Biology Reference
In-Depth Information
shorter transcript is light- and nitrogen-dependent whereas the longer transcript is synthesized
constitutively. A concomitant increase of NtcA protein inside the cells coincided with the accumulation
of the shorter transcript. The inhibition of NtcA accumulation in presence of electron transport
inhibitors and the accumulation of the two transcripts inside glucose-grown cells in darkness
emphasizes that it is electron transport and not light per se that regulates the expression of
ntcA
.
Primer extension analysis and gel mobility shift assays revealed that NtcA is bound to the shorter
transcript and not to the longer transcript. But the presence of three transcription start points (tsps)
at locations -49, -136 and -180 have been identifi ed in the promoter region of
ntcA
and these have
been designated as P1, P2 and P3, respectively (Ramasubramanian
et al
., 1996; Muro-Pastor
et al
.,
2002). Two putative NtcA-binding sites are located at -143.5 (GTAN
8
AAC) and -103.5 (GTAN
8
TAC)
in relation to tsp of
ntcA
and these are referred as 'distal' and 'proximal' NtcA-binding sites,
respectively. The distal NtcA-binding site overlapps with -10 mer sequence of the P2 promoter
region of
ntcA
(Muro-Pastor
et al
., 2002).
In vitro
binding assays performed with DNase protection
method revealed that NtcA is bound to the distal site (Ramasubramanian
et al
., 1996). The expression
of
ntcA
in relation to exogenous nitrogen source and heterocyst differentiation has been studied.
Transcripts of
ntcA
from P1 and P2 are produced in all nitrogen sources. The transcripts from P1 are
subjected to increase 6-12 h after a nitrogen step-down and are localized very strongly in the
heterocysts and those from P2 are constitutively expressed. The transcripts from P3 appear after
6-12 h of nitrogen step-down but the expression of
ntcA
from this tsp is very transient. However,
the expression of
ntcA
from P1 and P3 is dependent on NtcA and HetR (Muro-Pastor
et al
., 2002).
To identify which of these transcripts fi rst appears in the proheterocysts, Olmedo-Verd
et al
. (2006)
made three deletions from 5'-end of the promoter region of
ntcA
gene (involving the three tsps) and
introduced the altered versions of the
ntcA
promoter into the wild-type at a neutral site (the
nuiA-
nucA
region) of
Anabaena
α-megaplasmid. The integration of the respective altered promoter
constructs of
ntcA
by homologous recombination also altered the expression of wild-type
ntcA
gene.
The expression of
ntcA
from P1 and P3 was localized in the proheterocysts and heterocysts and these
observations have been additionally confi rmed by the expression of
ntcA
-
gfp
translational fusion.
Olmedo-Verd
et al
. (2008) studied the binding of NtcA to wild-type and mutated proximal and distal
NtcA-binding sites of the P2 promoter region of
ntcA
of
Anabaena
sp. strain PCC 7120. Mutation of
the proximal NtcA-binding site of
ntcA
from GTAN
8
TAC to CTAN
8
TAC abolished binding of NtcA
to the two sites but the mutation of distal NtcA-binding site from GTAN
8
TAC to CTAN
8
AAC did
not abolish NtcA binding to this site though more NtcA concentration was required than when
compared to the wild-type. However, mutation of both the proximal and distal NtcA-binding sites
still resulted in a lower NtcA-binding. These results are explained as follows. In nitrate-replete
medium,
Anabaena
sp. strain PCC 7120 shows expression of NtcA from P2 and as well as P1 that
would not require the presence of either NtcA or HetR (Muro-Pastor
et al
., 2002). When cells are
subjected to nitrogen deprivation, the low levels of NtcA present in the cells bind to the proximal
NtcA-binding site thus increasing the production of transcripts from P1. Then in presence of HetR
and other HetR-dependent elements, the transient expression from P3 takes place that in turn is
required for the localized induction of NtcA from P1 site in the prospective proheterocysts and
heterocysts. So the most important event during the nitrogen step-down is the binding of NtcA to
the proximal NtcA-binding site because this determines which of the promoters are to be utilized
for transcription of
ntcA
(Olmedo-Verd
et al
., 2008). As cyanobacteria perceive nitrogen status by
sensing intracellular 2-oxoglutarate levels (Muro-Pastor
et al.
, 2001), Valladares
et al.
(2008) reported
transcription activation by NtcA and 2-oxoglutarate of three genes
hetC
,
nrrA
and
devB
involved in
different stages of heterocyst differentiation.
