Inhibition of DNA Supercoiling-dependent
Transcriptional Activation by a Distant B-DNA to Z-DNA Transition*
Steven D.
Sheridan
§,
Craig J.
Benham¶, and
G. Wesley
Hatfield
From the
Department of Microbiology and Molecular
Genetics, College of Medicine, University of California, Irvine,
California 92697 and ¶ Department of Biomathematical Sciences,
Mount Sinai School of Medicine, New York, New York 10029
 |
ABSTRACT |
Negative DNA superhelicity can destabilize the
local B-form DNA structure and can drive transitions to other
conformations at susceptible sites. In a molecule containing multiple
susceptible sites, superhelicity can couple these alternatives
together, causing them to compete. In principle, these superhelically
driven local structural transitions can be either facilitated or
inhibited by proteins that bind at or near potential transition sites.
If a DNA region that is susceptible to forming a superhelically induced alternate structure is stabilized in the B-form by a DNA-binding protein, its propensity for transition will be transferred to other
sites within the same domain. If one of these secondary sites is in a
promoter region, this transfer could facilitate open complex formation
and thereby activate gene expression. We previously proposed that a
supercoiling-dependent, DNA structural transmission
mechanism of this type is responsible for the integration host
factor-mediated activation of transcription from the
ilvPG promoter of Escherichia coli
(Sheridan, S. D., Benham, C. J. & Hatfield, G. W. (1998)
J. Biol. Chem. 273, 21298-21308). In this report we
confirm the validity of this mechanism by demonstrating the ability of
a distant Z-DNA-forming site to compete with the superhelical
destabilization that is required for integration host factor-mediated
transcriptional activation, and thereby delay its occurrence.
 |
INTRODUCTION |
Negative supercoiling within DNA molecules can destabilize the
B-form duplex at locations where its thermodynamic stability is least.
If this superhelically induced DNA duplex destabilization (SIDD)1 is sufficiently
strong, it can drive transitions to locally unpaired structures such as
denaturation or cruciform extrusion (1, 2). Negative superhelicity also
can drive transitions to other helical conformations, such as to the
left-handed Z-form that requires an alternating purine-pyrimidine DNA
sequence (3).
In a negatively superhelical molecule containing two or more sites that
are susceptible to destabilizations or other transitions, superhelicity
induces a global competition among all the possible structural
alterations, with the energetically most favorable transition being the
first to occur (4-7). By absorbing negative superhelical turns, the
first transition induces a partial relaxation of the domain that delays
other transitions to more extreme superhelicities than would be needed
to drive them were the first transforming site not present. In
molecules containing a site that can occur in the Z-form, the B-Z
transition generally will be energetically favored over other
alternatives, primarily because the change from right-handed helix to
left-handed helix accommodates more negative superhelicity, and thereby
allows the balance of the domain to relax by a correspondingly larger
amount. So the insertion of a first Z-susceptible site into a molecule
will offset the threshold superhelix density required to drive other
destabilizations or structural transitions by an amount corresponding
to the number of negative superhelical turns absorbed by the B-Z
transition (7). In this way one can alter the destabilization
characteristics of other local regions without changing their base sequences.
Theoretical analyses predict that SIDD occurs within the A + T rich DNA
sequence extending from base pair +1 to base pair
160 in the
regulatory region of the ilvPG promoter of
Escherichia coli (8-10). Osmium tetroxide binding
experiments show that significant destabilization of the B-form DNA
duplex initiates at superhelical density
=
0.038 ± 0.003 around base pair
98 within UAS1, the upstream activating sequence
present in the 5' portion of this SIDD region (8, 11). As the negative
superhelix density of the DNA template becomes more extreme,
destabilization spreads in the 5' direction to base pair
153. When
this destabilization (or another transition) occurs, the free energy
associated with superhelicity is no longer uniformly distributed along
the sequence. Rather, the destabilized site constitutes a local
concentration of free energy. If this destabilization is sufficient to
drive a local transition, the resulting alternate structure also is a
local concentration of negative superhelical turns. In principle, such
local accumulations of free energy and torsional deformation could
serve functional purposes.
In previous studies (8, 12), we have investigated the role of
destabilization within the UAS1 region in the IHF-mediated transcriptional activation of the ilvPG
promoter. This activation was shown to require a supercoiled DNA
template having superhelical density
0.035 ± 0.005, coincident with the onset of UAS1 destabilization. Although IHF binds
to a site centered at base pair position
92 within UAS1, immediately
downstream of this superhelically destabilized region, its strength of
binding does not depend upon DNA superhelicity (8, 12, 13).
IHF-mediated activation of transcription from this promoter also does
not involve protein-protein interactions between IHF and the RNA
polymerase. However, IHF binding has been shown to stabilize the B-form
DNA helix within the UAS1 region (8). In the presence of IHF no duplex
destabilization is observed to occur in this region, even in highly
negatively supercoiled DNA templates. As a result, the free energy that
was localized by destabilization of this region in the absence of IHF
must, in the presence of IHF, be redistributed to other sites. In
particular, IHF binding to a supercoiled DNA template has been shown to
cause DNA helix destabilization in the
10 region of the downstream
promoter. This suggested a mechanism of activation whereby IHF binding
displaces superhelical destabilization from the UAS1 region to the
downstream 3' portion of the SIDD region that contains the
ilvPG promoter site (8). This
supercoiling-dependent, IHF-mediated destabilization of the
DNA helix in the
10 region lowers the energy of open complex
formation, which increases the rate of transcriptional initiation from
this promoter (8, 12, 14).
The experiments reported here were designed to test this DNA structural
transmission mechanism of IHF-mediated transcriptional activation. We
introduce a sequence with strong Z-DNA-forming potential into a site
distant from the UAS1 region, which alters the destabilization
characteristics of the UAS1 region without changing its base sequence.
If this structural transmission model for IHF-dependent
transcriptional activation is correct, then the presence of the
Z-forming site should delay activation until more extreme
superhelicities corresponding to a change of linking difference equal
to the number of turns absorbed by the B-Z transition.
 |
MATERIALS AND METHODS |
Chemicals and Reagents--
Restriction endonucleases and T4 DNA
ligase were purchased from New England Biolabs. E. coli RNA
polymerase was purchased from Amersham Pharmacia Biotech. Pancreatic
RNasin was purchased from Promega. Drosophila melanogaster
topoisomerase II was purchased from U. S. Biochemical Corp. Osmium
tetroxide (OsO4) and 2,2'-bipyridine were purchased from
Sigma Chemical Co. Radiolabeled nucleotides were obtained from NEN Life
Science Products. DNA sequencing was performed using the Sequenase kit
of U. S. Biochemical Corp. DNA oligonucleotides were purchased from
Operon Technologies. IHF was purified in this laboratory by the method
of Nash et al. (15).
Plasmids--
Plasmid DNA isolation, recombinant DNA
manipulations and construct verifications were carried out using
standard methods (16). Plasmid DNA containing the
(CG)13AATT(CG)22 Z-DNA forming sequence, pRW1554 (17), was isolated from the recA
E. coli strain XL-1 blue. Plasmid pDH
wt contains a 272-bp
EcoRI-BstBI (end-filled) restriction endonuclease
DNA fragment (E. coli ilv bp positions,
248 to +6) ligated
into the unique EcoRI and BamHI (end-filled)
sites of pDD3 (8) containing
rrnBT1T2 transcription terminating
sequences located 157 bp downstream of the ilvPG
transcriptional start site (12). The construction of pSS
Z is
described in Fig. 1. pDH
wt and pSS
Z contain 4,203 and 4,277 bp, respectively.
Generation of Plasmid DNA Topoisomers--
10 µg of each
plasmid was relaxed with D. melanogaster topoisomerase II in
40 µl reaction mixtures containing 0-60 µM ethidium bromide, as described previously (8). Topoisomers were purified and
their linking number deficiencies (
Lk) and superhelical densities (
) were determined by agarose gel electrophoresis (8, 18).
Two-dimensional Gel Electrophoresis of Plasmid DNA
Topoisomers--
Two-dimensional agarose gel electrophoresis was
performed with pooled topoisomers in 1.4% agarose gels, as described
previously (8). The first dimension (top to bottom) was run at 37 °C
in 0.5× TBE. The second dimension (from left to right) was performed in 1× TAE buffer containing 0.10 µg/ml ethidium bromide. DNA
supercoil-dependent structures were evaluated as described
by Bowater et al. (19).
In Vitro Transcriptions--
Closed-circular supercoiled
plasmids 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) 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
rrnBT1T2-terminated 157-base pair
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 (NEN Life Science Products).
Transcriptional rates were determined by quantitation of band intensity
versus reaction time using the public domain NIH IMAGE gel
quantitation software (ftp://zippy.nimh.nih.gov).
 |
RESULTS |
If the mechanism of IHF-mediated activation of the
ilvPG promoter involves modulation of
destabilization within the UAS1 region as proposed (8), then this
activation should be offset to a more extreme superhelix density by the
presence of a competing site that undergoes a B- to Z-DNA transition.
To test this prediction, the Z-DNA forming sequence
(CG)13AATT(CG)22 (10) was inserted into a unique SspI site 449 base pairs upstream of the
transcriptional start site of the ilvPG promoter
in pDH
wt to create pSS
Z (Fig. 1).

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Fig. 1.
The ilvPG
regulatory region and construction of plasmid
pSS Z. Plasmid pSS Z was constructed by
the insertion of a 74-bp BstY1 fragment from plasmid pRW1554
(10) containing the sequence (CG)13AATT(CG)22
into the unique SspI site 449 bp upstream of the
ilvPG transcriptional start site in plasmid
pDH wt (2). Arrows indicate the transcriptional start
sites of promoters in these plasmids. IHF binding site in the UAS1
region is indicated. The superhelically destabilized structure in the
5' portion of the SIDD region is indicated by a slashed box.
Numbers in parentheses indicate base pair positions relative to the
start of ilvPG transcription.
|
|
Characterization of DNA Supercoiling-dependent
Structural Transitions in pSS
Z and pDH
wt as Functions of
Superhelical Stress--
To determine the threshold superhelical
densities required for transitions in the pSS
Z and pDH
wt
plasmids, sets of DNA topoisomers of each plasmid were constructed
having defined linking number deficiencies in the range
62
Lk
0 (
0.16
0.00). These topoisomer sets
were pooled for each plasmid and analyzed by two-dimensional gel
electrophoresis. The gel migration pattern of the pDH
wt topoisomers is shown in Fig. 2A. The
superhelically induced destabilization of the A + T rich sequence in
the UAS1 region commences at linking difference
Lk =
15, (
=
0.038), which is comparable with the previously determined
threshold superhelical density required for this structure (8, 17). In
pSS
Z, however, the B- to Z-DNA transition is observed to initiate at
Lk =
10, (
=
0.025; Fig. 2B), a significantly
less extreme superhelix density than that required for the
destabilization observed in the UAS1 region (
=
0.038). The
migration pattern of the topoisomers in this gel also shows that, as
described previously (17), the B- to Z-DNA transition within the
74-base pair insert occurs in two steps, and absorbs approximately 13 superhelical turns. This observation is consistent with the loss of 1.8 turns of right-handed twist for every 10.5 base pairs of B-DNA that are
converted to Z-DNA (3). This shows that the B-Z transition in pSS
Z
occurs at a significantly less extreme threshold superhelical density
than does duplex destabilization of the SIDD site in UAS1.

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Fig. 2.
Characterization of structural transitions in
pDH wt and pSS Z by
two-dimensional agarose gel electrophoresis. Topoisomers of
plasmids pDH wt (A) and pSS Z (B) were
analyzed by two-dimensional agarose gel electrophoresis on 24-cm
square 1.4% agarose gels. The first dimension (top to bottom) was run
at 37 °C in 0.5× TBE. The second dimension (from left to right) was
performed in 1× TAE buffer containing 0.1 µg/ml ethidium bromide.
Graphical representations are presented on the right. Arrows
indicate threshold linking number deficiency ( Lk) for structural
transitions. The bracket in panel B indicates the
linking number range of the B- to Z-DNA transition.
|
|
The Effects of the B-Z Transition on the Topology of the
ilvPG Promoter Regulatory Region--
Because the B-Z
transition absorbs approximately 13 negative superhelical turns in
pSS
Z, it is predicted to offset the global plasmid linking number
required for duplex destabilization in the UAS1 region of that plasmid
by that amount. This prediction has been tested in experiments in which
OsO4 was used to probe both plasmids for destabilized
sites. The results of these experiments, shown in Fig.
3, confirm the prediction. In pDH
wt,
the plasmid lacking the Z-DNA insert, destabilization of the A + T rich
SIDD-susceptible site in the UAS1 region starts at linking differences
between
Lk =
11 ± 2 (
=
0.028 ± 0.005; Fig.
3A, lane 2) and
Lk =
18 ± 2 (
=
0.044 ± 0.005; Fig. 3A, lane 3). In
pSS
Z, the plasmid containing the Z-DNA insert, destabilization of
this site is observed to commence at linking differences between
Lk =
26 ± 2 (
=
0.064 ± 0.005; Fig.
3B) and
Lk =
31 ± 2 (
=
0.076 ± 0.005; Fig. 3B). Thus, the DNA template containing the Z-DNA
insert, pSS
Z, must be untwisted by 14 ± 2 additional turns to
reach a global superhelical density that initiates destabilization of the A + T rich SIDD-susceptible sequence in UAS1.

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Fig. 3.
Effect of template superhelicity on helix
destabilization in the UAS1 region of
ilvPG. Topoisomers of plasmids
pDH wt (A) or pSS Z (B) were treated with 2 mM osmium tetroxide and 2 mM 2,2-bipyridine,
and sites of modification were mapped by primer extension analysis.
Base pairs positions are shown with respect to the transcriptional
start site of the ilvPG promoter. Lanes
1-9 contain negatively supercoiled DNA topoisomers with average
linking number deficiencies ( Lk ± 2).
|
|
Z-DNA Formation Increases the Global Superhelix Densities Required
for Both Basal Level and IHF-activated Transcription from the
ilvPG Promoter--
We have shown that superhelically
induced duplex destabilization within the UAS1 region is offset to more
extreme superhelical densities in the pSS
Z plasmid that contains the
Z-DNA forming sequence. If, as we have proposed (8), destabilization of
this region of UAS1 is required for IHF-mediated transcriptional
activation, then activation in that plasmid should be offset to the
same extent. To test this prediction, we performed in vitro
transcription assays in the presence and absence of IHF on the same DNA
topoisomer sets of pDH
wt and pSS
Z that were used in the
experiments reported in Figs. 2 and 3. The results of these experiments
are shown in Fig. 4. In the absence of
both the Z-DNA insert and IHF, basal level transcription from the
ilvPG promoter in pDH
wt increases approximately 40-fold as the negative superhelicity of the DNA is
changed from
Lk =
18 ± 2 to the optimum value of
Lk =
49 ± 2 (Fig. 4A). In the presence of
IHF, activation of transcription from the ilvPG
promoter in pDH
wt is observed over this same range of superhelix
densities (12) (Fig. 4B). However, in the plasmid containing
the Z-DNA insert, the half-maximal level of basal transcription is
delayed by 11 ± 4 linking numbers (Fig. 4A), and
IHF-activated transcription is delayed by 12 ± 4 linking numbers
(Fig. 4, compare B and C). Thus, as predicted,
the presence of the Z-susceptible insert offsets the threshold
superhelicity required for IHF-mediated activation by approximately 13 turns, the amount of twist absorbed by the B-Z transition.

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Fig. 4.
Effects of superhelicity on basal level and
IHF-activated transcription from the ilvPG
promoter. Panel A, the ilvPG
basal transcriptional rates determined on topoisomers of pDH wt ( )
or pSS Z ( ) are plotted as a function of average linking number
deficiency ( Lk ± 2). Panel B, the
ilvPG basal and IHF-activated transcriptional
rates determined on topoisomers of pDH wt in the absence ( ) and
presence ( ) of IHF. Panel C, the
ilvPG basal and IHF-activated transcriptional
rates determined on topoisomers of pSS Z in the absence ( ) and
presence ( ) of IHF. Arrows indicate DNA templates of the
lowest | Lk| that demonstrate OsO4 reactivity in the
UAS1 region. Transcriptional rates (±S.D. of three experiments) were
normalized by setting the maximum level of transcription for each DNA
template in each plot equal to 1.0.
|
|
 |
DISCUSSION |
Transitions to superhelically induced non B-DNA structures can
either be facilitated or inhibited by proteins that bind at or near
potential transition sites (8, 9). If a DNA region that is favored to
form a superhelically induced alternate structure is stabilized in the
B-form by a DNA-binding protein, its propensity for transition will be
transferred to other sites within the same superhelical domain. If one
of these secondary sites is in a promoter region, where strand
separation is required for transcriptional initiation, this transfer
could facilitate open complex formation and thereby activate
transcription. We have previously demonstrated that IHF binding
inhibits the superhelically induced destabilization of the B-form DNA
helix in an upstream activating sequence (UAS1) of the
ilvPG promoter of E. coli, and
simultaneously facilitates duplex destabilization of its downstream
10 region (8, 14). We also have shown that this downstream
destabilization decreases the energy required for open promoter complex
formation, which activates transcriptional initiation (14). We have
proposed that the superhelical energy required for this transcriptional activation was derived from the IHF-mediated inhibition of
superhelically induced duplex destabilization within the upstream UAS1
region (8). This novel DNA structural transmission mechanism explains the observations that IHF-mediated activation of transcription from the
ilvPG promoter requires a supercoiled DNA
template, and occurs in the absence of specific interactions between
IHF and RNA polymerase.
We emphasize that, although this model involves the transfer of the
free energy of destabilization, it does not require DNA denaturation,
either within the SIDD site of UAS1 before IHF binding or around the
10 region after binding. Indeed, transcriptional activation was
assayed at moderate salt concentrations, a condition where denaturation
at normal superhelix densities is inhibited (20). (We note that
OsO4 binds to structures in which the B-form is
destabilized but not necessarily denatured (21).) We have performed
sample calculations to illustrate the transmission of destabilization
energy under these conditions. The methods used in these calculations
have been described previously (10, 22, 23). Their results are plotted
in Fig. 5. Fig. 5A displays
the calculated destabilization energy profiles for a 500-bp portion of
the plasmid sequence that contains the UAS1 region, both in the absence
(dotted line) and in the presence (solid line) of bound IHF. Here G(x) is the incremental free
energy that is required to guarantee that the base pair at position
x is open (23). Smaller values of G(x)
correspond to more strongly destabilized sites. This calculation
assumes that the plasmid has an overall superhelix density of
=
0.035. In the absence of IHF binding, the low basal level of
transcription is regarded as slightly reducing the local superhelix
density upstream of the polymerase, to
=
0.045. IHF binding is
modeled as constraining the DNA to B-form throughout the 30-bp region
where DNA-protein contacts are seen in the co-crystal structure (24).
This binding causes an extreme bend, which we regard as strongly
affecting the manner in which transcriptionally generated incremental
negative superhelicity is accommodated. Specifically, when the wake of
negative superhelicity generated by transcription (25, 26) encounters
the strongly bent region, its untwisting torsional deformations (the
component of superhelicity that directly drives transitions) act to
physically rotate the DNA·IHF complex, thereby transducing
superhelical torsional deformations into writhing deformations. Because
this transduction requires rotation of a relatively large complex
through a medium that is highly viscous on this size scale, the
torsional stresses that drive it must accumulate on the 3' side of the
IHF. Thus, although bound IHF is not a barrier to the passage of
superhelicity (26), the torsional component that drives transitions
will still be lower on its 5' side and higher on its 3' side. The
increased rate of transcription consequent on activation will produce a further augmentation of the negative superhelicity 5' to the RNA polymerase complex. We model these events in our calculation by assuming that the component of superhelicity that drives transitions reverts to its basal level on the 5' side of the IHF·DNA complex, and
has twice that value in the region between that complex and the
polymerase. Fig. 5B shows the calculated change in the
destabilization free energy G(x) in this region
consequent on IHF binding. The largest decrease, corresponding to the
greatest destabilization, occurs around the
10 and
35 regions of
the promoter. (The
10 region is denoted by a bar in both parts of the
graph in Fig. 5.) Under the conditions assumed by this calculation,
approximately 4 kcal/mol less free energy is needed to open this region
when IHF is bound than when it is not bound.

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Fig. 5.
Sample calculations were performed to
illustrate the transmission of destabilization energy consequent
on IHF binding from the UAS1 region to the 10 region. The
energies of denaturation used were experimentally measured at [Na] = 0.1 M (30), close to the ionic strength used in the
transcription assays reported here. Panel A displays the
calculated destabilization energy profiles for a 500-bp portion of the
plasmid sequence that contains the UAS1 region, both in the absence
(dotted line) and in the presence (solid line) of
bound IHF. Here G(x) is the incremental free
energy that is required to guarantee that the base pair at position
x is open (23). Smaller values of G(x)
correspond to more strongly destabilized sites. This calculation
assumes the plasmid has an overall superhelix density of = 0.035.
In the absence of IHF binding, the low basal level of transcription
further reduces the local superhelix density upstream of the polymerase
to = 0.045. IHF binding constrains the DNA to B-form throughout
the 30-bp region where DNA-protein contacts occur. The torsional stress
component that drives superhelical transitions is modeled as being
lower on the 5' side of the bound IHF and higher on its 3' side, as
described in the text. Panel B shows the calculated change
in the destabilization free energy G(x) in this
region consequent on IHF binding. A substantial decrease in the
stability around the 10 and 35 regions of the promoter arise when
IHF binds, even though no region is predicted to denature under these
circumstances. In both parts of this figure the 10 region is denoted
by a bar.
|
|
This model predicts that superhelically induced DNA structural
destabilization, not the presence of specific DNA sequences, is the
primary determinant of IHF-mediated activation. To directly test this
prediction, we inserted a distant, superhelically induced Z-DNA-forming
sequence that is capable of inhibiting the destabilization of UAS1
without altering the DNA sequence in any part of the
ilvPG regulatory-promoter region. The inserted
(CG)13AATT(CG)22 sequence undergoes a
superhelically driven B-Z transition, which absorbs 13 negative
superhelical turns (Fig. 2), and thereby relaxes the global superhelix
density of the remainder of the supercoiled DNA template by a
corresponding amount (17). Because this B-Z transition occurs at a
lower threshold superhelical density than does the destabilization of
UAS1, it inhibits UAS1 destabilization until approximately 13 additional negative superhelical turns are added to the DNA template
(Fig. 3). If the energy required for IHF-mediated transcriptional
activation is indeed derived by transfer from the initially
destabilized UAS1 region upon IHF binding, then the superhelicity
required for IHF activation should be offset by approximately 13 turns
in the plasmid containing the Z-susceptible site. The results of
transcription assays on DNA templates of defined superhelix densities
show this to be the case (Fig. 4). The superhelicities required both
for half-maximal basal level and for IHF-activated transcription are
indeed offset by approximately 13 turns. These experiments clearly
demonstrate the involvement of superhelically induced DNA duplex
destabilized structure of UAS1 in IHF-mediated activation. Because we
have previously shown that IHF binding to its target site in UAS1 is unaffected by superhelical density (12), these experiments: (i) support
our previous demonstration that IHF-mediated activation occurs in the
absence of interactions between IHF and RNA polymerase (14), even on a
supercoiled DNA template; (ii) explain the requirement for a
supercoiled DNA template for IHF-mediated activation; and (iii) confirm
the predictions of the protein-mediated DNA structural transmission
mechanism of transcriptional activation (8).
It is important to emphasize that the effects of the Z-DNA structure on
basal level transcription and IHF-mediated activation reported here are
facilitated by altering the structure of the DNA helix in the
ilvPG promoter-regulatory region without
altering its natural DNA sequence. This type of experiment establishes a competition between DNA structures to separate the regulatory roles
of structural transitions from those of base sequence (9). We suggest
that this approach can be applied to dissect the effects of these
factors in a wide variety of other DNA
supercoiling-dependent biological processes.
The biological significance of superhelically induced DNA secondary
structures is becoming an increasingly active area of research, and a
great deal of information suggesting that DNA assumes a variety of
structures in living cells is emerging (27-29). Our work demonstrates
that these DNA supercoiling-dependent biological processes
can be regulated by distant or nearby DNA
supercoiling-dependent structures, and that these effects
can be modulated by DNA-binding proteins through their influence on the
formation of these structures (8). This realization presents an
important paradigm for future studies on the regulation of many
biological processes, such as gene expression, that are sensitive to
the superhelical state of DNA.
 |
ACKNOWLEDGEMENTS |
We are grateful to Elaine Ito for expert
technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by grants from the National
Institutes of Health (GM55073 and GM47012) and the National Science Foundation (MCB9723452).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 Predoctoral Training
Grant (GM07311). Present address: Dept. of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA
02138-2092.
To whom correspondence should be addressed. Tel.: 949-824-5858;
Fax: 949-824-8598; E-mail: gwhatfie{at}uci.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
SIDD, superhelically
induced DNA duplex destabilization;
IHF, integration host factor;
bp, base pair(s).
 |
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