Plant Molecular Biology 36: 649–659, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Promoter recognition by a cyanobacterial RNA polymerase: in vitro studies
with the Calothrix sp. PCC 7601 transcriptional factors RcaA and RcaD
ese Coursin, Nicole Tandeau de Marsac and Jean Houmard
Physiologie Microbienne (CNRS URA 1129), D
epartement de Biochimie et G
Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France (
author for correspondence);
Department of Microbiology & Immunology, School of Medicine, Emory University, 3001 Rollins Research
Center, Atlanta, GA 30322, USA
Received 15 May 1997; accepted in revised form 6 October 1997
Key words: complementary chromatic adaptation; in vitro transcription, lac promoters, RcaA, transcription factor.
To study the transcriptional apparatus and the mechanisms that control gene expression in cyanobacteria, the RNA
polymerase was puriﬁed from the ﬁlamentous Calothrix sp. PCC 7601 and used in in vitro transcription assays.
Conditions required for speciﬁc transcription initiation to occur were analyzed with the eleven Calothrix PCC
7601 genes for which the 5
ends have been mapped. Most of the transcripts directly obtained did not have the
expected size, providing a test for looking at speciﬁc transcription factors. Addition of RcaA, a protein that binds
to the promoter region of the phycobiliprotein cpeBA operon, restored accurate initiation of transcription in the in
vitro system for three phycobiliprotein promoters. RcaA thus is a transcription factor that allows to mimick in vivo
transcription. In parallel, the functional properties of the Escherichia coli and cyanobacterial RNA polymerases
were compared. The enteric enzyme could not precisely initiate transcription at the promoter of a phycobiliprotein
gene and, reciprocally, the cyanobacterial RNA polymerase could initiate transcription at P
, but not from
promoters. The different behaviours of the enzymes are discussed in the light of the structural
differences that exist between subunits of the RNA polymerases.
Cyanobacteria are peculiar organisms because of their
wide variety and ecological distribution which result
from very diverse adaptation capabilities . The
regulatory mechanisms that are employed to with-
stand changes occurring in natural environments are,
however, only poorly documented at present. Getting
insights into the regulatory networks is necessary to
unravel gene expression, deﬁne the requirements for
controlling expression of a gene in a foreign back-
ground, or induce gene expression at a deﬁned stage
of cell growth, for example.
Transcriptional controls of gene expression, either
positive or negative, are very common in prokaryotes
[1, 12, 19, 20]. Sigma factors are main actors in many
of these mechanisms, being in charge of recruiting
the RNA polymerase core enzyme and recognizing
speciﬁc promoter elements. The gene that encodes
the major sigma factor, sometimes designated ‘house-
keeping’ sigma, is usually referred to as rpoD or sigA.
Cyanobacterial sigma factors have only recently star-
ted to be characterized [2–4, 18, 28, 32]. Only a few
studies have yet been conducted to determine the dif-
ferent partners required for the transcription of a gene
to occur in cyanobacteria, and most of the molecular
effectors and DNA elements involved in the regulation
of gene expression remain to be characterized [7, 8,
11, 13, 21].
Because cyanobacteriaare photoautotrophic proka-
ryotes, light plays a major role in their metabolism
both through its intensity and spectral quality, and
important changes occur in gene expression that imply
both transcriptional and post-transcriptional regula-
tions [33, 36]. The size and composition of the light-
harvesting apparatus, in particular the phycobilisomes,