CIRCADIAN RHYTHMS - Cyanobacteria - page 315

Although the data obtained overwhelmingly support the idea that the regulation of clock genes
in S . elongatus PCC 7942 resembles autoregulatory feed back models put forward for eukaryotes but
the recent work focuses on the aspect that the mechanism seems to be more complex (Iwasaki et
al ., 2002; Xu et al ., 2003; Tomita et al ., 2005). Ditty et al . (2005) detected that the rhythmic expression
pattern of kaiA or kaiBC operon in certain of the mutants was altered but the rhythms persisted. Even
if expression of kaiA or kaiBC occurred 12 h out of phase from the normal (and thus 12 h out of phase
from other Kai locus) it did not affect the time required for one cycle. These results signify that the
Cis elements within the promoters of the kai genes are not necessary to sustain clock functions.
On the basis of enhancement in KaiA-KaiB interaction in vitro by KaiC, Iwasaki et al . (1999)
suggested the formation of a heteromultimeric complex containing all the three Kai proteins. Further,
immunoprecipitation analysis at two points of the day revealed the existence of interactions between
KaiC-SasA and KaiB-SasA (Iwasaki et al ., 2000). By studying a number of arrhythmic mutants of
S . elongatus PCC 7942 that lack each functional Kai protein or SasA, Kageyama et al . (2003)
demonstrated that KaiC forms multimeric protein complexes with other Kai proteins and SasA in
a circadian fashion. That is the size of the complex varies from ~350 and 400-600 kDa during the
subjective day and night respectively. This signifi es that KaiC functions as a scaffold protein.
i) Phosphorylation Cycle : As phosphorylation of KaiC reaches its maximum rate at late night, it
coincides with the association of all the component proteins (KaiA, KaiB, KaiC and SasA) into a
periodosome. This assembly and disassembly of the component proteins coincides with the 24 h
cycle and also with the rhythmicity in the phosphorylation of KaiC. When key residues in KaiC are
mutated no phosphorylation takes place. Due to this the periodosome formation does not occur and
the rhythms are abolished. Golden (2004) put forward a simple model explaining these facts.
Mori et al. (2002) included KaiC in the superfamily of RecA/DnaB of proteins that has a bearing
on its enzymatic activity and its functional role in bringing global changes in gene expression patterns.
These observations were based on analytical and ultracentrifugational analyses of the association
of KaiC molecules into hexameric rings in presence of ATP. Hayashi et al . (2003) reported Mg-ATP
induced hexameric ring structure of KaiC whereas in the absence of Mg-ATP KaiC remained as
a monomer. SDS-PAGE analysis revealed a ladder of six bands at 56, 120, 173, 232, 287 and 340
kDa corresponding to cross-linked species ranging from monomer to hexamer. KaiC existed as a
monomer below 1µM concentration of Mg-ATP and with the increase of concentration above this
hexameric ring structure was favoured with no monomer existing at concentrations of 100 µM.
This was further confi rmed by the studies on single and double mutations in the Walker's motif A.
Each single mutation resulted in a reduced affi nity for ATP to different extents and greatly reduced
hexamerization. The KaiC hexamer has the shape of a hexagonal pot with a large internal cavity.
The diameter and the height of the hexameric KaiC are equal, i.e. 100 Å. It has only one opening
and the cavity has a depth of 73 Å. However, the cavity has a width of 34 Å at its widest part and its
entry is narrowed to 18 Å. Two molecules of KaiA can interact with one molecule of KaiC hexamer.
However, the interaction of one molecule of KaiA with KaiC hexamer will suffi ce to enhance KaiC
phosphorylation (Hayashi et al. , 2006). Of the two ATP-binding sites of KaiC, one at the N-terminal
end is a high affi nity site where as the C-terminal ATP-motif is a low affi nity site. It is the N-terminal
motifs that are responsible for hexamerization while the C-terminal motifs are responsible for both
stabilization and phosphorylation of the KaiC hexamer. These studies are supported by ATPχS
fi lter-binding assay of KaiC derived from T . elongatus BP-1 and its mutants (Hayashi et al ., 2004).
KaiC phosphorylation can be reconstituted in vitro by using purifi ed KaiA, KaiB, KaiC proteins
with ATP. The phosphorylation and dephosphorylation cycles of KaiC in vitro were self-sustained