Nucleic Acids Research, 1993, Vol. 21, No. 16
3667-3670
c^-dependent promoters in Escherichia coli are located in
DNA regions with intrinsic curvature
Manuel Espinosa-Urgel and Antonio Tormo
Departamento de Bioqufmica y Biologfa Molecular I, Facultad de Ciencias Qufmicas,
Universidad Complutense, Madrid 28040, Spain
Received May 27, 1993; Revised and Accepted July 12, 1993
ABSTRACT
Expression of a number of genes during stationary
phase In Escherichia coll Is controlled by the alternative
sigma factor a* (KatF). Promoters recognized by o* do
not present a well-defined consensus sequence In their
- 1 0 and - 35 regions. By polyacrylamlde gel electrophoresls of DNA fragments performed at different
temperatures, and by computer prediction analyses, we
have found that o*-regulated promoters are located in
regions where DNA shows Intrinsic curvatures. This
feature does not appear In a statlonary-phase-lnduced
promoter which Is not controlled by a*. We propose
that DNA bending may help In recognition and/or
binding of a* to stationary-phase-induced promoters.
INTRODUCTION
In the last few years an increasing number of studies in E. coli
have focused on stationary phase, as it is possibly the state in
which this bacterium spends most of its life in natural conditions
(1). The onset of stationary phase in E.coli implies a series of
metabolic and morphological variations. Cells change their shape
from bacillar to spherical, as the result of modifications in cell
wall and membrane composition. Changes in chromosome
topology and compactation take also place in stationary phase
(2). General metabolism decreases, although several processes
are activated in response to starvation. A considerable number
of proteins are known to be synthesized during cessation of
growth. The expression of at least 30 of these proteins is under
the control of a novel sigma factor, a*, encoded by rpoS
(previously called katF) (3,4,5). Among the o*-regulated genes
are katE (encoding for catalase HPII (6,7)), xthA (exonuclease
m (7))» glgS, involved in glycogen synthesis (8), the osmorregulatory pathway genes otsBA (9), dps, which encodes for a
protein that binds non-specifically to DNA (10), and mcc,
encoding for microcin C7 (11).
Three other genes show increased expression levels associated
to cessation of growth, as a consecuence of what has been called
'gearbox' regulation, in which expression rate is inversely
proportional to growth rate (12). These genes are the morphogen
bolA (13), the division cluster ftsQAZ (12), and mcbA, which
encodes for microcin B17 (14,15). Promoters responsible for
gearbox regulation show high sequence homology at the —10
and - 3 5 regions. In spite of their sequence similarities, the way
in which these promoters are regulated is different. Expression
from the gearbox promoter bolApl is a*-dependent (16,17), as
is the ftsQpl promoter (M.Vicente et al., personal communication). Surprisingly, mcbAp, although nearly identical in
its —10 and —35 regions to bolApl and ftsQpl, is not under
the control of a*, but requires a70 for in vitro expression (16).
Although bolApl and ftsQpl show sequence homologies, other
a*-regulated promoters do not share these similarities. What is
more, some typical o^-type promoters can be recognized by a6,
as has been recently demonstrated for lacUV5 and trp (18). Thus,
against what would be expected for promoters controlled by the
same sigma factor, it is difficult to find a consensus sequence
for as-regulated promoters.
These two facts, the absence of sequence homologies between
a*-regulated promoters, and the different effects of a" upon
otherwise similar gearbox promoters led us to study other
structural features appearing in these promoters. Here we show
that all the o*-regulated promoters that we have analyzed are
located in intrinsically curved DNA regions. Computer analysis
of other a*-regulated promoters predicts them to be also in bent
DNA regions. We propose that DNA bending may play an
important role in regulation by a*, maybe compensating the
absence of a well-defined recognition sequence.
MATERIAL AND METHODS
DNA and plasmids
Plasmids used in this work are listed in Table 1. mcbA promoter
DNA was a kind gift from Felipe Moreno.
Computer analysis of DNA bending
Computer prediction of DNA bending was made with DNAstar
computer programs (DNASTAR, Inc), using the wedge model,
with the angle values calculated by Trifonov (19). Unless
otherwise indicated, the analysis was made upon the sequence
of a 200bp fragment containing the promoter region.
DNA isolation and manipulation
Plasmid DNA was obtained following classical methods (20).
DNA was always phenol extracted before and after digestion with
3668 Nucleic Acids Research, 1993, Vol. 21, No. 16
endonucleases, except when extracted from agarose gels, and
dialyzed before performing electrophoresis. Restriction enzimes
were purchased from Boehringer-Mannheim and New England
Biolabs, and used following supplier's instructions.
Polyacrylamide gel electrophoresis of DNA fragments
Analysis of differential fragment migration was performed in 5 %
polyacrylamide gels, run at temperatures of 4°C and 60°C, at
a constant voltage of 8 V/cm in 40mM Tris (pH 8.0), 5mM
sodium acetate, lmM EDTA buffer.
RESULTS
Intrinsically curved DNA in the regions encompassing o*regulated promoters
First evidences of the presence of intrinsic curvatures in DNA
regions encompassing cr'-regulated promoters were obtained by
computer analysis. To confirm these predictions, polyacrylamide
gel electrophoresis at different temperatures was performed, as
described above. Bent DNA fragments show lower mobility than
what would be expected for their size in polyacrylamide gel
electrophoresis when run at low temperatures; at high temperature
fragments migrate closer to its size (21).
Plasmid pTGVl was cleaved with BamHI. This digestion
renders a 680bp fragment containing the two promoters—the Deregulated promoter bolApl and the regular promoter bolAp2—and
the predicted bent region of bolA. At 4°C this fragment migrates
to an apparent size of — 1300bp, and close to its actual size at
60°C (Figure 1), indicating that this fragment is curved. A similar
assay was performed using plasmid pMAV103. This plasmid
carries only the bolApl promoter, in a 225bp fragment. This
construction was the one used in previous works for studying
the effects of rpoS mutations on the bolApl promoter (16). This
fragment also had altered electrophoretic mobility, migrating to
an apparent size of ~26Obp when run at 4°C, and to its actual
size at 60°C (data not shown).
To test DNA bending in the katE promoter, plasmid pRSkatE16
was digested with BamHI and EcoRI. The resulting 1.4 Kb
fragment, containing the katE promoter region was isolated and
digested with Hpall. As it is shown in Figure 2, the 444bp
fragment in which katEp is located presented lower mobility than
expected for its size when electrophoresis was performed at 4°C.
Two other fragments also showed altered mobility at low
4°C
temperature, thus indicating static bending in different regions
of katE. Computer analysis of the complete 1.4 Kb EcoRl/BamHI
fragment is shown (Figure 3), illustrating the fact that katEp is
located in a strongly curved DNA region.
The same electrophoretic analysis was performed with the
promoter region of ftsQ. Plasmid pTGV13 was cleaved with
EcoRI and Hpal. This digestion renders two fragments of an
identical size of 549bp, one of them encompassing the promoter
region of ftsQ, and the other belonging to the vector, plasmid
pRS550 (22). This digest was run in polyacrylamide gels at 4°C
and 60°C. As a control, an EcoRI/Hpal digest of pRS550 was
also loaded. Results are shown in Figure 4. At 4°C the 549bp
containing the ftsQ promoter migrates less than the vector's
fragment of the same size, indicating curvature in the promoter
region of ftsQ.
Computer analysis of bending pattern in other o*-regulated
promoters
Computer predictions of DNA bending were performed upon the
sequences of other (^-regulated promoters. Results of these
analyses were compared to the predicted structure of the bolA
4°C
60°C
Ml
445
417
Figure 2. Polyacrylamide gel electrophoresis of the EcoRI/BamK 1.4 Kb fragment
from pRSkatE16 cleaved with Hpall (lanes 2, 4), at temperatures of 4°C and
60°C. T7 DNA cut with Hpall was used as molecular weight marker (lanes 1,
3). Sizes are indicated in bp. Arrows indicate the fragment containing katE
promoter.
60°C
Figure 1. Polyacrylamide gel electrophoresis of pTGVl cleaved with BamHI
(lanes 1, 3), at temperatures of 4°C and 60°C. T7 DNA cut with Hpall was
used as molecular weight marker (lanes 2, 4). Sizes are indicated in bp. Arrows
indicate the fragment containing bolA promoter region.
Figure 3. Computer prediction of DNA structure in the 1.4 Kbfragmentcontaining
katE promoter. Arrow indicates promoter location.
Nucleic Acids Research, 1993, Vol. 21, No. 16 3669
and katE promoter regions. Both the xthA promoter and the dps
putative promoter region are predicted to be curved (Figure 5).
Two other o'-regulated genes, glgS and treA, have been
studied and could present a bent promoter region (data not
shown). Yet, a more detailed analysis of the structure of these
and other o*-regulated genes requires starting point and
promoter location being well-determined.
Mobility of the gearbox promoter mcbAp in polyacrylamide
gels
To determine a possible correlation between a* regulation and
DNA bending, mobility oimcb promoter in polyacrylamide gels
was tested. Expression from this gearbox promoter is also induced
in stationary phase, but it is regulated by a70 and not by a* (16).
A 24Obp Smal/Kpnl fragment containing mcbA promoter was
run at temperatures of 4°C and 60°C. As shown in Figure 6,
no significant differences in fragment migration were observed.
6CFC
4°C
DISCUSSION
549*
The alternative sigma factor o* is a major regulatory element,
essential for survival of E.coli in stationary phase (23), which
may replace a70 in its binding to the core of RNA-polimerase.
Expression from a wide number of promoters has been described
as being controlled by a*. Yet, when the best characterized of
J49*
4°C
6CTC
Figure 4. Polyacrylamide gel elecrophoresis of pTGV13 (lanes 1, 3), and pRS550
(lanes 2, 4) at 4°C and 60°C. Sizes are given in bp. Fragment containing ftsQ
promoter is indicated (•).
Figure 6. Polyacrylamide gd electrophoresis of the 240 bp Smal/Kpnl fragment
containing mcb promoter (lanes 1, 3), at temperatures of 4°C and 60°C. T7DNA
cut with Hpall was used as molecular weight marker (lanes 2, 4). Sizes are
indicated in bp.
Table 1. Plasmids
Plasmid
Marker
Source/Reference
pTGVl
pMAV103
pRSkatE16
pTGV13
pRS350
bolA::lacZ; Amp8
bolApl::lacZ; Amp11
katEr.lacZ; Amp*
JisQplp2::lacZ; Amp11
AmpR; Kan*; lacZYA
M.Vicente (12)
M.Vicente (15)
R.Kolter (6)
(15)
(22)
Table 2. DNA curvature in o^-regulated promoters
Promoter
1
pi ft
1
f—
E
1
••
1
1
1 1
H
•
• H
H
F
Figure 5. Computer-generated structure of promoter regions in dpsA (A; putative
promoter region) and xthA (B), compared to bolAp] (C) and kasEp (D). Arrows
indicate promoter location. The structure of katEp was rotated in order to ftrilitiitr.
the comparison with the other promoters. Physical maps of the 680bp bolAp
fragment (E) and the 1.4Kb katEp fragment (F) are depicted, showing promoter
location (arrows) and fragments used for computer predictions (shaded boxes).
Restriction sites are indicated as follows: E, EcoRI; B, BamHl; H, Hpall.
Predicted
DNA curvature
Observed
fizQpl
bolApl
katEp
map1
xthAp
dpsp2
2
ggp
trcAp2
1
F.Moreno et al, personal communication.
DNA fragments that include a putative promoter,
nd: not determined.
2
nd
nd
nd
nd
3670 Nucleic Acids Research, 1993, Vol. 21, No. 16
these promoters were searched for a sequence motif which could
be recognized by a*, no good consensus could be found at the
—10 and —35 regions. A tentative consensus has been proposed,
based upon certain sequence homologies between katEp, xthAp
and bolApl (17). Other o'-regulated promoters do not share
these homologies. As an explanation to these differences, it has
been proposed an indirect effect of a1 upon some of these
promoters (16). Yet, no other regulatory factor has been
identified, at least for bolA and katE. Thus, it seemed that the
only common feature for (^-dependent promoters was the
absence of common features.
Here we have shown that these promoters seem to be located
in DNA regions with intrinsic curvature. This structure is
computer-predicted for the best characterized o*-dependent
promoters, those of katE, xthA, and bolA, and has been confirmed
experimentally for bolAp, katEp and JisQp. It may be present
also in other (^-regulated promoters, such as the apsA promoter;
curvature has been found in the promoter region of mcc (F.
Moreno et al., personal communication), which is also under
the control of a*. Thus, four out of four studied ^-dependent
promoters are located in intrinsically curved DNA regions, and
some other are computer-predicted to be so (Table 2). On the
contrary, a growth-phase-dependent promoter which is not under
the control of <f but of a70, me gearbox promoter mcbAp, does
not show bending. This promoter is otherwise strikingly similar
to bolApl, not only in sequence but in transcription pattern (12).
DNA bending has already been described as an important
element for regulation of transcription, being associated to
promoter strength (24,25). Results presented here may indicate
that DNA bending could be related to modulation by a1. As (independent promoters lack a good consensus sequence, they could
require additional features such as DNA bending for promoter
recognition by and interaction with RNA-polimerase. The
possibility of RNA-polymerase binding to curved DNA upstream
the - 1 0 region has already been stated (24).
However, the role of the promoter sequences has to be taken
on account, as bolApl has sequence requirements at the - 1 0
region for growth-dependent induction (12) and control by o*
(M.Espinosa-Urgel and A.Tormo, unpublished results). The
question arises, then, on werther curved DNA is essential for
o* regulation of transcription, or it is just a feature related to
promoter strength. Further research following results presented
here, may allow to state the importance of this bending pattern
in promoter recognition and activity.
ACKNOWLEDGEMENTS
We gratefully acknowledge Dr M.Espinosa for allowing us to
use laboratory material and computer programs, and for helpful
discussions and critical revision of the manuscript. The original
idea of computer searching for predicted bending in the gearboxes
was his. We thank Felipe Moreno, Miguel Vicente and Roberto
Kolter for strains and plasmids, and for sharing results prior to
publication; and Miriam Moscoso for valuable help and
discussion of results. This work was supported by grant
PB89-O030 from the DGICYT.
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