Activation of s28-dependent transcription by CRP 1103
or s28. This indicates that CRP cannot activate transcrip-
tion from the aer promoter when bound at a Class II
location. Moving the CRP site to position -61.5 (aer211)
results in a twofold decrease in promoter activity, but,
while the residual promoter activity is dependent on
CRP, it is independent of s28. The most likely explanation
for this is that, here, CRP is activating transcription from
an alternative cryptic s28-independent promoter. For
example, in the aer211 fragment, a 6 bp sequence,
5'-TAAAGA-3', is located 32 bp downstream of the DNA
site for CRP and this may well generate a weak Class II
CRP-dependent promoter served by Es70 (recall that the
consensus -10 hexamer for Es70 is 5'-TATAAT-3').
Next, we used the same system to monitor the effects of
making a 5 bp deletion or insertion between the DNA site
for CRP and the -35 element at the aer promoter. Both the
deletion, which moved the DNA site for CRP to position
-44.5 (aer226), and the insertion, which moved the DNA
site for CRP to position -54.5 (aer227), resulted in a
reduction in promoter activity in the CRP+ FliA+ strain to the
basal level observed in the absence of CRP (Fig. 4, lower
two panels). This indicates that CRP is unable to activate
transcription from the aer promoter when its DNA site is
moved to the opposite face of the DNA helix. These experi-
ments argue that optimal activation of s28-dependent tran-
scription requires CRP binding at position -49.5.
Location of RNA polymerase a C-terminal domains at
the aer promoter
Activation by CRP at both Class I and Class II s70-
dependent promoters requires a contact between CRP
activating region 1 (AR1) and aCTD. Previous work
showed that CRP-dependent activation at the aer pro-
moter also requires AR1 (Hollands et al., 2007), which
likely functions by contacting aCTD in Es28. Because the
organization of the aer promoter is unlike that at Class I or
Class II CRP-dependent promoters, it is unclear whether
the interaction between AR1 and aCTD occurs via the
upstream or downstream subunit of dimeric CRP bound at
the promoter. To address this, we mapped the location of
aCTD binding at the aer promoter using purified RNA
polymerase that had been labelled with the chemical
nuclease reagent iron [S]-1-[p-bromoacetamidobenzyl]
ethylenediaminetetraacetate (FeBABE) on a single cys-
teine residue at position 302 in the aCTDs (see Experi-
mental procedures). Transcriptionally competent open
complexes were formed using the end-labelled aer200
promoter fragment, purified CRP and FeBABE-tagged
Es28, and DNA cleavage by FeBABE was triggered.
Analysis of the pattern of DNA cleavage by gel electro-
phoresis reveals the location of the aCTDs at the aer
promoter. Note that, in this assay, in most cases, a single
Fe-BABE-labelled aCTD will give rise to cleavages in two
adjacent minor grooves, as a wave of hydroxyl radicals
generated from the Fe-BABE impinges on the target DNA
(Lee et al., 2003).
Results presented in Fig. 5A show that, in the presence
of CRP and Es28 (lane 3), DNA cleavage on the template
strand of the aer promoter is enhanced around positions
-72 and -64 upstream of the DNA site for CRP, and around
positions -38 and -30 downstream of the CRP site. This
indicates that the aCTDs can contact the DNA both
upstream and downstream of the bound CRP dimer. In the
presence of Es28, but in the absence of CRP (lane 2), the
pattern of DNA cleavage is similar to the background
detected in the absence of any protein (lane 1). This
suggests that the two aCTDs are positioned at their targets
on the DNA only in the presence of CRP. Interestingly, the
spacing between the centre of the DNA site for CRP and
the downstream FeBABE-induced DNA cleavage at the
aer promoter is identical to that observed by Lee et al.
(2003) at a Class I CRP-dependent promoter served by
Es70 (Fig. 5B and C). Similarly, the spacing between the
centre of the DNA site for CRP and the upstream FeBABE-
induced DNA cleavage is identical to that seen at a Class II
CRP-dependent promoter served by Es70 (Lee et al.,
2003). Thus, the juxtaposition between the downstream-
bound aCTD and CRP in open complexes at the aer
promoter appears to be identical to the AR1-mediated
juxtaposition between downstream-bound aCTD and CRP
at a Class I CRP-dependent promoter. Similarly, the juxta-
position between the upstream-bound aCTD and CRP at
the aer promoter appears to be identical to the AR1-
mediated juxtaposition between upstream-bound aCTD
and CRP at a Class II CRP-dependent promoter (Fig. 5B
and C).
In the crystal structure of the CRP-aCTD-DNA complex,
aCTD contacts approximately 6 bp of DNA spanning a
minor groove, centred 18-19 bp from the centre of the
DNA site for CRP (Benoff et al., 2002). The locations of
the specific DNA cleavages at the aer promoter are con-
sistent with binding of the aCTDs at sites centred 18.5 bp
both upstream and downstream of the DNA site for CRP
(Fig. 5B). These sequences are also AT-rich, a feature
associated with DNA binding by aCTD (Gourse et al.,
2000).
Regulation by CRP at another s28-dependent promoter
To investigate whether CRP directly regulates Es28-
dependent transcription at other promoters, we used elec-
tromobility shift assays to compare the binding of CRP to
end-labelled DNA fragments covering the regulatory
regions of aer and the seven other s28-dependent operons
from E. coli K-12 strain MG1655 described by Zhao et al.
(2007). The results, illustrated in Fig. 6, show that CRP
binds to a single site in the aer200 fragment, but binding
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 75, 1098-1111