at Ser431 and Thr432 (ST-KaiC) which oscillated with a circadian period but
with different phases. When unphosphorylated KaiC was incubated with KaiA, phosphorylation fi rst
occurred at Thr432 resulting in the formation of T-KaiC followed by the phosphorylation of Ser431.