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(Received for publication, March 3, 1995; and in revised form, June 23, 1995) From the
SoxR protein of Escherichia coli is activated by
superoxide-generating agents or nitric oxide as a powerful
transcription activator of the soxS gene, whose product
activates
Excessive production or inadequate disposal of reactive
derivatives of oxygen, such as superoxide (O) and hydrogen peroxide
(H Genetic
analysis in Escherichia coli has helped define responses that
are triggered by distinct signals of oxidative stress. One group of
genes is activated by H SoxR,
isolated from bacteria that overproduce the protein, contains an FeS
cluster(s) essential for in vitro transcriptional activation
of the soxS promoter (Hidalgo and Demple, 1994). The metal is
not required for the binding of SoxR to the soxS promoter nor
for the subsequent binding of RNA polymerase (Hidalgo and Demple,
1994). These observations suggest that the critical effect of activated
SoxR on transcription occurs at a later stage, perhaps by specific
conformational effects of Fe-SoxR ( Although protein FeS centers have been
most commonly associated with electron transfer and some enzymatic
dehydratase reactions (Johnson, 1994), iron has recently been proposed
as an important regulatory component of other genetic responses
(Beinert, 1990). In the cytoplasmic form of mammalian aconitase, for
example, the iron center regulates the activity of the protein as an
RNA-binding factor: the apoprotein binds mRNAs encoding ferritin
(blocking its translation) and the transferrin receptor (stabilizing
the message), while the [4Fe-4S] and [3Fe-4S] forms
do not (Klausner et al., 1993). This protein is thus linked to
the iron status of the cell and coordinates key constituents of iron
assimilation, utilization, and storage. The Fnr protein of E. coli coordinates transcription of a large number of genes that allow
cells to take advantage of electron acceptors other than oxygen (Green
and Guest, 1993). Although iron seems to be required for in vitro DNA binding by Fnr, the structure of its metal center has not been
elucidated. The structure of the iron center in SoxR is of
importance both for the transcriptional activity described above and
for the role of this protein as a sensor of oxidative stress. We
describe here experiments that define [2Fe-2S] clusters in
SoxR, and we show that only the metalloprotein activates in vitro transcription and is apparently associated with formation of the
open complex at the soxS promoter.
The transcription products were quantified by primer
extension analysis. Primer 1 (5`-CTGAATAATTTTCTGATGGG-3`; Nunoshiba et al.(1992)) hybridized 64 bp downstream of the
transcriptional start site of the soxS gene. As an internal
control for transcription activity,
Iron concentration was determined by two different methods:
by inductively coupled plasma emission spectrometry (Hidalgo and
Demple, 1994) and colorimetrically by using the iron chelator ferrozine
(Stookey, 1970). For the colorimetric determinations, 460-µl
samples of SoxR were mixed with 100 µl of ultrapure concentrated
HCl (Baker analyzed), and the mixture was incubated at 80 °C for 20
min with occasional vigorous shaking. After centrifugation at 15,000
Labile inorganic sulfide was determined by
using a modification of the basic methylene blue procedure (Fogo and
Popowsky, 1949) as described by Beinert(1983).
Figure 1:
SoxR-dependent in vitro transcription of the soxS gene. Increasing amounts of
either Fe-SoxR or apo-SoxR (1-100 ng; 3-300 nM SoxR monomer) were incubated with a constant concentration of RNA
polymerase (100 nM) in in vitro transcription
reactions. The primer extension products for the bla and soxS transcripts are indicated.
Figure 2:
5-Phenyl-1,10-phenanthroline-copper(I)
(CuPPA) footprinting of the soxS promoter by SoxR. A,
a 180-bp fragment was labeled in the transcribed strand (right
panel) or the nontranscribed strand (left panel) and
incubated with various combinations of RNA polymerase (50 nM),
Fe- or apo-SoxR (25 nM), as indicated. The binding reactions
were incubated with the cleavage reagent CuPPA as explained in the
text. The sites of hypersensitivity are indicated by arrows.
The -10 and -35 sites of the soxS promoter are
indicated, as well as the transcriptional start site (+1). Each
strand was also chemically digested with dimethyl sulfate and
piperidine in a ``G-specific'' reaction (Sambrook et
al., 1989) to generate sequence markers (G in the
figure). For the left panel, the marker positions were
established from a longer autoradiographic exposure. B,
comparison of the DNase I (Hidalgo and Demple, 1994) and CuPPA
footprints and hypersensitive sites. The -10 and -35 sites
of the soxS promoter are indicated with brackets. The
9-bp inverted repeat is shown in bold, the center of the dyad
symmetry being labeled with a dot. The outlined areas show the protection exerted by SoxR against cleavage by DNase I (closed box, upper) or CuPPA (horizontal
lines, lower). The hypersensitive sites are indicated by arrows.
CuPPA-hypersensitive sites were observed in the vicinity of the soxS transcription start site only with Fe-SoxR and RNA
polymerase together, in both the nontranscribed (Fig. 2A, left) and the transcribed strand (Fig. 2A, right). CuPPA-hypersensitive sites
at -3 to -7 in the transcribed strand and at +4 and
+5 of the nontranscribed strand have been associated with open
complex formation at other promoters (Sigman et al., 1991;
Thederahn et al., 1990). CuPPA-hypersensitive sites were also
reported at +4 to +6 in the transcribed strand of the lacUV5 open complex (Thederahn et al., 1990), but
were not apparent in our experiments. These sites may have been
difficult to detect because they are much weaker than those seen at
-3 to -7 in the same strand (Sigman et al., 1991;
Thederahn et al., 1990). Taken together, these data indicate
that Fe-SoxR specifically leads to open complex formation by RNA
polymerase. In addition, three sites in the nontranscribed strand,
in the center of the SoxR binding site, became unprotected against
CuPPA only with Fe-SoxR and RNA polymerase together (Fig. 2A, left side). This deprotection was
observed consistently when the background cleavage by CuPPA was high,
but was difficult to detect under milder cleavage conditions. We
therefore determined whether the footprinting reagents had specific
effects on the in vitro transcription of soxS. CuPPA
at 0.16 mM inhibited soxS-specific transcription
(relative to bla transcription) by 30%, but 6.7 mM mercaptopropionic acid eliminated detectable transcription of both bla and soxS. Because of the general inhibition of
transcription by mercaptopropionic acid, we checked for specific
effects on Fe-SoxR. Fe-SoxR retained 90% of the visible absorption
characteristic of the intact metalloprotein (Hidalgo and Demple, 1994).
Therefore, the integrity of Fe-SoxR was not strongly compromised by the
footprinting reagents, although partial effects were noted. The
CuPPA footprinting results are compared in Fig. 2B with
those found previously for DNase I (Hidalgo and Demple, 1994). The
cleavage sites in the nontranscribed strand that became unprotected
with Fe-SoxR and RNA polymerase are in the center of dyad symmetry of
the SoxR binding site (Fig. 2B). Interestingly,
CuPPA-hypersensitive sites were described for Hg-MerR, also positioned
in the center of the protein binding site (Frantz and O'Halloran,
1990).
Figure 3:
Pattern for SoxR sedimentation in sucrose
gradients. Fe-SoxR (60 µl of 0.167 mg/ml) (A) or apo-SoxR
(80 µl of 0.125 mg/ml) (B) were loaded on 5-20%
sucrose gradients together with 10 µg of carbonic anhydrase (29
kDa) and 10 µg of soybean trypsin inhibitor (20.1 kDa). After
centrifugation, fractions were collected and analyzed for protein by
SDS-PAGE, silver staining, and quantitative densitometry. The figure
shows the percentage of each protein contained in each fraction for the
gradients with Fe-SoxR (A) or apo-SoxR (B).
The amount of iron in freshly
purified SoxR was quantified by two independent methods:
colorimetrically with the iron chelator ferrozine and by plasma
emission spectrometry. As summarized in Table 1, the ratio of Fe
to SoxR monomer averaged 2.7 ± 0.3.
For the determination of
inorganic sulfide, the low solubility of SoxR necessitated using the
assay described by Beinert(1983). These measurements revealed ratios of
S
Figure 4:
Spectroscopic analysis of Fe-SoxR. A, UV/visible absorption. SoxR protein,
Figure 5:
Effect of DTT treatment on the visible
spectrum, transcriptional activity, and physical properties of Fe-SoxR. A, visible spectra of untreated Fe-SoxR (solid line)
and Fe-SoxR incubated with 100 mM DTT (dashed line). B, effect of DTT on in vitro transcription. Reactions
were as described for Fig. 1B, except that SoxR treated
first with 100 mM DTT was then diluted into the transcription
reactions either containing 100 mM DTT or omitting the DTT (to
yield a final concentration of 5 mM DTT). C, gel
filtration of Fe-SoxR in the absence of DTT (upper panel) or
Fe-SoxR treated with 100 mM DTT and chromatographed with 100
mM DTT in the elution buffer (lower panel). Fractions
were collected and analyzed for SoxR protein (by SDS-PAGE and Coomassie
Blue staining) and iron (using ferrozine).
Although the visible absorption spectrum of Fe-SoxR was changed by
the DTT treatment (Fig. 5A), DTT-treated SoxR did not
show an EPR spectrum (data not shown). Therefore, DTT-treated Fe-SoxR
does not correspond to the form of the protein with reduced
[2Fe-2S] centers. We further analyzed the effect DTT had on
Fe-SoxR by gel filtration chromatography in the absence or the presence
of 100 mM DTT in the column buffer. The amounts of SoxR
protein and Fe were determined in the eluted fractions. SoxR and Fe
co-eluted at a 1:2 ratio in the samples without DTT (Fig. 5C). Chromatography in the presence of DTT
yielded a peak of SoxR associated with diminished amounts of iron (a
1:0.9 ratio in the experiment shown) accompanied by a considerable
amount of iron of slower mobility (Fig. 5C). These data
indicate that DTT reversibly blocks the transcriptional activity of
Fe-SoxR by destabilizing the [2Fe-2S] centers, which can then
be physically removed in the continuing presence of high levels of DTT. The studies presented here indicate that SoxR protein is a
homodimer that, in its activated form, contains a pair of
[2Fe-2S] centers. These metal clusters are not required to
maintain the overall structure of SoxR, since the apoprotein is also a
homodimer that binds the soxS promoter with high affinity
(Hidalgo and Demple, 1994). It remains to be established whether the
two SoxR [2Fe-2S] clusters are arranged as one per subunit or
as a pair of clusters coordinated between the subunits. With respect to
the latter possibility, it is interesting to note that a single Hg is
coordinated to the homologous MerR protein by distinct cysteine ligands
from each subunit of a dimer (Helmann et al., 1990). Two of
these cysteine residues of MerR are positioned identically in
alignments with the SoxR protein (Amábile-Cuevas
and Demple, 1991). At least one intersubunit iron-sulfur cluster has
been described, the [4Fe-4S] center of Azotobacter
vinelandii nitrogenase (Georgiadis et al., 1992). The
[2Fe-2S] centers of SoxR are necessary for the
protein's function as a transcriptional activator. The features
of CuPPA footprinting of Fe-SoxR and RNA polymerase at the soxS promoter show that only active SoxR leads to open complex
formation by the polymerase. For the homologous MerR protein,
transcriptional activation has been proposed to result from localized
underwinding that compensates for the suboptimal spacing (19 bp)
between the -10 and -35 elements of the merT promoter (Ansari et al., 1992). The SoxR-regulated soxS promoter also seems to be overwound, with 19-bp spacing
(Hidalgo and Demple, 1994). The FeS centers of many proteins are
damaged when cells are exposed to intracellular superoxide-generating
agents or to nitric oxide. Superoxide-sensitive FeS proteins typically
contain [4Fe-4S] centers (Gardner and Fridovich, 1991;
Liochev and Fridovich, 1992). Tetranuclear FeS centers, as found in
aconitase, are inactivated in mammalian cells exposed to nitric oxide
(Drapier et al., 1993; Weiss et al., 1993), perhaps
by peroxynitrite (Hausladen and Fridovich, 1994) formed from the
combination of NO and O (Koppenol et al., 1992). In contrast,
Fe-SoxR must remain active when E. coli is exposed to high
intracellular fluxes of O (Nunoshiba et al., 1992; Wu and
Weiss, 1992) or NO (Nunoshiba et al., 1993, 1995). Perhaps
binuclear [2Fe-2S] clusters are well suited to this
requirement. The [2Fe-2S] clusters of SoxR seem to be quite
stable in the oxidized form that is rapidly generated upon exposure of
the protein to air (Hidalgo and Demple, 1994). ( Although
the metal centers of SoxR are clearly essential for the transcriptional
activity of the protein (Hidalgo and Demple, 1994; this work), the
mechanism that activates SoxR in vivo is unknown. As
demonstrated by the EPR experiments present here, the oxidized form of
the protein is certainly active. The question then is whether the
nonactivated state for SoxR is the reduced form or the apoprotein, or
perhaps some other species. Some recent experiments So
far, apo-SoxR has been isolated only following purification of the
protein in buffers containing
Volume 270,
Number 36,
Issue of September 08, pp. 20908-20914, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
2Fe-2S
Clusters in the Escherichia coli SoxR
Protein and Role of the Metal Centers in Transcription (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
10 other promoters. SoxR contains non-heme iron
essential for abortive initiation of transcription in vitro.
Here we show that this metal dependence extends to full-length
transcription in vitro. In the presence of E. coli
RNA polymerase, iron-containing SoxR mediates
open complex formation at the soxS promoter, as determined
using footprinting with Cu-5-phenyl-1,10-phenanthroline. We
investigated the nature of the SoxR iron center by chemical analyses
and electron paramagnetic resonance spectroscopy. Dithionite-reduced
Fe-SoxR exhibited an almost axial paramagnetic signature with g values
of 2.01 and 1.93 observable up to 100 K. These features, together with
quantitation of spin, iron, and S
, and hydrodynamic
evidence that SoxR is a homodimer in solution, indicate that
(SoxR)
contains two [2Fe-2S] clusters. Treatment
of Fe-SoxR with high concentrations of dithiothreitol caused subtle
changes in the visible absorption spectrum and blocked transcriptional
activity without generating reduced [2Fe-2S] centers, but
was also associated with the loss of iron from the protein. However,
lowering the thiol concentration by dilution allowed spontaneous
regeneration of active Fe-SoxR.
O), is called oxidative stress (Sies, 1991).
Cells respond to sublethal levels of oxidative stress by coordinately
activating batteries of antioxidant genes (Demple, 1991; Hidalgo and
Demple, 1995). The molecular signals that activate these
multifunctional defense systems have been the objects of considerable
recent interest (Meyer et al., 1993; Gounalaki and Thireos,
1994; Nunoshiba et al., 1993;
González-Flecha and Demple, 1994).O-imposed oxidative
stress and is controlled by the oxyR gene (Demple, 1991; Storz et al., 1991). In contrast, the soxRS system governs
an inducible response to superoxide-generating agents (Nunoshiba et
al., 1992; Wu and Weiss, 1992) or nitric oxide (Nunoshiba et
al., 1993, 1995). The soxRS response occurs in two
stages: an intracellular signal of oxidative stress converts existing
SoxR protein into a potent transcriptional activator of the soxS gene; the resulting increase in SoxS levels then triggers
expression of the various regulon genes
(Amábile-Cuevas and Demple, 1991).
)on DNA, as proposed for
the homologous MerR protein (Ansari et al., 1992). It seems
likely that the FeS center(s) of SoxR is involved in the signal
transduction mechanism that links O or NO stress to gene activation in
the soxRS system.
Purification of SoxR
Purification of SoxR
protein from E. coli containing the SoxR expression plasmid
pKOXR was performed as described previously (Hidalgo and Demple, 1994).
Fractions eluted from heparin-agarose columns (Life Technologies, Inc.)
(purity of 65-80%) were used for these studies; the more
purified material obtained by DNA-affinity chromatography (Hidalgo and
Demple, 1994) could not be obtained in a high enough concentration for
the physical studies conducted.
In Vitro Transcription
All the solutions and
reagents used during the assay were prepared RNase-free (Sambrook et al., 1989). Protein-DNA binding reactions were performed in
19 µl containing 75 mM KCl, 2 mM dithiothreitol
(DTT), 10% glycerol, 15 mM MgCl, 10 mM Tris-HCl, pH 7.5, 200-400 ng of pBD100 DNA, and the
indicated amounts of SoxR. Plasmid pBD100 is a pBR322 derivative
containing an E. coli genomic insert of
4 kilobases that
includes the whole soxRS locus
(Amábile-Cuevas and Demple, 1991). After a 5-min
incubation at room temperature, 1 µl of 1 mg/ml E. coli
-containing RNA polymerase (
300 units/ml;
kindly provided by Drs. Linda D'Ari and Michael Chamberlin,
University of California, Berkeley) was added to each sample to achieve
a final concentration of 0.1 µM, and the reactions were
incubated for 15 min at 37 °C. One µl of a mixture of the four
NTPs (25 mM each) was then added, and the polymerase extension
reaction proceeded for another 5 min at 37 °C. The in vitro transcription reaction was then stopped by mixing into each sample
330 µl of a mixture of a solution of 73% ethanol, 7 µg/ml tRNA,
0.11 M sodium acetate. After a 45-min precipitation at
-20 °C, the samples were centrifuged for 20 min at 10,000
g at room temperature. The precipitates were
resuspended in 10 µl of H
O treated with diethyl
pyrocarbonate.
-lactamase (bla) gene
transcript, also directed by pBD100
(Amábile-Cuevas and Demple, 1991), was quantified
in parallel by primer extension analysis using primer pBR-1
(5`-GGGTGAGCAAAACAGGAA-3`), which hybridizes to a site 105 bp
downstream from the 5`-end of this message (Russell and Bennett, 1981).
The primers were labeled at the 5`-end with
[-
P]ATP (3,000 Ci/mmol; DuPont NEN) and T4
polynucleotide kinase (New England Biolabs). Fifty fmol of the
indicated
P-labeled primer was annealed to 5-µl
samples from the in vitro transcription reactions, and primer
extension reactions were performed with avian myeloblastosis
virus-reverse transcriptase (Promega) as recommended by the
manufacturer. Samples corresponding to 40% of each reaction were
analyzed by electrophoresis on an 8% polyacrylamide, 6 M urea
gel (Sambrook et al., 1989).
P-Labeled
X174
DNA digested with HinfI (Promega) was used for size
calibration.
5-Phenyl-1,10-phenanthroline-Copper(I) (CuPPA)
Footprinting
A 180-bp DNA fragment containing the soxS promoter was isolated and labeled in one strand as described
previously (Hidalgo and Demple, 1994). For binding, labeled DNA
(0.5-1 nM final concentration;
10,000 cpm) and
SoxR protein (
25 nM in the reaction mixture) were
incubated in an 18-µl reaction containing 10 mM
Tris
HCl, pH 8.0, 50 mM KCl, 2 mM mgCl, 5% glycerol, 0.05% Nonidet P-40, 0.1 mM EDTA, pH 8.0, 50 µg/ml bovine serum albumin, 10 µg/ml
salmon sperm DNA, and 1 mM 3-mercaptopropionic acid. After 10
min at room temperature, 1 µl of E. coli RNA polymerase
(130 units/ml; Promega) was added (final concentration 50 nM),
and the incubation continued for 15 min at 37 °C. The cleavage
reagent was freshly prepared by mixing 10 µl of 32 mM
5-phenyl-1,10-phenanthroline (Sigma) in ethanol with 10 µl of 7.2
mM cupric sulfate (Sigma) and immediately diluting it with 80
µl of HO. One µl of the cleavage reagent was added
to each 19-µl binding reaction, and strand scission was initiated
by addition of 1.2 µl of 100 mM 3-mercaptopropionic acid
(Sigma). After 2 min at 37 °C, the reactions were quenched by
adding 1.3 µl of 50 mM 2,9-dimethyl-1,10-phenanthroline
(Sigma) (dissolved in ethanol) and further incubation for 2 min at room
temperature. The DNA was then precipitated by the addition of a
100-µl aliquot of the following mixture: 1 ml of ethanol, 50 µl
of 3 M sodium acetate, pH 5.2, and 10 µl of 1 mg/ml yeast
tRNA. After centrifugation, the precipitated DNA was redissolved in 5
µl of formamide loading buffer and analyzed by electrophoresis in
5% polyacrylamide, 6 M urea gel (Sambrook et al.,
1989). The same 180-bp probe was cleaved in a Maxam-Gilbert
guanine-specific reaction (Sambrook et al., 1989) and used as
a DNA sequence ladder.Sedimentation Analysis
SoxR protein was analyzed
by zonal centrifugation as described by Freifelder(1973) in 5-20%
sucrose gradients in 0.5 M NaCl, 50 mM HEPES, pH 7.6.
The relatively high salt concentration was necessary to maintain SoxR
solubility. Bovine carbonic anhydrase (29.0 kDa; Sigma) and soybean
trypsin inhibitor (20.1 kDa; Sigma) were used as markers. After
sedimentation for 24 h at 50,000 rpm in a Beckman SW50.1 rotor, 0.3-ml
fractions were collected from the bottoms of the tubes using a
peristaltic pump. Samples were analyzed by SDS-PAGE, silver staining,
and quantitative densitometry (BioImage system, Millipore).Protein, Iron, and Labile Sulfide
Determinations
Protein concentrations were determined by
Coomassie staining of SDS-polyacrylamide gels using as a standard SoxR
previously quantified by amino acid analysis (Hidalgo and Demple,
1994). g for 5 min to remove denatured protein, 510 µl of
the supernatant was mixed with 20 µl of 10 mM ferrozine
and 20 µl of 75 mM ascorbic acid. The mixture was
neutralized by the addition of 120 µl of saturated ammonium acetate
to allow ferrozine chelation. After a 20-min incubation at room
temperature, the absorbance at 562 nm was determined and iron
concentration was calculated using
= 27,900 M
cm
(Stookey, 1970).
Electron Paramagnetic Resonance (EPR)
Spectroscopy
Samples of purified Fe-SoxR (500 µl at 10
µM) inside an anaerobic chamber (Vacuum Atmospheres Co.
model HE-493) at an O
concentration of 0.5-1 ppm
were incubated with 10 µl of 3 mg/ml dithionite (freshly dissolved;
this amount contained
25 reducing equivalents (Hidalgo and Demple,
1994)). Oxidized and reduced SoxR were sealed in cuvettes and removed
from the chamber, and the spectra were recorded with a Hewlett-Packard
model 8452A spectrophotometer to determine the extent of reduction. The
cuvettes were then returned to the anaerobic chamber, opened, and 300
µl of each sample was placed inside a 4-mm EPR sample tube. Tubes
were sealed with rubber septa, removed from the anaerobic chamber, and
immediately frozen in a bath of liquid nitrogen until analysis. EPR
spectra at X-band were recorded at 10-100 K on a Bruker model ESP
300 spectrometer maintained at constant temperature either with an
Oxford Instruments ESR 910 continuous flow cryostat or with a Bruker
ER4111VT variable temperature controller. Spin quantitations were
carried out under nonsaturating conditions and were calculated by
comparison of the integrated intensity of samples with that of a 1
mM Cu
prepared as described by
Malmström et al.(1970). Intensities of
samples and the standard were corrected for differences in g value as
described previously (Aasa and Vänng,
1975).
DTT Treatment of Purified Fe-SoxR Fractions
SoxR
protein was incubated aerobically with increasing concentrations of DTT
(10 to 100 mM) for 10 min at room temperature. The spectra of
the oxidized and DTT-treated preparations were recorded in a
Perkin-Elmer Lambda 3A UV/VIS spectrophotometer. In vitro transcription reactions for the DTT-treated samples were performed
under the conditions described above, except that the same
concentration of DTT used for the SoxR treatment was maintained in the
transcription reaction. Reversibility of the DTT treatment was analyzed
by omitting DTT from the in vitro transcription reaction.
Stability of the FeS center in DTT-treated SoxR was analyzed by gel
filtration chromatography of 100-µl samples using NICK columns
(Pharmacia), following the manufacturer's instructions. Protein
concentration in the eluted fractions (150 µl) was measured as
described above. Colorimetric determination of iron was performed by
adding 0.1 M DTT to the eluted fractions (except for those
which already contained it), and a further addition of 10 µl of 10
mM ferrozine. The absorption at 562 nm was determined after 20
min of incubation, and iron concentrations were estimated as described
above.
Fe-SoxR-dependent Transcription on a Supercoiled
Template
We previously showed that purified Fe-SoxR, but not
apo-SoxR, was able to initiate abortive transcription from a linear
template containing the soxS promoter (Hidalgo and Demple,
1994). However, we had not examined whether this difference is retained
in the more relevant situation of a supercoiled template and
transcription of the intact soxS gene in the presence of
competing promoters. A supercoiled plasmid containing the whole soxRS locus (pBD100; Amábile-Cuevas and
Demple, 1991) was used as the template for these in vitro reactions and allowed for the simultaneous detection of bla (-lactamase gene) transcription, which was expected to be
SoxR-independent. In the presence of E. coli -containing RNA polymerase and Fe-SoxR, a
64-nucleotide primer extension product was detected that corresponded
to the size expected for soxS mRNA, while a 105-nucleotide
product corresponded to the size expected for the bla transcript. In these experiments (Fig. 1), the amount of soxS mRNA increased strongly with the amount of Fe-SoxR added
to the in vitro transcription reaction and was near-maximal
with 10 ng of this protein. In contrast, even with 100 ng of apo-SoxR,
only a small increase in the production of soxS transcript was
observed compared to control reactions with RNA polymerase alone. Thus,
the transcriptional activity of SoxR in this assay also depends
strongly on its iron content.
5-Phenyl-1,10-phenanthroline-Copper(I)
Footprinting
Our previous studies (Hidalgo and Demple, 1994)
showed that Fe-SoxR has only a small effect on the binding of RNA
polymerase to the promoter, which suggested that Fe-SoxR acts at a
subsequent step, such as conformational changes leading to the
initiation of transcription. We addressed this issue by conducting
footprinting studies with 5-phenyl-1,10-phenanthroline-copper(I)
(CuPPA), a reagent that is also sensitive to protein-induced changes in
DNA structure (Sigman et al., 1991). Footprints of both
Fe-SoxR and apo-SoxR across the -10 to -35 region of both
strands of the soxS promoter were apparent in the absence and
in the presence of RNA polymerase (Fig. 2A). These
footprints were not as sharply defined as those observed with DNase I
(Hidalgo and Demple, 1994), but were detected consistently.
Iron and S
Before determining the iron stoichiometry of SoxR, we
first established the oligomeric state of the protein. Two features
seemed to indicate that SoxR could exist in solution as a dimer (or
higher oligomer). First, the SoxR homologue, MerR, is dimeric both in
the presence and in the absence of the HgContent of Dimeric
SoxR
(Helmann et al., 1990). Second, SoxR binds a DNA site in the soxS promoter that has perfect dyad symmetry (Hidalgo and Demple,
1994). This hypothesis was tested by sedimentation analysis of both Fe-
and apo-SoxR. In these experiments, a clear peak of SoxR protein was
observed sedimenting just slower than carbonic anhydrase (29 kDa) and
significantly faster than soybean trypsin inhibitor (20 kDa) (Fig. 3). These data indicate that both Fe-SoxR and apo-SoxR are
predominantly homodimers at 0.5 M NaCl, even in dilute
solution (<1 µM).
:SoxR monomer near 2 in 11 independent, freshly
prepared samples ( Table 1and data not shown). Together with the
iron determinations described above, these data indicate that
(SoxR)
might contain either a single [4Fe-4S]
cluster or a pair of [2Fe-2S] centers.EPR Spectroscopy of Fe-SoxR
EPR spectroscopy was
used to obtain more information about the structure and properties of
the SoxR FeS center(s). Both oxidized Fe-SoxR and the protein reduced
with an excess of dithionite (Fig. 4A) were analyzed by
EPR. No signal was observed for oxidized SoxR at temperatures between
10 and 100 K (Fig. 4B). However, the dithionite-treated
protein showed a nearly axial signature with apparent g values of 2.01
and 1.93 (Fig. 4B). This signal was observed at
temperatures as high as 100 K. The integrated intensity indicated the
presence of 1 spin/2.2 Fe (1.3 spin/SoxR monomer). Of the common types
of FeS clusters, the symmetry, temperature dependence, and integrated
intensity of the SoxR EPR signal are consistent only with a
[2Fe-2S] cluster (Orme-Johnson and Orme-Johnson, 1982). A
[3Fe-4S] cluster would be expected to exhibit an isotropic
signal in its oxidized state, which would integrate to <1 spin/3 Fe.
A [4Fe-4S] cluster should exhibit a rhombic signal, which
should be observed only at low temperature (<20 K) and which should
integrate to <1 spin/4 Fe. In contrast, the data are entirely
consistent with the presence in (SoxR) of two
[2Fe-2S] clusters. The fact that the spin/SoxR monomer ratio
exceeds 1 suggests that the SoxR concentration is routinely
underestimated (by 30%) by our method.
10 µM in 50 mM HEPES-NaOH, pH 7.6, 0.5 M NaCl, was
reduced with dithionite under anaerobic conditions (see text for
details). The absorption spectrum over the range 300-600 nm was
recorded immediately after transfer of the sample to a sealed cuvette (reduced SoxR). An untreated Fe-SoxR sample is also shown (oxidized SoxR). The strong absorption below 380 nm is due to
the relatively large amount of dithionite used for reduction in this
experiment (25 eq relative to SoxR, compared to 5 eq in previous work
(Hidalgo and Demple, 1994)). B, EPR spectroscopy. Oxidized or
dithionite-reduced SoxR (300-µl samples) were processed for EPR
spectroscopy as described under ``Materials and Methods.'' Upper trace, EPR spectrum of
10 µM reduced
Fe-SoxR at 40 K; the g values are indicated. Lower
trace, oxidized (not dithionite-treated) Fe-SoxR. The upper
spectrum was recorded at 40 K with the following spectrometer
conditions: microwave frequency, 9.42 GHz; microwave power, 200
microwatts; modulation amplitude, 1 millitesla (mT); receiver
gain, 10
. The lower spectrum was recorded at 10 K, with a
modulation amplitude of 0.4 millitesla, and all other conditions
identical with those described for the upper spectrum. No signal was
observed for oxidized SoxR at temperatures between 10 and 100 K, and
magnetic fields between 290 and 370
millitesla.
Destabilization of the SoxR [2Fe-2S] Center by
Thiols
Some transcriptional regulators are thought to be
controlled by reversible redox reactions of the protein (Storz et
al., 1990; Abate et al., 1990). We therefore tested
whether reduction of the [2Fe-2S] centers of SoxR might
modulate its transcriptional activity. Preliminary experiments showed
that the dithionite-treated protein could not be readily used for this
purpose, owing to its easy reoxidation (Hidalgo and Demple, 1994). We
therefore examined the effects of treating Fe-SoxR with DTT.
Concentrations of this thiol 
50 mM did not alter the
visible spectrum of SoxR. However, incubation of Fe-SoxR with DTT at
75 mM decreased the absorption peaks at 414,462 and 548 nm
and generated a new maximum at 424 nm (Fig. 5A). The
activity of these samples was analyzed in in vitro transcription reactions also containing DTT. In the presence of
100 mM DTT, the transcriptional activity of Fe-SoxR was
abolished (Fig. 5B, lane 3 versus lane 2).
However, if the DTT concentration was diminished either by gel
filtration chromatography in elution buffer lacking the thiol (data not
shown) or by omitting the thiol from the transcription reaction (Fig. 5B), Fe-SoxR transcriptional activity was
completely recovered. The high DTT concentrations did not affect the
transcription of the bla gene (Fig. 5B).
)Such
stability is shared by the spinach dihydroxyacid dehydratase, which has
a [2Fe-2S] cluster that is stable in the presence of O, while
the [4Fe-4S] cluster of the E. coli dehydratase is
exquisitely sensitive to O (Flint et al., 1993). suggest
that dithionite-reduced Fe-SoxR is still active as a transcription
factor (in contrast to DTT-treated Fe-SoxR), but the ease with which
this protein is reoxidized by Oin vitro indicates
that caution should be applied in this interpretation. It is also
possible that apo-SoxR is the physiologically relevant inactive state.
If this is so, the [2Fe-2S] centers would be reconstituted
adventitiously during extraction of the protein, even from cells not
treated to activate SoxR. This possibility is being explored.-mercaptoethanol (Hidalgo and
Demple, 1994). Since the mere addition of -mercaptoethanol to
Fe-SoxR did not affect its spectroscopic or transcriptional
properties, it seemed likely that this thiol destabilizes
the [2Fe-2S] centers and allows their removal during
chromatography (Hidalgo and Demple, 1994). A similar destabilizing
effect seems to be mediated by DTT, high concentrations of which also
abolish the transcriptional activity of Fe-SoxR. DTT-treated Fe-SoxR
and apo-SoxR are the only two transcriptionally inactive forms of the
protein thus far identified. This opens the possibility that the
DTT-treated protein could correspond to the inactive form of SoxR in vivo, perhaps with partially disassembled
[2Fe-2S] centers. However, the high thiol concentrations
necessary to generate this inactive state do not prevail in cells,
which typically contain 5 mM glutathione accounting for
most of the low molecular weight thiol (Meister and Anderson, 1983).
))
We are grateful to Beatriz
González-Flecha for helpful discussions and to Ed
Voelkel for his help in setting up the anaerobic chamber. We also thank
Linda D'Ari and Michael Chamberlin (University of California) for
providing us with purified E. coli RNA polymerase and Becky
Auxier (University of Georgia) for performing the ICP analysis. We are
especially grateful to W. Orme-Johnson and the Dept. of Chemistry,
Massachusetts Institute of Technology, for their generosity in allowing
us use their EPR spectrometer.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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