(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 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.
Excessive production or inadequate disposal of reactive
derivatives of oxygen, such as superoxide (O) and hydrogen peroxide
(HO
), 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).
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 HO
-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).
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 ()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.
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, -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.
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
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).
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: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.
Figure 4:
Spectroscopic analysis of Fe-SoxR. A, UV/visible absorption. SoxR protein, 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.
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). ()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).
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 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 O
in 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.
So
far, apo-SoxR has been isolated only following purification of the
protein in buffers containing -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).