Provided by University of Birmingham Research Archive, E-prints Repository
Molecular Microbiology (2009) 75(5), 1098-1 1 1 1 ■
doi:10.1111/j.1365-2958.2009.06913.x
First published online 4 November 2009
Activation of s28-dependent transcription in Escherichia coli
by the cyclic AMP receptor protein requires an unusual
promoter organizationmmi_69131098..1111
Kerry Hollands, David J. Lee, Georgina S. Lloyd and
Stephen J. W. Busby*
School of Biosciences, University of Birmingham,
Edgbaston, Birmingham, UK.
Summary
The Escherichia coli aer regulatory region contains a
single promoter that is recognized by RNA polymerase
containing the flagellar sigma factor, s28. Expression
from this promoter is dependent on direct activation
by the cyclic AMP receptor protein, which binds to a
target centred 49.5 base pairs upstream from the tran-
script start. Activator-dependent transcription from
the aer promoter was reconstituted in vitro, and a
tethered inorganic nuclease was used to find the posi-
tion of the C-terminal domains of the RNA polymerase
a subunits in transcriptionally competent open com-
plexes. We report that the ternary activator-RNA
polymerase-aer promoter open complex is organized
differently from complexes at previously character-
ized promoters. Among other E. coli promoters recog-
nized by RNA polymerase containing s28, only the trg
promoter is activated directly by the cyclic AMP recep-
tor protein. The organization of the different promoter
elements and the activator binding site at the trg pro-
moter is the same as at the aer promoter, suggesting a
common activation mechanism.
Introduction
The cyclic AMP receptor protein (CRP, also known as the
catabolite activator protein, CAP) is a global transcription
factor, which plays a central role in the control of metabo-
lism in Escherichia coli and other enteric bacteria (Kolb
et al., 1993; Barrett et al., 2005). CRP, which is functional
as a homodimer, recognizes 22 bp target sequences, with
the consensus 5'-AAATGTGATCTAGATCACATTT-3'. At
Accepted 30 September, 2009. *For correspondence. E-mail s.j.w.
[email protected]; Tel. (+44) 121 414 5439; Fax (+44) 121 414
5925.
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most target promoters studied to date, CRP activates
transcription by making one or more direct contacts with
RNA polymerase, and there appear to be two major
classes of simple CRP-activated promoters (Busby and
Ebright, 1999). At Class I promoters, CRP binds upstream
of the promoter -35 element, at a site centred at position
-61.5 (i.e. between base pairs 61 and 62 upstream from
the transcript start), or further upstream, and an activating
region (AR1) in the downstream subunit of the CRP dimer
makes contact with the C-terminal domain of one of the
two RNA polymerase a subunits (aCTD). At Class II pro-
moters, CRP binds at a target that overlaps the promoter
-35 element and is usually centred at position -41.5. AR1
in the upstream subunit of the CRP dimer interacts with
aCTD, while a second activating region (AR2) in the
downstream subunit interacts with the N-terminal domain
of one of the two RNA polymerase a subunits (aNTD)
(Busby and Ebright, 1999).
Although the mechanisms of activation by CRP at both
classes of promoter have been scrutinized in detail, most
studies have focused on a small number of natural and
synthetic model promoters, so it is unclear whether
the findings apply to all target promoters. Genomic
approaches have now identified scores of new targets for
CRP (Tan et al., 2001; Brown and Callan, 2004; Gosset
et al., 2004; Zheng et al., 2004; Grainger et al., 2005).
This affords an opportunity to study CRP-dependent regu-
lation at a range of naturally occurring promoters, and to
uncover novel mechanisms of regulation by CRP. Previ-
ously, Hollands et al. (2007) investigated the action of
CRP at 11 such uncharacterized targets in the E. coli K-12
genome. One of these was located in the regulatory
region of the aer gene, which encodes an aerotaxis
sensor protein that controls movement of bacterial cells in
response to the availability of oxygen and other electron
acceptors in the environment (Bibikov et al., 1997; Reb-
bapragada et al., 1997; Taylor et al., 1999). CRP binding
upstream of aer was first detected by Grainger et al.
(2005) in a whole genome chromatin immunoprecipitation
analysis, and it was subsequently shown that CRP acti-
vates transcription by binding to a single DNA site in the
aer regulatory region (Hollands et al., 2007).
Recent transcriptome analyses have indicated that
expression of aer in both E. coli and Salmonella enterica
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd