CIRCADIAN RHYTHMS - Cyanobacteria - page 319

They suggested that a conformational change in KaiC molecule makes the A-loops to be in an
exposed or buried state. When A-loops in KaiC are exposed, the autokinase activity is favoured
due to its interaction with KaiA at its binding site, moving ATP closer to the site. This results in a
stabilization of the A-loops and phosphorylation continues. The autophosphatase activity of KaiC
is prevalent when A-loops are in a buried state thereby disallowing the interaction of A-loops with
KaiA. These conformational changes in KaiC molecule are suggested to be brought about by KaiB
which interacts with KaiC at the A-loops. The type of conformational change KaiC undergoes during
autophosphorylation and dephosphorylation cycles at Ser431 and Thr432 is explained by Chang et
al . (2011). According to them autophosphorylation at Ser431 stabilizes the CII ring making it rigid
but autophosphorylation at Thr432 relieves this rigidity to some extent. In presence of KaiA and
KaiB the rigid and fl exible states of CII ring of KaiC are rhythmically controlled and at the same
time this sets the rhythm for the ATPase activity of the CI ring too. The evidences for such dynamic
interactions of KaiA, KaiB and KaiC have been provided by Pattanayek et al . (2008) on the basis of
X-ray crystallography, cryo-electron microscopy and gel-electrophoresis studies. The simultaneous
binding of KaiA and KaiB to KaiC explains the role of KaiB in bringing conformational changes in
KaiC and its role in phosphorylation and dephosphorylation of KaiC. Qin et al . (2010) studied the
intermolecular associations between wild-type and mutant Kai proteins. They used KaiC mutant
proteins in a hyperphosphorylated (with a change in Ser431Glu and Thr432Glu or Ser431Asp
and Thr432) and hypophosphorylated (Ser431Ala and Thr432Ala or Ser431Ala and Thr432) state.
The KaiB proteins with a change in Arg22 to Cys22 and Arg74 to Cys74 were used. The important
fi ndings are that: (i) KaiA and KaiB proteins rhythmically interact with KaiC poteins; (ii) KaiA has
a higher affi nity to hyperphosphorylated KaiC; (iii) hyperphosphorylated KaiC mutant proteins
formed stable complexes with the three proteins; (iv) in the absence of KaiB, hyperphosphrylated
KaiC could not form stable complex with KaiA; (v) mutant forms of KaiB interacted with KaiC to
form KaiB.KaiC complexes or in presence of KaiA formed KaiA.KaiB.KaiC complexes very much
in a similar fashion as those of the wild-type proteins; (vi) free and associated forms of the three
proteins oscillated in phase and anti-phase, respectively and in this way the observations of van
Zon et al . (2007) are confi rmed. The KaiC phosphorylation rhythm has been confi rmed in vitro at
varying concentrations of Kai proteins and the regulation of phosphorylation cycle by KaiA and KaiB
proteins has been termed as “parameter-tuning” and “state-switching”, respectively by Nakajima
et al . (2010). They suggested that the clock proteins oscillate in vivo in a circadian fashion and a
possible entrainment mechanism is operative. A mathematical model presented by Brettschneider
et al . (2010) could reproduce the phosphorylation dynamics of KaiC in vitro and identifi ed KaiA
inactivation as the primary event in the phosphorylation cycle. The inactivation of KaiA has been
suggested to be due to its sequestration by KaiBC complex and evidences for a possible temperature
entrainment mechanism have been presented with a temperature-dependent oscillation of KaiAC
and KaiBC complexes.
ii) Relationship between PPC and transcriptional-translational feed-back loop (TTFL) : When
S. elongatus PCC 7942 was grown in LL, it exhibited a turnover of KaiC protein in a circadian phase-
dependent manner (Imai et al ., 2004). Proper circadian changes in KaiC accumulation involved
transcriptional, translational and post-transcriptional processes. Inhibition of translation led to KaiC
degradation and phosphorylation proceeded within at least 4 h in a circadian phase-dependent
manner. Due to the reconstitution of a self-sustained circadian oscillator in vitro , Nakajima et
al . (2005) put forward the idea that the circadian oscillator in vivo also is an autonomous KaiC
phosphorylation unit that is not dependent on transcription and translation feedback loop. In