Activation of s28-dependent transcription in Escherichia coli by the cyclic AMP receptor protein requires an unusual promoter organization

Activation of s²⁸-dependent transcription by CRP 1101

Table 1. Effect of mutations in the -10 and -35 elements on aer promoter activity.

b-Galactosidase activity

The table lists b-galactosidase activities (in Miller units) measured in strain M182 DfliA containing pKXH100 (CRP⁺ FliA⁺), strain M182 DfliA Dcrp
containing pKXH100 (CRP^- FliA⁺) or strain M182 DfliA containing ‘empty’ pET21a (CRP⁺ FliA^-), each carrying different aer promoter::IacZfusions
cloned in pRW50 and grown to late exponential phase (OD650 0.9-1.1) in LB medium. The aer200 fragment carries the wild-type aer promoter, the
aer206 fragment carries three point mutations in the proposed -10 element, and the aer213, aer214 and aer224 fragments carry single point
mutations in the proposed -35 octamer. The sequence of the -10 and -35 elements of the s²⁸-dependent aer promoter is listed for each fragment,
and the location of base changes in each of the mutant promoter derivatives is underlined. The consensus sequence for a s²⁸-dependent promoter
is shown below the table. Data listed are averages from at least three independent experiments, shown ± one standard deviation.

stitutions in the -10 element had the greatest effect on
promoter activity, reducing expression from the aer regu-
latory region to the level observed in the absence of
s²⁸. Mutations at positions -32, -30 and -28 in the -35
element also severely reduced promoter activity. We con-
clude that the proposed -10 and -35 elements are essen-
tial for s²⁸-dependent transcription of aer, and, together
with the transcript start site data, this argues that aer is
expressed from a single promoter, at least under the
conditions tested here.

Transcription initiation at the aer promoter in vitro

Next, we sought to confirm our in vivo findings by exam-
ining the s factor selectivity and CRP dependence of the
aer promoter in vitro. We began by cloning the aer200
fragment upstream of the loop terminator in plasmid pSR,
and tested the ability of purified Es²⁸ and Es⁷⁰ to drive
transcription from the aer promoter in an in vitro multi-
round transcription assay, in the presence and absence of
purified CRP and cAMP (Fig. 3A). In this system, tran-
scription initiating at the aer promoter terminates at the
loop terminator to generate a 158-base transcript that
can be identified by electrophoresis. In the presence of
Es²⁸, a single transcript was observed (Fig. 3A, lanes
3-12). At low Es²⁸ concentrations, this transcript is
detected only in the presence of CRP (lanes 3-6),
although some transcript is generated in the absence of
CRP as the RNA polymerase concentration is increased
(lanes 7-12). At even higher concentrations of Es²⁸, tran-
scription becomes completely independent of CRP (data
not shown). The aer transcript generated by Es²⁸ is not
detected in reactions using Es⁷⁰ (Fig. 3A, lanes 13 and
14). Instead, a single CRP-independent transcript is pro-
duced, which corresponds to the 108-base RNAI control
transcript that originates from the pSR replication origin.

To confirm that in vitro transcription initiates from the
same promoter defined in our in vivo experiments, pro-
moter unwinding by RNA polymerase was monitored by
using KMnO4 to probe for single-stranded regions of DNA
(Fig. 3B). In the presence of Es²⁸ (lanes 3 and 4), KMnO4-
reactive bands appeared from positions -10 to +4, indica-
tive of promoter melting around the -10 element of the
s²⁸-dependent promoter highlighted in Fig. 2B. This was
observed both in the presence and in the absence of CRP,
which is consistent with our finding that transcription ini-
tiation by Es²⁸ is independent of CRP in vitro at the high
RNA polymerase concentrations used in these reactions.
Incubation with Es⁷⁰ did not result in promoter melting
around the aer transcript start site, either in the presence
or in the absence of CRP (Fig. 3B, lanes 5 and 6). Taken
together, the in vitro data confirm that aer is transcribed
from a single, s²⁸-dependent promoter that is activated by
CRP when the RNA polymerase concentration is limited.
The observation that the aer promoter becomes less
dependent on CRP at higher RNA polymerase concentra-
tions suggests that CRP activates transcription by recruit-
ment of RNA polymerase (Rhodius et al., 1997).

Transcription activation at the aer promoter requires
CRP binding at an atypical location

Mutational analysis showed that CRP-dependent activa-
tion of the aer200::lacZ fusion requires CRP binding to
the single DNA target indicated in Fig. 2B (Hollands
et al., 2007). This target site is centred 49.5 bp upstream
from the transcript start site, which falls between the
typical Class I location of position -61.5 and the Class II
location of position -41.5. To investigate whether CRP
can activate s²⁸-dependent transcription from positions
-41.5 or -61.5 at the aer promoter, we constructed a
deletion or insertion in the aer200 fragment to make the

© 2009 The Authors

Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 75, 1098-1111

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