CIRCADIAN RHYTHMS - Cyanobacteria - page 312

patterns. Thus KaiA and KaiC are suggested to be positive and negative regulators of kaiBC expression
(Ishiura et al ., 1998). In both situations, i.e. due to elevated kaiC gene expression and its repression,
the cells experience an arrhythmic state. This pointed toward the existence of a feed back regulatory
mechanism required for a cyclic pattern of gene expression regulated by the circadian clock.
Iwasaki et al . (1999) reported a yeast two-hybrid system that was useful for demonstrating the
interaction of KaiA with KaiC both in vitro as well as in Synechococcus cells. Two KaiA-binding domains
(C KABD1 and C KABD2 ) in KaiC have been identifi ed by Taniguchi et al . (2001) by the use of yeast two-
hybrid system and further confi rmed by in vitro interactions. These two domains, i.e. C KABD1 and C KABD2
were found on corresponding C-terminal regions CI and CII, respectively. Many of the mutations
reported earlier by Ishiura et al . (1998) in these two domains of KaiC exhibited altered interaction of
KaiC with KaiA. Similar studies were made by Vakonakis et al . (2004a) and Vakonakis and LiWang
(2004) on T. elongatus BP-1. NMR structural studies revealed that the C-terminal domain of KaiA
that interacts with KaiC is a novel α-helical homodimeric structure. This is shown to interact with
the linker region connecting the two globular KaiC domains. A KaiC region of ~12 kDa interacting
with C-terminal domain of KaiA (residues 180-283; ThKai A180C) from T . elongatus BP-1 has also
been identifi ed by Vakonakis et al . (2004b). Vakonakis and LiWang (2004) identifi ed a KaiC derived
peptide (residues 488-518, CII ABD ) that interacts with terminal domain of KaiA (residues 180-230;
ThKai A180C). Studies on NMR structure of the complex (~32kDa) revealed that CII ABD binding to
ThKai A180C alters the ThKai A180C dimerization angle suggesting that KaiA-KaiC affi nity can be
modulated by changes in dimerization geometry of the KaiA C-terminal domain. These protein-
protein interactions have been explained in relation to the crystal structures of the three Kai proteins
(Egli et al ., 2007).
Williams et al . (2002) explained the functional aspects of the three proteins of the circadian
oscillator, probable role of cikA gene product in signal perception and the perception of the signal by
KaiA protein that triggers autophosphorylation of KaiC protein. Additionally, KaiC phosphorylation
rate determines (i) chromosome condensation (Mori and Johnson, 2001a); (ii) its aggregation state into
homo and heterotypic interactions (Iwasaki et al ., 1999; Nishiwaki et al ., 2000) and (iii) its interaction
with regulatory proteins such as SasA protein kinase (Iwasaki et al., 2000) (Fig. 2). Another similarity
of cyanobacterial circadian clock to post-transcriptional control of eukaryotic clock is the existence
of KaiC in phosphorylated forms in vivo (Iwasaki et al ., 2002). KaiA is known to stabilize KaiC in its
phosphorylated form and KaiB antagonizes the effect of KaiA (Iwasaki et al ., 2002; Williams et al .,
2002; Kitayama et al ., 2003; Ye et al ., 2004). Phosphorylated KaiC forms a tight complex with KaiA
that is found at maximum level at clock time of 16 to 18 h (Kageyama et al ., 2003; Kitayama et al .,
2003). The ratio of phosphorylated to unphosphorylated KaiC is correlated with the period at which
the clock runs (Ye et al ., 2004). Kitayama et al. (2003) estimated that KaiB and KaiC oscillate around
an average of approximately 10,000 molecules per cell, while KaiA is present at an almost constant
level of nearly 500 molecules per cell throughout the circadian cycle. While KaiC is present in the
cytosol (near to the nucleoid region), KaiB exists in a bounded state to the membrane as well as
occurs freely in the cytosol. Around clock time of 20 h, KaiB seems to be released from membrane into
the cytosol there by its cytosolic concentration increases. The released KaiB attaches to KaiA-KaiC
complex to form large clock complex (Kitayama et al ., 2003). Regulation of KaiC phosphorylation is
proposed to be achieved due to the existence of clock proteins KaiA and KaiB in multiple states. This
has been explained on the basis that even when kaiBC gene is regulated by KaiC protein, sustained
oscillation of gene expression occurred and sustained oscillation of KaiC phosphorylation continued
in the absence of transcription and translation processes (Kurosawa et al ., 2006a). A novel mutant of
KaiC that was unable to reset the circadian clock has been isolated after mutagenesis of S . elongatus