CIRCADIAN RHYTHMS - Cyanobacteria - page 309

adjacent genes designated as kaiA , kaiB and kaiC regulate the circadian clock in S . elongatus PCC 7942.
The meaning of ' Kai ' in Japanese means “rotation” or “cycle”. It is further interesting to know that
these three genes could be rescued by a plasmid carrying the entire kaiABC cluster. Mapping studies
by DNA sequencing revealed that at least 19 mutations could be assigned to the three kai genes.
All these mutations were suggested to be mis-sense mutations due to single nucleotide exchanges.
Further analysis revealed that (i) the three genes had two mutations mapped to each one of them,
(ii) all possible clock phenotypes belonged to kaiC, (iii) there is no signifi cant similarity in between
the kai genes or any other known gene sequence from prokaryotes or eukaryotes except that gene
sequences similar to kaiC have been noted in certain archaebacteria and (iv) the kai group of genes
appear to be clock-specifi c because deletion of either one of the genes or the entire cluster does not
affect the viability of the strain but results in arhythmicity.
Ishiura et al . (1998) proposed a feedback model to explain the working of circadian oscillator. A
promoter situated upstream of kaiA is responsible for the production of monocistronic kaiA mRNA
where as the promoter located upstream of kaiBC cistrons produces a dicistronic transcript for kaiB
and kaiC genes. It has also been observed that the transcripts of kaiA and kaiBC show rhythmicity.
Kutsuna et al . (2005) studied the transcriptional regulation of kaiBC and identifi ed a region of 56 bp
sequence from transcription start site (upstream of the operon that is located from -55 to +1). This
region has been designated as the suffi cient promoter region (SPR). Of this, especially the sequence
from -52 to -28 bp was found to be essential for kaiBC transcription. Deletion of SPR from upstream
or downstream sequences almost completely eliminated promoter activity. At the same time, a
constitutive negative regulatory region upstream of SPR (base pairs -897 to -56) was also identifi ed
that extended into the coding region of kaiA .
Inactivation of one or the other of the Kai genes abolishes the rhythms and lowers kaiBC promoter
activity. This has been termed as negative feed back where as kaiA overexpression enhanced kaiBC
promoter activity and this has been termed as positive feed back. kaiC overexpression resets the
phase of the rhythms. Accordingly, the kaiC expression is directly linked to the phase of oscillation.
Nishimura et al . (2002) introduced mutations in kaiA region by PCR mutagenesis, a technique that
was earlier reported in case of bacteria (Diaz et al ., 1991) and Synechocystis sp. strain PCC 6803
(Narusaka et al ., 1999).
C) Properties of Kai proteins : KaiA possesses an amino-terminal domain (KaiA 135N) that has
no function in the autophosphorylation and on the basis of NMR structure determination it has
been designated as a pseudo-receiver domain. KaiA protein exists at least as long and short types.
Amino acid sequence of long type consists of approximately 300 amino acid residues. This has been
confi rmed in case of S . elongatus PCC 7942, Synechocystis sp. strain PCC 6803 and Synechococcus sp.
strain WH 8102. The conservation in the amino acid sequence in the amino-terminal region (200
amino acids) of the long type of KaiA protein is very limited whereas a high degree of conservation
persists in the 100 amino acid residues of the carboxy-terminal region. The short type of KaiA protein
(from Anabaena sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133) is independent of carboxy-
terminal domains and these lack the amino-terminal regions. Proteolytic studies of KaiA (284 amino
acid residues) protein yielded three major protein species with molecular weights of 22kDa, 17kDa
and 13kDa consisting of 1-189, 1-154 and 164-284 amino acid residues, respectively. The C-terminal
domain of KaiA consisting nearly of 100 amino acid residues is suffi cient for binding to KaiC. Crystal
and solution structures of KaiA domains reveal it to be a dimer with tight interactions (Williams et
al ., 2002; Uzumaki et al ., 2004; Ye et al ., 2004; Garces et al ., 2004; Vakonakis et al ., 2004; Fig. 1A).