From the Department of Microbiology and Molecular
Genetics, College of Medicine, University of California, Irvine,
California 92697 and the ¶ Department of Biomathematical Sciences,
Mount Sinai School of Medicine, 1 Gustave Levy Place,
New York, New York 10029
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ABSTRACT |
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We have previously demonstrated that integration
host factor (IHF)-mediated activation of transcription from the
ilvPG promoter of Escherichia coli
requires a supercoiled DNA template and occurs in the absence of
specific interactions between IHF and RNA polymerase. In this report,
we describe a novel, supercoiling-dependent, DNA structural
transmission mechanism for this activation. We provide theoretical
evidence for a supercoiling-induced DNA duplex destabilized (SIDD)
structure in the A + T-rich, ilvPG regulatory
region between base pair positions +1 and 160. We show that the
region of this SIDD sequence immediately upstream of an IHF binding
site centered at base pair position
92 is, in fact, destabilized by
superhelical stress and that this duplex destabilization is inhibited
by IHF binding. Thus, in the presence of IHF, the negative superhelical twist normally absorbed by this DNA structure in the promoter distal
half of the SIDD sequence is transferred to the downstream portion of
the SIDD sequence containing the ilvPG promoter
site. This IHF-mediated translocation of superhelical energy
facilitates duplex destabilization in the
10 region of the
downstream ilvPG promoter and activates
transcription by increasing the rate of open complex formation.
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INTRODUCTION |
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Negative superhelicity imposed on a DNA domain can drive local transitions to several types of alternate DNA conformations. These include cruciform extrusion at inverted repeat sequences, Z-DNA at alternating purine-pyrimidine sequences, and denaturation (also called local melting) that concentrates at A + T-rich sites (1-3). Each of these transitions requires free energy, and each releases free energy because the change of local helicity that occurs upon formation partially relaxes the imposed superhelicity. In a domain containing a single susceptible site, the transition will occur when the energy released exceeds the energy required to perform the transition (4).
In domains that contain multiple sites that are susceptible to transition, a complex competition among them will occur (1, 5). Whether a specific transition occurs depends not just on its local sequence but also on how well that transition competes with all others elsewhere in the domain. For example, David Lilley and colleagues (2) showed that the melting transition of an A + T-rich region contained within a supercoiled DNA plasmid required a significantly higher level of negative supercoiling when a (TG)12 region able to form left-handed Z-DNA under superhelical tension was inserted into the plasmid. This shows that the presence of a more susceptible site can inhibit less favorable transitions at other positions. In other words, superhelix-induced structures compete with one another for the negative superhelical energy required for their formation.
The transition behavior of superhelical domains also may be influenced
by proteins that bind at or near these transition sites. For example,
if a DNA region that is favored to form a superhelix-induced alternate
structure is trapped in the B-form by the binding of a protein, the
transition may be transferred to another region in the domain. If this
second site is the 10 region of a promoter, this translocation of
destabilization of the B-form can be used to activate transcription
initiation by facilitating open promoter complex formation. In this
report, we present evidence for this type of a DNA structural
transmission mechanism for the regulation of gene expression in
Escherichia coli.
Integration host factor
(IHF)1 binds to an upstream
activating sequence (UAS1) and activates transcription from the
downstream ilvPG promoter of the
ilvGMEDA operon of E. coli. This activation occurs in the absence of specific protein interactions between IHF and
RNA polymerase, is not the consequence of a DNA looping mechanism, and
requires a superhelical DNA template (6-8). IHF binding in the UAS1
region of a superhelical DNA template results in duplex destabilization
in the 10 region of the downstream ilvPG
promoter site. This DNA structural change at the downstream promoter
site is correlated with an increase in the rate of open complex
formation and a concomitant increase in the rate of transcription initiation (7). In this report, (i) we provide theoretical evidence for
a supercoiling-induced DNA duplex destabilized (SIDD) structure in the
A + T-rich, ilvPG regulatory promoter region between bp positions +1 and
162; (ii) we present experimental evidence to show that the local B-form DNA structure in the UAS1 region
immediately upstream of the IHF binding site is destabilized by
superhelical stress; (iii) we show that IHF binding prevents this
duplex destabilization; (iv) we show that the threshold superhelical densities required for IHF activation and for the destabilization of
this upstream portion of the SIDD region in UAS1 are similar; and (v)
we demonstrate that the presence of this upstream destabilized structure is required for IHF activation and IHF-mediated duplex destabilization in the
10 region of the downstream promoter. These
observations suggest a novel, superhelically dependent DNA structural
transmission mechanism of the type described above; i.e.
binding of IHF prevents the superhelical destabilization of the
upstream region but promotes duplex destabilization in the
10 region
of the ilvPG promoter at the other end of the
SIDD structure. This IHF-mediated duplex destabilization in the
10 region of the ilvPG promoter lowers the energy
of activation for open complex formation and increases the rate of
transcription initiation (7). This mechanism accounts for the facts
that IHF activation of transcription from the
ilvPG promoter is DNA supercoiling-dependent and occurs in the absence of
specific interactions with RNA polymerase.
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MATERIALS AND METHODS |
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Chemicals and Reagents-- Restriction endonucleases, T4 DNA ligase and T4 polynucleotide kinase were purchased from New England Biolabs. E. coli RNA polymerase, pancreatic RNasin, and DNase I were purchased from Boehringer Mannheim. Radiolabeled nucleotides were obtained from NEN Life Science Products. Osmium tetroxide (OsO4), potassium permanganate (KMnO4), and 2,2'-bipyridine were purchased from Sigma. DNA probes were radiolabeled using a nick translation kit purchased from Amersham Pharmacia Biotech. DNA sequencing was performed using the SequenaseTM kit of U.S. Biochemical Corp. DNA oligonucleotides were purchased from Operon Inc. Integration host factor was purified in this laboratory by the method of Nash et al. (28).
Plasmids and Bacterial Strains-- Plasmid DNA isolation and all recombinant DNA manipulations were carried out using standard methods (29, 30). Plasmids used in this study are described in Table I.
Determination of IHF Binding Affinity to the Wild Type and Mutant
ilvPG UAS1 Regions--
Gel mobility shift assays with
linear DNA fragments containing the IHF target site in the UAS1 region
upstream of the ilvPG promoter were employed to
determine the binding affinity of IHF. For the wild type UAS1 region, a
471-bp EcoRI-BamHI DNA fragment containing the
ilvPG promoter region from ilv bp
position 248 to +6 (9) was isolated from plasmid pDH
wt (Table I)
and radiolabeled at each 5'-end with T4 polynucleotide kinase and 10 µCi of [
-32P]ATP (6,000 Ci/mmol). The binding
affinity of IHF to the UAS1 region with the upstream half of the A + T-rich sequence in the UAS1 region deleted was determined on a 269-bp
BstYI-BamHI DNA fragment containing the
ilvPG promoter region from
98 to +6 (9) isolated from plasmid pSS
98 (Table I). The radiolabeled DNA (1 × 10
11 M final concentration) was
preincubated with purified IHF in a 20-µl assay mixture (40 mM Tris-HCl (pH 8.0), 4 mM MgCl2,
70 mM KCl, 0.1 mM EDTA, and 0.1 mM
dithiothreitol). The free IHF concentration in each sample was assumed
to be the same as the total IHF concentration, since the DNA template
concentration was significantly lower than the Kd
for IHF binding. DNA fragments and IHF were incubated at 25 °C for
20 min, and the free and IHF-bound DNA fragments were separated by
electrophoresis on a 5% polyacrylamide gel (4.83% acrylamide, 0.17%
N,N'-methylenebisacrylamide) in TAE buffer (40 mM Tris acetate (pH 8.0), 1 mM EDTA) (29). Electrophoresis was performed at 25 °C. Free and IHF-bound DNA fragments were visualized by autoradiography following multiple time
exposures of the dried gels to Kodak XAR-5 film at
70 °C in the
presence of a Cronex Quanta III intensifying screen (DuPont). Quantitation of band intensity on autoradiographic film was performed utilizing the public domain NIH Image gel quantitation
software.2 Determination of
equilibrium binding constants (KB) was performed by
fitting the binding curve to the Langmuir isotherm, Y = k[P]/1 + k[P], where k is the
equilibrium binding constant and [P] is the free protein
concentration with Sigmaplot version 4.17 software by Jandel Scientific
Corp.
DNase I Footprinting of IHF Binding to Wild Type and Mutant UAS1
Regions of ilvPG--
DNase I footprinting assays were
performed to determine the location of IHF binding in the
ilvPG promoter-regulatory region of various
constructs contained on a negatively supercoiled DNA template.
Supercoiled plasmid DNA (1 × 109 M
final concentration;
=
0.06) was incubated for 20 min at 25 °C
in the absence or presence of purified IHF (30 nM final concentration) in the same assay mixture used for the gel mobility shift assays. The IHF/DNA mixture was treated with 5 ng of DNase I for
exactly 2 min to ensure single-hit kinetic conditions (8). DNase I
reactions were stopped by placing the samples in boiling water for 5 min. The DNA was collected by precipitation with 2-propanol and
resuspended in distilled water. Sites of DNase I-specific cleavage were
visualized by the primer extension analysis described below with
single-stranded DNA oligonucleotide primers that anneal to
ilv DNA sequences 5' (ilv bp positions
207 to
187 or
160 to
132) or 3' (vector-specific bp positions +62 to +32
relative to the ilvPG transcriptional start
site) (9) of the UAS1 and ilvPG promoter regions
on the nonsense and sense strands, respectively.
Mung Bean Nuclease Assays--
1.0 µg of supercoiled plasmid
DNA template was digested with mung bean nuclease (0.1 units) in a
50-µl reaction (45 mM Tris borate (pH 7.6), 1 mM ZnCl2, and 1 mM EDTA) for 15 min
at 37 °C as described (3). The reaction was quenched by two
extractions with phenol/chloroform/isoamyl alcohol followed by a single
extraction with chloroform. The DNA was collected by precipitation with
2-propanol and resuspended in distilled water. The sites of mung bean
nuclease sensitivity were resolved using primer extension mapping as
described below with single-stranded DNA oligonucleotide primers that
anneal to ilv DNA sequences 5' (ilv bp positions
207 to
187) (9) of the UAS1 and ilvPG
promoter regions on the nonsense strand.
Osmium Tetroxide Probing Assays-- 1.0 µg of supercoiled plasmid DNA template was treated with 2 mM osmium tetroxide (OsO4) in the presence of 2 mM 2,2'-bipyridine in a 50-µl reaction (45 mM Tris borate (pH 7.6) and 1 mM EDTA) at 37 °C. After 5 min, the reaction was stopped by precipitation with isopropyl alcohol, and the DNA pellet was collected by centrifugation. The pellet was dried, resuspended in distilled water, and reprecipitated with isopropyl alcohol. The collected pellet was washed with 70% ethanol, collected, and dried. The final pellet was resuspended in distilled water. The sites of OsO4 modification were resolved using primer extension mapping as described below with the single-stranded DNA oligonucleotide primers described above.
Potassium Permanganate Probing Assays--
1.0 µg of
supercoiled plasmid DNA was treated with 3 mM potassium
permanganate in a 50-µl reaction (45 mM Tris borate (pH 7.6) and 1 mM EDTA) at 37 °C. After exactly 4 min, the
reaction was stopped by the addition of 3 µl of -mercaptoethanol.
The modified DNA was precipitated with isopropyl alcohol, collected by
centrifugation, washed twice with 70% ethanol, and dried. The final
pellet was resuspended in distilled water. The sites of KMnO4 modification were resolved using primer extension
mapping as described below with the single-stranded DNA oligonucleotide primers described above.
Base Pair Resolution Mapping of Chemical Modification and
Nuclease-specific Cleavages--
The sites of chemical modification
and nuclease cleavage described above were mapped by primer extension
using T7 DNA polymerase and ilv-specific oligonucleotides.
After nuclease or chemical treatment, the DNA plasmid template was
denatured in the presence of 200 mM NaOH for 10 min at
37 °C. 100 ng of an oligonucleotide primer was added, and the DNA
was renatured with 300 mM NaOAc (pH 5.2). The reannealed
DNA was precipitated with isopropyl alcohol, washed once with 70%
EtOH, collected, and dried. The DNA pellet was resuspended in reaction
buffer (40 mM Tris-HCl (pH 7.5); 20 mM
MgCl2; 50 mM NaCl; 300 nM each
dGTP, dCTP, and dTTP; 1 mM dithiothreitol; and 10 µCi of
[-32P]dATP (3,000 Ci/mmol)). 1 unit of T7 DNA
polymerase was preincubated in this reaction mixture for 5 min. The
polymerization reaction was initiated with the addition 2.5 µl of
buffer containing a 100 µM concentration of each dNTP in
distilled water. The reaction was allowed to continue for 5 min at
25 °C until it was stopped by the addition of an equal volume of
stop buffer (95% formamide, 5 mM EDTA, 0.025% each
bromphenol blue and xylene cyanol).
In Vitro Transcriptions--
The closed circular supercoiled
plasmids pDHwt and pSS
98 (Table I) were used as DNA templates for
in vitro transcription assays performed in the absence and
presence of purified IHF protein. RNA polymerase-plasmid DNA complexes
were formed by preincubating 0.5 units (1.2 pmol) of RNA polymerase and
250 ng of plasmid DNA (0.1 pmol) in a 45-µl reaction mixture (0.04 M Tris-HCl (pH 8.0), 0.1 M KCl, 0.01 M MgCl2, 1.0 mM dithiothreitol, 0.1 mM EDTA, 200 µM CTP, 20 µM UTP,
10 µCi of [
-32P]UTP (3,000 Ci/mmol), 100 µg/ml
bovine serum albumin, and 40 units of RNasin) for 10 min at 25 °C.
Transcription reactions were initiated by the addition of 5 µl of a 2 mM ATP, 2 mM GTP solution. Reactions were
terminated after 2, 4, 6, 8, and 10 min by removing a 10-µl sample
into 10 µl of stop solution (95% formamide, 0.025% bromphenol blue,
0.025% xylene cyanol). The reaction products were separated by
electrophoresis on an 8% denaturing polyacrylamide gel (7.6%
acrylamide, 0.4% N,N'-methylenebisacrylamide) containing 8 M urea in TBE buffer and visualized by autoradiography
following exposure of the gels to Kodak XAR-5 film at
70 °C in the
presence of a Cronex Quanta III intensifying screen (DuPont).
Generation of Plasmid DNA Topoisomers--
10 µg of each
plasmid was treated with 20 units of Drosophila melanogaster
topoisomerase II in a 40-µl reaction mixture (10 mM
Tris-HCl (pH 7.9), 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 µg/ml
bovine serum albumin, 1 mM ATP, and ethidium bromide (0-20
µM)) for 5 h. Each plasmid DNA sample was extracted
three times with phenol to remove the ethidium bromide, precipitated with 2 volumes of isopropyl alcohol, and resuspended in 100 µl of TE
(10 mM Tris (pH 8.0) and 1 mM EDTA). The
plasmid DNA topoisomers were resolved by electrophoresis on four 1.4%
agarose gels in TAE buffer containing 0.006, 0.02, 0.04, or 0.08 µg/ml ethidium bromide. The average linking number difference of the
DNA plasmid in each sample (Lk) was determined by the
band counting methods of Keller (31) and Singleton and Wells (32). The
average superhelical density (
) was calculated using the equation
=
10.5(
Lk/N), where N is the
number of base pairs in the plasmid (pDH
wt and pSS
98 contain 4203 and 3860 bp, respectively).
Two-dimensional Gel Electrophoresis of Plasmid DNA
Topoisomers--
A 20-µl sample containing a mixture of 0.2 µg of
each topoisomer set created above was mixed with 2 µl of a stock
loading buffer containing 50% glycerol and 10 mg/ml xylene cyanol.
This topoisomer mixture typically contained plasmid DNAs with
superhelical densities ranging from = 0.00 to approximately
=
0.10. The sample was loaded into a single circular well (radius = 2 mm) of 24 × 24-cm 1.4% agarose gels prepared with 0.5× TBE
(45 mM Tris borate, pH 8.0, 0.5 mM EDTA).
Electrophoresis of each gel in the first dimension was performed in
0.5× TBE buffer at 2.5 V/cm at 37 °C for 28 h with constant
buffer recirculation at a rate of at least 1 liter/h. Each gel was
removed and soaked in 1× TAE buffer (40 mM Tris acetate,
pH 8.0, 1.0 mM EDTA) containing different amounts of
interchelating dye (0.02-0.08 µg/ml ethidium bromide) for 6-8 h.
Each gel was electrophoresed in a direction 90° to the first
dimension in the running buffer used to soak the gel. DNA
supercoil-dependent structures were evaluated as described
by Bowater et al. (12).
Generation of Predicted Stress-induced Duplex Destabilization Profiles-- The transition properties of superhelical DNA molecules were calculated using a statistical mechanical analysis that evaluates the equilibrium properties of a population of identical, superhelically stressed DNA molecules in which every base pair is regarded as being susceptible to denaturation (1, 5, 10, 33). All states are examined individually, and the cumulative influence of the high energy states is estimated by a density of states calculation. In each state, the superhelical constraint is divided among three deformations, the change of helicity consequent to denaturing the collection of base pairs that are open in that state, helical interwinding of the two strands comprising the resulting denatured region(s), and the residual superhelicity that remains to stress the molecule. The free energies associated with these deformations have been determined by experimental observation. Copolymeric denaturation energetics are assumed, in which the free energy of opening a base pair depends only on whether it is AT or GC. The other energy parameters are given the values that have been found to pertain under experimental conditions in which T = 37 °C, pH = 7.0, and the DNA is placed in a low ionic strength buffer containing 10 mM Tris-HCl and 1 mM Na2EDTA. Under these experimental conditions, comparison with experimental observations has shown the results of these calculations to be quantitatively accurate. We note that the pH and ionic conditions of the OsO4 and nuclease digestion experiments reported here are somewhat different that those assumed in the calculations.
Here the results of calculations are depicted as destabilization profiles. The free energy G(x) that is required to assure opening of the base pair at position x is calculated for each position in the DNA sequence. Low values of G(x) occur at positions where the base pair is substantially destabilized, whereas high values are found when the base pair is not destabilized by the imposed superhelical stresses. Destabilization profiles provide more information than plots of opening probability versus position, because they also show sites where the duplex is substantially destabilized but not sufficiently to cause denaturation with high probability. Such sites can be biologically important if another process or molecule can provide only an incremental amount of free energy to the opening reaction. ![]() |
RESULTS |
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SIDD Is Predicted to Occur in the UAS1 Region at a Physiological
Superhelical Density--
The base pair composition of the
ilvPG promoter-regulatory region is
exceptionally A + T-rich (Fig. 1). The
80-bp segment from bp positions 67 to
153 is approximately 88% A + T (9). In order to predict the stability of this region under
superhelical stress, DNA SIDD profiles (1, 10) were calculated for
plasmid pDH
wt. This plasmid contains the
ilvPG promoter region from bp position
248 to
+6 (9) in a pBR322-based vector containing transcriptional terminators
located downstream of the ilvPG start site
(Table I). Calculations were performed
using the energy parameters appropriate for the nuclease digestion
procedure of Kowalski (1, 3). The results of these calculations are
presented in Fig. 2, A and
B. These are destabilization profiles, plots of the
incremental free energy, G(x), required to
guarantee denaturation of the base pair at position x. The
smaller the value of G(x), the greater the
destabilization experienced by that base pair. Values near 0 occur at
sites where the B-form DNA base pair interactions are almost completely
destabilized. The calculation profiles show that the DNA duplex in the
UAS1 region is predicted to be destabilized in this plasmid at a
physiological superhelical density of
=
0.05. In fact, the
predicted DNA destabilization of the UAS1 region in this plasmid
context is even more pronounced than the well characterized melting
transition of the A + T-rich sequence around bp position 3100 in the
3'-region of the
-lactamase gene (3). A close up view of the
UAS1-ilvPG promoter region of this SIDD profile
is shown in Fig. 2C. These data show that the DNA duplex in
the promoter region is predicted to be more stable than it is in the
upstream UAS1 region.
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Physical Evidence for Superhelically Induced Duplex Destabilization
in the UAS1 Region--
The SIDD profile for pDHwt (Fig. 2),
predicts duplex destabilization in the ilvPG
promoter-regulatory region at a physiological superhelical density (
=
0.05). This prediction was tested by chemically and enzymatically
probing the DNA structure of this region in a set of pDH
wt DNA
topoisomers, each prepared at a defined superhelical density. Each
plasmid topoisomer was treated in the absence or presence of IHF with
osmium tetroxide (OsO4), which modifies thymine residues in
DNA regions where the helix is in a form that exposes C-5-C-6 bonds
(11) or with mung bean nuclease, which cleaves single-stranded DNA (3).
OsO4 reacts with destabilized sites, including premelted or
"breathing" regions of A + T-rich DNA sequences, as well as
with melted DNA. Nuclease digestion, in contrast, appears only to
detect stably denatured regions. The locations of the chemically
modified or enzyme-cleaved sites were determined by primer
extension analysis as described under "Materials and Methods."
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Similar Superhelical Density Thresholds Are Required for
IHF-mediated Activation and Superhelically Induced DNA
Duplex Destabilization--
The superhelical density threshold
for IHF-mediated activation of transcription from the
ilvPG promoter in pDHwt occurs between superhelical densities of
=
0.03 and
0.04 (8). If
superhelically induced duplex destabilization in the UAS1 region and
IHF-mediated activation are functionally correlated, then the threshold
superhelical densities required for these two events should be similar.
To test this prediction, we employed two-dimensional agarose gel electrophoresis. This method has the ability to accurately determine the threshold superhelical density required to initiate the formation of a superhelically dependent DNA secondary structure to a precision of
±
Lk of 1 or, in the case of plasmid pDH
wt, ±
= 0.003 (12). For this measurement, a set of plasmid pDH
wt topoisomers
was created, pooled, and resolved by two-dimensional electrophoresis as
described under "Materals and Methods." Fig.
4, A and B, shows that pDH
wt exhibits a gradual, DNA superhelically dependent
structural transition, beginning at a linking number of
Lk =
15. No increase in writhe is observed from a
linking number of
16 to the limit of resolution of the gel. This lack
of change in writhe over a range of linking numbers is consistent with
a gradual untwisting of the DNA helix. However, since the pBR322-based
construct, pDH
wt, contains other superhelically dependent structures
(3), we could not be certain that the extension of this transition was due solely to the unwinding of the DNA helix in the UAS1 region. We
therefore placed the ilvPG promoter DNA region
contained in pDH
wt (ilv bp positions
248 to +6) into
the unique BamHI site of pUC19 to create pSS19
wt (Table
I). Since pUC19 does not contain any supercoiling-dependent
DNA structures (Fig. 4C), only the DNA superhelically
destabilized structure in UAS1 is detected in pSS19
wt (Fig.
4D). This UAS1-specific structural transition initiates at a
threshold superhelical density of
=
0.032. Thus, these
experiments demonstrate the initiation of a DNA superhelically induced
structural transition in the UAS1 sequence at a threshold superhelical
density that correlates well with the superhelical density required for
IHF-mediated activation (8).
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Deletion of the A + T-rich UAS1 Region Upstream of the IHF Binding
Site Eliminates Superhelically Induced Duplex Destabilization in the
UAS1 Region--
The data presented above demonstrate that the
threshold superhelical densities required for IHF-mediated activation
of transcription from the ilvPG promoter and the
DNA supercoiling-induced duplex destabilization in the UAS1 region are
similar. This correlation suggests that destabilization of the DNA
duplex in the UAS1 region might be required for IHF-mediated
activation. In order to investigate the possibility that these events
are functionally related, we created a deletion in which the
superhelically destabilized DNA sequence in the UAS1 region was removed
without altering the downstream IHF binding site. The UAS1 region
upstream of the IHF site in this plasmid (pSS-98; Table I) was
replaced with vector DNA of an essentially random base composition
(Fig. 5).
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IHF Binds to Its Target Site in the UAS1 Region of
pSS-98--
Binding sites for IHF consist of a core consensus
sequence, WATCAANNNNTTR (where W represents A or T, N represents any
base, and R represents pyrimidine), and frequently contain a flanking dA-dT element located immediately upstream of this core sequence (13,
14). Although the functional role of this dA-dT element is unclear, it
has been suggested to influence the binding of IHF through indirect
contacts with the minor groove of the DNA helix (13, 15). Rice et
al. (15) have recently analyzed the structure of a co-crystal with
the H' site of phage
DNA and IHF. They showed that the interaction
of IHF with an A tract on the 5'-side of the core sequence is
stabilized by the presence of a narrow minor groove created by the
poly(dA-dT) sequence and suggested that this interaction is facilitated
by structural rather than by sequence specificity. The IHF binding site
in the UAS1 region also contains a dA-dT sequence
(5'-TATTTATTTTTAAA-3') on the 5'-side of its core binding sequence.
While this sequence is disrupted in pSS
-98, an upstream 5'-AATAAA-3'
sequence is retained (Fig. 5B). In order to ensure that we
had not disrupted the ability for IHF to bind to its core binding site
in the UAS1 region, gel mobility shift assays were performed, and the
affinities of IHF binding to its target site in the UAS1 region of
plasmids pDH
wt and pSS
-98 were compared (Fig.
7). The Kd value for
IHF binding was found to be 2.2 × 10
9 M
for pDH
wt and 4.0 × 10
9 M for
pSS
-98, respectively. In addition, the results of DNase I
footprinting experiments on supercoiled plasmids confirmed that IHF
binds to the same site on both plasmids.3 Thus, the
deletion of the sequences upstream of the IHF binding site does not
eliminate the binding of IHF to its target binding site. We have
previously shown that IHF also binds with nearly identical affinities
to relaxed and supercoiled DNA templates (8).
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Deletion of the A + T-rich UAS1 Region Upstream of the IHF Binding
Site Eliminates IHF-mediated Superhelically Induced Duplex
Destabilization in the 10 Region of the ilvPG
Promoter--
The results of the experiments described above
demonstrate that the superhelically destabilized structure in UAS1 is
required for IHF-mediated activation. Our proposed mechanism suggests
that the IHF-mediated inhibition of the formation of this structure activates transcription by shifting some of the superhelical stress normally absorbed by this structure in the absence of IHF to the downstream ilvPG promoter portion of the SIDD
region described above (Fig. 1). If this is the case, then the binding
of IHF to plasmid pSS
-98, which does not contain the upstream
superhelically destabilized structure, should not destabilize the DNA
duplex in the
10 region of the downstream
ilvPG promoter. This prediction was tested by
chemically probing the DNA structure in the
10 region of the
ilvPG promoter with KMnO4 and
OsO4 in the presence and absence of IHF on supercoiled
pDH
wt and pSS
-98 DNA templates. The results shown in Fig.
8A were obtained by primer
extension using an oligonucleotide that anneals to ilv bp
positions
160 to
132 on the transcribed DNA strand. In the presence
of IHF, KMnO4 sensitivity is observed at thymines
located on the transcribed strand at bp positions
11 and
12 in the
promoter region (Fig. 8A, lane 1). In
the absence of IHF, no KMnO4 sensitivity is observed in
this region (Fig. 8A, lane 2).
IHF-induced duplex destabilization at these same nucleotide positions
is detected with the more sensitive structural probing reagent
OsO4 (Fig. 8A, lane 3).
Again, no destabilization is observed in the absence of IHF (Fig.
8A, lane 4). Since the upstream primer
binding site is not present in pSS
-98, a DNA oligonucleotide that
binds to a downstream sequence at bp positions 32-62 relative to the
ilvPG transcriptional start site on the nontranscribed strand was used for primer extension through the promoter region from the other direction. On this strand, IHF-induced sensitivity on pDH
wt (
=
0.06) is observed at all thymines located between bp positions +7 and
18 (Fig. 8B,
lane 1). However, in the absence of IHF (Fig.
8B, lane 2) or with pSS
-98 in the presence (Fig. 8B, lane 3) or absence
of IHF (Fig. 8B, lane 4), OsO4 reactivity at these thymine sites is greatly
diminished. These results provide strong evidence that IHF inhibition
of duplex destabilization in UAS1 is responsible for IHF activation of
duplex destabilization in the ilvPG promoter
region.
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Superhelically Induced DNA Duplex Destabilization in the UAS1
Region Is Required for IHF-mediated Activation--
The results
presented above demonstrate that the B-form structure in the upstream
half of the UAS1 region is strongly destabilized by negative
superhelicity and that this destabilization is inhibited by IHF binding
to its target site in the downstream part of this region. A functional
correlation between the superhelicity-dependent formation
of this structure and the superhelicity dependence of IHF-mediated
activation is suggested by the observation that both events occur at
similar superhelical densities. This correlation suggests that the
formation of this destabilized region might be required for
IHF-mediated activation of transcription. The results presented above
also demonstrate that a plasmid DNA (pSS-98) with the upstream
portion of the UAS1 region deleted in a way that does not eliminate IHF
binding cannot form this structure and that IHF binding to this DNA
template does not facilitate duplex destabilization in the
10 region
of the ilvPG promoter. Therefore, to
finally determine whether or not this DNA
supercoiling-induced structure is required for IHF activation, we
performed quantitative in vitro transcription experiments in
the absence and presence of a saturating concentration of IHF (30 nM) with either pDH
wt or pSS
-98 DNA templates at a
superhelical density of
=
0.07. The data presented in Fig.
9 show that IHF activates transcription from the downstream ilvPG promoter on pDH
wt
but is unable to activate transcription on plasmid pSS
-98, which
does not contain a superhelically destabilized site in the upstream
UAS1 region and does not exhibit IHF-mediated duplex destabilization in
the
10 region of ilvPG, the promoter.
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DISCUSSION |
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In previous reports, we have shown that IHF binding in UAS1 causes
the formation of a nucleoprotein structure on a superhelical DNA
template that facilitates the destabilization of the DNA duplex in the
10 region of the downstream ilvPG promoter and
increases the rate of the isomerization step of the transcription
initiation reaction (7). We have also shown that (i) in the absence of IHF, promoter activity increases with increasing DNA supercoiling; (ii)
in the presence of IHF, the effect of DNA supercoiling on basal level
promoter activity is amplified 5-fold; (iii) IHF binding does not alter
the superhelical density of the DNA template; (iv) in the presence or
absence of IHF, the relative transcriptional activities of the promoter
remain the same at any given superhelical density; (v) IHF activation
cannot be replaced by simply increasing the negative superhelical
density of the DNA template; and (vi) IHF binds to relaxed and
negatively supercoiled DNA templates with nearly identical affinities
(8). These results demonstrated that although IHF activation requires a
supercoiled DNA template, activation by DNA supercoiling and IHF are
effected by separate mechanisms. Therefore, to accommodate these
observations and because IHF-mediated activation was shown to occur in
the absence of protein interactions between IHF and RNA polymerase, we
considered the possibility of activation by a DNA structural
transmission mechanism (7, 8).
The experimental results described in this report demonstrate a
superhelically induced destabilization of the DNA helix in UAS1. IHF
binding is shown to prevent these DNA structural changes. Furthermore,
destabilization of this region in the absence of IHF binding is shown
to occur at a similar superhelical density threshold required for
IHF-mediated activation of transcription from the downstream
ilvPG promoter (8). These correlations suggest a
DNA structural transmission mechanism for this activation that involves
DNA secondary structural transitions (Fig.
10). According to this mechanism, IHF
binding in the UAS1 region of a superhelical DNA template inhibits the
formation of an untwisted, superhelically induced, alternate DNA
structure in which the B-form of the duplex is locally destabilized. In
the absence of IHF, this structure is formed under negative
superhelicity. In the presence of IHF, the formation of this structure
is inhibited. Thus, the superhelicity normally absorbed by this
structure in the absence of IHF must be accommodated by transitions at
other DNA sites in the presence of IHF. Our experimental results
demonstrate that one of these sites is located in the 10 region of
the downstream ilvPG promoter where duplex
destabilization facilitates open complex formation to activate
transcription initiation. This mechanism is consistent with our
previous demonstration that IHF binding alters the distribution but not
the absolute level of the linking deficiency of a superhelical domain
containing the UAS1 region. The experimental evidence reported here in
support of this DNA structural transmission mechanism is discussed
below.
|
Theoretical analysis of superhelically induced DNA duplex
destabilization (Fig. 2) predicts strong destabilization in the A + T-rich promoter-regulatory region at physiologically relevant superhelical densities. Chemical and enzymatic probing of the structure
of the DNA helix demonstrated that duplex destabilization initiates at
a low, physiological, superhelical density around bp positions 98 and
116 in the UAS1 portion of this region and spreads about 50 bp to bp
position
153 as the negative superhelical density of the DNA template
becomes more extreme (Fig. 3A). These experiments also
showed that although the DNA helix in this region is destabilized, it
is not entirely stably unwound (Fig. 3B). It is interesting
that this superhelically destabilized region is confined to the
promoter-distal half of the UAS1 region located immediately upstream of
the IHF binding site. It does not extend into the IHF binding site or
the farther downstream ilvPG promoter region
(Fig. 1). In the presence of IHF, however, no duplex destabilization in
the UAS1 region was observed (Fig. 3C). In this case, the
superhelical deformation absorbed in the absence of IHF by
destabilizing this region must be redistributed to other sites.
Therefore, since IHF binding has been shown previously to cause DNA
superhelicity-dependent duplex destabilization in the
10
region of the downstream promoter (7), it was plausible that this
downstream destabilization was caused by the IHF-induced inhibition of
transitions in the upstream UAS1 region. If this hypothesis is correct,
then no activation would be expected in the absence of this structure.
Since the region that experiences this destabilization is upstream from the IHF binding site in UAS1 (Figs. 1 and 3), it was possible to
replace it with a superhelically stable DNA sequence without affecting
the DNA sequence of the IHF binding and promoter sites (Fig. 5) or the
ability of IHF to bind to its target site in the UAS1 region (Fig. 8).
When this superhelically destabilized DNA sequence in UAS1 was replaced
with a superhelically stable one, it was observed that, as expected,
IHF could not activate transcription initiation from the
ilvPG promoter, even on a highly supercoiled DNA
template (Fig. 9). Furthermore, in the absence of the superhelically destabilized structure in UAS1, IHF binding to a supercoiled DNA template does not destabilize the DNA duplex in the
10 region of the
downstream ilvPG promoter (Fig. 7). These
results demonstrate that IHF-mediated inhibition of the formation of
this structure is responsible for the downstream duplex destabilization
that leads to activation of transcription initiation from the
ilvPG promoter.
This DNA structural transmission mechanism further predicts that the
threshold superhelical densities required for duplex destabilization in
the UAS1 region and for IHF activation of transcription initiation from
the ilvPG promoter should be similar. No
transmission would be expected at less extreme superhelical stress
levels than those required for duplex destabilization in UAS1.
Two-dimensional gel electrophoresis of topoisomer sets of the pDHwt
plasmid containing the ilvPG promoter-regulatory
region suggest that this is the case. Negative superhelical densities
of
>
0.032 ± 0.003 are required for duplex destabilization
in the UAS1 region of this plasmid (Figs. 4 and 6). This value agrees
well with our previously estimated threshold negative superhelical
density of
>
0.035 ± 0.005 for IHF activation (8). It
should be emphasized, however, that these values are not expected to be
identical. This is because the conditions required for each of these
measurements are different. The two-dimensional gel experiments were
performed at a lower ionic strength (45 mM Tris borate)
than the transcription reactions (100 mM KCl). Stable
unwinding of A + T-rich regions is known to be suppressed at ionic
strengths greater than 50 mM NaCl (16). Thus, a negative
superhelical density more extreme than the threshold value measured by
the two-dimensional electrophoresis experiment is expected in a
transcription reaction that occurs at a higher salt concentration. On
the other hand, destabilization of the UAS1 region at a less extreme
threshold is predicted by the fact that additional negative
supercoiling is generated in this region during basal level
transcription from the downstream ilvPG promoter (17-19). For example, Rahmouni and Wells (20) have shown the formation
of a supercoiling-dependent B-DNA to Z-DNA transition upstream of an active promoter, although the average superhelical density present in vivo should not have been high enough to
induce this structural transition (19).
It has been documented that small sequence changes can radically alter the transition behavior of a supercoiled molecule if they change the relative competitiveness of different regions (5). Two examples of this behavior are relevant to this report. First, deleting a portion (even a small portion) of a strongly destabilized site can radically alter its competitiveness, causing the transition to shift to another location although most of the original site remains intact. In the case described here, removal of part of the UAS1 region destroys the ability of the remaining downstream promoter portion to become destabilized by superhelicity. Second, it is possible to alter transition behavior by constraining a portion of an easily destabilized DNA region to remain in the B-form. This also can have the effect of altering the competitiveness of the region involved sufficiently to shift the transition to another part of the same destabilized region or to another easily destabilized site. This is the essence of the mechanism proposed in this report; i.e. IHF binding forces the superhelically destabilized site in the UAS1 region to remain in the B-form, which shifts part of the transition energy to the promoter site of this SIDD region and the rest to other easily destabilized sites.
Another IHF-mediated, supercoiling-dependent model for the
regulation of transcription has been reported. Higgins et
al. (34) have suggested that IHF activates transcription from the
phage µ PE promoter by forcing the promoter site to the
outside of a superhelical node where it is in an ideal location for
interaction with RNA polymerase. In support of this model, Van Rijn
et al. (35) demonstrated that this activation is dependent
on the face of the helix and correlated with increased RNA polymerase
binding affinity. In contrast, the IHF-mediated activation of
transcription from the ilvPG promoter is
independent of the face of the helix and affects the kinetic step of
open complex formation (7). Thus, a super-loop type model can be
excluded for IHF-mediated regulation of transcription from the
ilvPG promoter. It was also noted that both IHF
binding in the UAS1 region and the pSS-98 construct eliminate
in vitro transcription from the
ilvPG1 promoter (Fig. 9). This suggested the
possibility that IHF might act to facilitate downstream promoter
selection by repressing transcription from the upstream promoter.
However, previously published data demonstrate that this is also not
the case (7). Point mutations in the UAS1 region that abolish
transcription from ilvPG1 but do not affect
IHF-binding do not activate transcription from the downstream
ilvPG promoter. Furthermore, these point
mutations do not affect IHF-mediated activation.
It is of interest to note that DNA sequences having higher than average
A + T content are observed upstream of several strong 70
promoters and that alterations in these upstream A + T-rich regions affect the rates of transcription initiation from downstream promoters (21, 22). Since a considerable proportion of the negative supercoiling
in the bacterial nucleoid is unconstrained, these A + T-rich regions
can serve as local sites that are susceptible to destabilization. The
results reported here demonstrate that regulatory proteins that bind to
these upstream regions can affect the susceptibility of sequences to
become destabilized by imposed superhelicity and transfer this
susceptibility to nearby promoter sites. Therefore, the mechanism of
protein-mediated, superhelicity-dependent, regulation of
transcription initiation in prokaryotes that is reported here may be of
general significance. For example, Fyfe and Davies (36) have recently
reported that the deletion of an A + T-rich DNA sequence upstream of an
IHF binding in the regulatory region of the Neisseria
gonorrhoreae pilEp1 promoter, which has a
striking resemblance to the ilvPG
promoter-regulatory region described here, eliminates IHF-mediated
activation in this system. However, the DNA supercoiling dependence of
this activation has not yet been examined.
Recent reports indicate that DNA superhelicity and superhelically
destabilized structures also are important for the regulation of
transcription in eukaryotes. For example, Emerson and colleagues (23)
have shown that supercoiled DNA templates are required for
enhancer-mediated in vitro regulation of the chicken
A-globin and mouse T-cell receptor genes. Also,
Michelotti et al. (24) have shown that the basal level of
transcription from the c-myc gene generates negative DNA
superhelicity that destabilizes upstream A + T sequences. These
destabilized regions are targets for single-stranded DNA-binding
proteins that are required for enhanced in vivo
transcription from this promoter. Thus, it appears that
protein-stabilized DNA duplex destabilization might serve a range of
roles in in vivo regulatory mechanisms in both prokaryotes and eukaryotes.
In conclusion, it should be stressed that it is essential to separate the strands of the DNA duplex for many biological processes such as DNA replication, recombination, and transcription initiation in both prokaryotes and eukaryotes (25-27). Negative superhelicity lowers the energy of activation for strand separation. The data presented in this report demonstrate a level of regulation for these processes that involves a competition between potential destabilized sites that is mediated by protein binding. Therefore, since it is now apparent that DNA supercoiling is an integral component of many mechanisms involved in the regulation of cell growth and metabolism, a further elucidation of these types of protein-mediated, DNA supercoiling-dependent effects should enhance our understanding of a large number of regulatory systems.
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ACKNOWLEDGEMENTS |
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We are grateful to Elaine Ito for technical assistance and to Stuart Arfin, She-Pin Hung, and our colleagues at the University of California, Irvine, for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Science Foundation Grants MCB-9723452 (to G. W. H.) and BIR-93-10252 (to C. J. B.) and National Institutes of Health Grants GM55073 (to G. W. H.) and GM47012 (to C. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by U.S. Public Health Service Training Grant GM07311.
To whom correspondence should be addressed. Tel.:
949-824-5858; Fax: 949-824-8598; E-mail: gwhatfie{at}uci.edu.
The abbreviations used are:
IHF, integration
host factor; UAS, upstream activating sequence; SIDD, supercoiling-induced duplex destabilization; bp, base pair(s); Lk, linking number difference
, superhelical
density.
2 Available on the Internet at ftp://zippy.nimh.nih.gov.
3 S. D. Sheridan and G. W. Hatfield, unpublished data.
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REFERENCES |
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