Recruitment of
54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of
Subunit Carboxyl-terminal Domain on an UP-like Element*
Raffaella Macchi,
Lorena Montesissa,
Katsuhiko Murakami
,
Akira Ishihama
,
Víctor de Lorenzo ¶ and
Giovanni Bertoni ||
From the
Dipartimento di Genetica e Biologia dei Microrganismi, Università
degli Studi di Milano, via Celoria 26, 20133 Milan, Italy, the
Nippon Institute for Biological Science, Ome,
Tokyo 198-0024, Japan, and the ¶Department of
Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo
Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049
Madrid, Spain
Received for publication, March 25, 2003
, and in revised form, May 8, 2003.
 |
ABSTRACT
|
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The interactions between the
54-containing RNA polymerase
(
54-RNAP) and the region of the Pseudomonas putida
Pu promoter spanning from the enhancer to the binding site for the
integration host factor (IHF) were analyzed both by DNase I and hydroxyl
radical footprinting. A short Pu region centered at position
104 was found to be involved in the interaction with
54-RNAP, both in the absence and in the presence of IHF
protein. Deletion or scrambling of the 104 region strongly reduced
promoter affinity in vitro and promoter activity in vivo,
respectively. The reduction in promoter affinity coincided with the loss of
IHF-mediated recruitment of the
54-RNAP in vitro.
The experiments with oriented-
54-RNAP derivatives
containing bound chemical nuclease revealed interchangeable positioning of
only one of the two
subunit carboxyl-terminal domains (
CTDs)
both at the 104 region and in the surroundings of position 78.
The addition of IHF resulted in perfect position symmetry of the two
CTDs. These results indicate that, in the absence of IHF, the
54-RNAP asymmetrically uses only one
CTD subunit to
establish productive contacts with upstream sequences of the Pu
promoter. In the presence of IHF-induced curvature, the closer proximity of
the upstream DNA to the body of the
54-RNAP can allow the
other
CTD to be engaged in and thus favor closed complex formation.
 |
INTRODUCTION
|
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Bacterial promoters are modular DNA regions able to establish productive
interactions both with subunits of RNA polymerase holoenzyme
(RNAP1; subunit
composition:
2
'
) and regulatory
proteins
(14).
The core promoter elements are signature tags for
factor selectivity
(4). The major sigma factor
70 generally directs RNAP to interact with the core DNA
elements 10 and 35 (hexamers with consensus
5'-TATAAT-3' and 5'-TTGACA-3', respectively)
(5). The core promoter elements
can be both overlapped and flanked by protein-bound DNA sites involved in the
fine modulation of promoter activity
(2,
4). In the last decade, a
considerable amount of attention has been given to a A + T-rich promoter
sequence, the UP element, located upstream of the core promoter region and
consisting of two distinct subsites, each of which, by itself, can be bound by
the carboxyl-terminal domain of the RNAP
subunit (
CTD)
(3,
610).
CTD recognizes and interacts with the backbone structure in the minor
groove of the UP element. The A + T-rich sequence of the UP element are needed
to provide the optimum width of minor groove for interaction with
CTD
(7,
11). Several lines of evidence
showed that the role of the UP elements is to stimulate transcription in an
activator protein-independent manner and to a different extent (from 1.5 to
90-fold) depending on the similarity with the consensus UP element
sequence (3,
9). It is currently believed
that the transcription stimulation by an UP element has to be traced mainly to
the cooperation of the sigma factor and the
subunit in RNAP binding to
the promoter (1,
12). Thus, the presence of an
UP element in a promoter plays the major role of increasing the initial
equilibrium constant of closed complex formation between RNAP and promoter DNA
(13,
9). However, influences of
-UP element interaction on later steps in transcription initiation were
also reported (13,
14). The location of the UP
element with respect to the transcription start site can influence the degree
of transcription stimulation
(15). In the Escherichia
coli rrnB P1 promoter, the UP element is located in a region spanning
form the 40 and 60 positions and is able to increase
transcription from 30- to 70-fold
(6,
13). The artificial upstream
re-location of the rrnB P1 UP element by a single turn of DNA helix
decreases but does not prevent transcription stimulation, while further
displacements abolish UP element-dependent transcription
(15). The ability of
CTD to contact DNA and/or activator molecules at different locations
upstream of the core promoter
(8,
1521)
has been attributed to the flexibility of the linker connecting
CTD to
the
amino-terminal domain (
NTD)
(8,
22) assembled in the body of
RNAP. This linker flexibility also accounts for the ability of the two copies
of
CTD to function interchangeably with respect to the subsite
recognition within the UP element
(10). Sufficient length of the
linker between
CTD and
NTD is also needed for UP
element-dependent transcription activation. The linker is flexible but
structured to a certain extent to facilitate the positioning of the
CTD
to a proper location for interaction with the UP element
(23,
15).
UP-like elements were also found in promoters recognized by alternative
sigma factors, such as the
D-dependent flagellin promoter of
Bacillus subtilis
(24) and the
54-dependent Pu promoter of Pseudomonas
putida (Fig. 1)
(25,
26). The latter drives
transcription of TOL plasmid upper operon for the degradation of
toluene (27) and shows the
typical modular structure of the
54-dependent promoters (for
review, see Refs. 28 and
29) that consists of: (i) the
12/24 region (consensus: TGGCAC N5 TTGCa/t located
between positions 11 and 26)
(30) recognized by
54 and considered the functional analogue of
10/35 core promoter bound by
70
(31), (ii) DNA enhancer
sequences (known as upstream activating
sequences or UAS) targets for the activators of the
54-RNAP, usually located at >100 bp from the
transcription start site, and (iii) an intervening sequence between UAS and
12/24 motif that may contain a target site of the IHF
(32), which, by its ability to
bind and bend DNA sequences, assists the looping out required to bring closer
together the
54-activator prebound at UAS and
54-RNAP assembled in a closed complex with
12/24 DNA region. The productive contact between
54-activator and
54-RNAP closed complex
triggers promoter opening (open complex) and eventually transcription
initiation (33,
34).
Within this typical modular scheme for
54-dependent
promoters, the P. putida Pu promoter presents unique features. In
fact, our previous results showed the additional IHF role of stimulating the
otherwise limiting step of closed complex formation between
54-RNAP and Pu DNA
(26,
35). We also showed that the
recognition of Pu promoter by
54-RNAP involves not
only the 12/24 region but also a functional equivalent of an UP
element located in the intervening region, upstream to the IHF binding site.
Furthermore, our data strongly suggested that the Pu UP element could
play a key role in the IHF-mediated stimulation of closed complex formation by
54-RNAP. In this work, we closely inspected the
interactions, both in the presence and absence of IHF, between
54-RNAP and the Pu intervening region located
upstream the IHF binding site. The data strongly support the notion of a
non-canonical arrangement of the stimulating DNA sequences functioning as UP
element.
54-RNAP upstream interactions concentrate on two
sites located in the surroundings of positions 104 and 78,
respectively, thus being distant about 25 bp. In the absence of IHF and
probably due to asymmetrical positioning of the upstream DNA, the two sites
can be contacted interchangeably only by one
CTDs of the
2
'
54 complex
constituting the
54-RNAP. On the contrary, the bending by
IHF apparently introduces symmetry to the nucleoprotein complex allowing the
other
CTD to interact with the two sites. Thus, the IHF-mediated
stimulation of closed complex would result from curvature-dependent increased
probability of wide range upstream interactions by
54-RNAP
through the
CTDs.
 |
MATERIALS AND METHODS
|
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Bacterial Strains, Plasmids, and General ProceduresPlasmid
pEZ9 (25) contains the entire
Pu promoter sequence as a 312-bp EcoRI-BamHI insert
in pUC18 spanning positions 208 to +93. P. putida strains
KT2442 hom.fg/xylRS and its derivative HFPu
(Pu::lacZ, xylR+) have been described previously
(36,
37). KT2442PuXhoCla
(PuXhoCla::lacZ, xylR+), KT2442PuScra1
(PuScra1::lacZ, xylR+), and
KT2442PuScra2 (PuScra2::lacZ, xylR+)
carrying mutant Pu::lacZ fusions in the same location of the
chromosome as HF Pu were obtained as follows. The Pu
version, cloned in pUC-PuClaI-79, derived from pEZ9 and bearing a
ClaI site engineered within positions 79 and 84
(26), was subjected to
site-directed mutagenesis by the QuikChangeTM site-directed mutagenesis
kit (Stratagene) to engineer a XhoI site within nucleotides
121 to 126. This procedure generated the plasmid
pUC-PuClaXho. The replacement of the 47-bp
XhoI-ClaI fragment of pUC-PuClaXho for synthetic
XhoI-ClaI fragments harboring scrambled sequences from
nucleotides 105 to 120 and from 95 to 120 gave
rise to plasmids pUC-PuScra1 and pUC-PuScra2, respectively.
The Pu versions present in pUC-PuClaXho,
pUC-PuScra1, and pUC-PuScra2, respectively, were rescued as
312-bp EcoRI-BamH fragments, fused to lacZ by
cloning in the corresponding sites of pBK16 vector
(36) and recombined with the
homology fragment inserted in the chromosome of KT2442
hom.fg/xylRS as described previously
(36). All cloned inserts and
DNA fragments were verified before use by automated DNA sequencing.
Recombinant DNA manipulations were carried out according to published
protocols (38).
Proteins and Protein TechniquesAccumulation of
-galactosidase raised by lacZ fusions was measured in P.
putida KT2442 cells permeabilized with chloroform and sodium dodecyl
sulfate (SDS) as described by Miller
(39) under the conditions
specified in each case. Purified
54 factor and IHF were
kindly donated by B. Magasanik and S. Goodman, respectively; native core RNAP
was purchased from Epicenter Technology. Reconstitution of RNAP carrying
(p-bromoacetamidobenzyl)-EDTA·Fe (Fe·BABE) on
-associated
,
(Fe)
·
',
'-associated
,

·
(Fe)
', and simultaneously on both
subunits,
(Fe)
·
(Fe)
', was
carried out as described by Murakami et al.
(17,
40).
DNA Binding AssaysThe DNA fragments used in DNA
footprinting experiments of Fig. 2
(A and B) and
Fig. 5 were excised from pEZ9
as 390-bp EcoRI-PvuII and end-labeled at their
EcoRI sites by in-filling the overhanging ends as described below.
The collection of Pu promoter sequences (Pu-126,
Pu-114, Pu-105, Pu-85) having the same downstream
end at +45 and different upstream ends at 126, 114, 105,
and 85 were generated by separate PCR amplification from pEZ9 with the
common reverse primer 5'-GAGAAAATACAACATTGAAGGGTCACCACT-3' and
forward primers
5'-GCTCTAGA(XbaI)TACAGCCAGCGTGCTGTAGA-3',
5'-GAAGGCCT(StuI)GCTGTAGATTTTCTCTCATAC-3',
5'-GCTCTAGA(XbaI)TTTTCTCTCATACCCCCCCT-3',
5'-GCTCTAGA(XbaI)TTCTTTTTTACAAAGAAAAT-3',
respectively.

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FIG. 5. Monitoring the positioning of the two CTDs of
54-RNAP along upstream sequences of the Pu
promoter. The same end-labeled fragment as
Fig. 2 was incubated separately
with the (Fe·BABE)-labeled 54-RNAPs indicated at the
top, both in the presence and in the absence of IHF, and then run in
a sequencing gel. No protein, IHF alone, and unlabeled
54-RNAP were used as negative controls. The location of the
IHF binding site, some coordinates along the promoter sequence, and the
regions corresponding to the UP-likePuI and
UP-likePuII sites are indicated to the
sides, using the Maxam and Gilbert A + G reaction as a reference.
Nucleotides, which become hypersensitive to the cleavage with hydroxyl
radicals, are indicated with closed arrows (see
Fig. 1). The concentration of
IHF used was 100 nM. RNAPs contained 60 nM, core enzyme
mixed, in each case, with a 3-fold molar excess of purified
54.
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For gel retardation assays, the PCR-amplified fragments described above
were end-labeled with [
-32P]ATP and T4 polynucleotide
kinase. Radioactive nucleotides not incorporated in DNA were removed by
centrifuging briefly in small Sephadex G-25 columns. Binding reactions were
performed in a total volume of 25 µl of transcription buffer containing 35
mM Tris acetate, 70 mM KAc, 5 mM
MgAc2, 20 mM NH4Ac, 2 mM
CaCl2,1mM dithiothreitol, 3% glycerol, and 40 µg/ml
of poly[d(I-C)]. Labeled fragments, added to the buffer at a final
concentration of 5 nM, were incubated with 100 nM IHF,
60 nM core RNAP, and a 3-fold molar excess of
54
factor for 25 min at 30 °C. The entire reaction volume was loaded onto
non-denaturing 4% polyacrylamide gels (acrylamide:bis ratio 80:1) in
0.5x TBE buffer (45 mM Tris borate, pH 8.3, 0.1 mM
EDTA, 5 mM MgCl2), electrophoresed at 12 mM
at 4 °C for 6 h, and dried. Bands were visualized and quantified by
Typhoon 8600 variable mode imager (Amersham Biosciences) upon storage phosphor
autoradiography. DNA footprinting assays were performed in a total volume of
50 µl and with similar concentrations of end-labeled fragments and proteins
used in the gel mobility-shift assays. For DNase I footprinting, after
preincubation of end-labeled Pu DNA and proteins in transcription
buffer for 25 min at 30 °C, 3 ng of DNase I were added to each sample and
further incubated for 3.5 min. Reactions were stopped by addition of 25 µl
of STOP buffer containing 0.1 M EDTA, pH 8, 0.8% SDS, 1.6
M NH4Ac, and 300 µg/ml sonicated salmon sperm DNA.
Nucleic acids were precipitated with 175 µl of ethanol, lyophilized and
directly resuspended in denaturing loading buffer (7 M urea, 0.025%
bromphenol blue, and 0.025% xylene cyanol in 20 mM Tris, pH 8)
prior to loading on a 7% DNA sequencing gel. A + G Maxam and Gilbert reactions
(41) were carried out with the
same fragments and loaded onto the gels along with the footprinting samples.
For hydroxyl radical footprinting, after preincubation of end-labeled
Pu DNA and proteins for 25 min at 30 °C in hydroxyl
radical buffer containing 25 mM HEPES, 70 mM KAc, 5
mM MgAc2, 19 mM NH4Ac, 0.7
mM DTT, 1% glycerol, and 40 µg/ml of poly[d(I-C)], 3 µl each
of [Fe(EDTA)]2 (125 mM
(NH4)2Fe(SO4)2·6H2O,
250 mM EDTA), 28 mM ascorbate, and 0.84% hydrogen
peroxide were added to the samples and then incubated for a further 5 min.
Reactions were stopped by addition of 15 µl of 0.1 M thiourea
and 25 µl of STOP buffer (as above), respectively. Nucleic acids were
precipitated with 200 µl of ethanol and treated for separation and
visualization as described above for DNase I footprinting. For DNA
footprinting in the presence of (Fe·BABE)-RNAPs, after preincubation of
end-labeled Pu DNA and proteins for 25 min at 30 °C in
hydroxyl radical buffer, 1 µl each of 100 mM ascorbate
and 0.6% hydrogen peroxide were added to the samples and then incubated for a
further 15 min. Reactions were stopped by addition of 10 µl of 0.1
M thiourea and 25 µl of STOP buffer (as above), respectively.
Nucleic acids were precipitated with 200 µl of ethanol and treated for
separation and visualization as described above for DNase I footprinting.
 |
RESULTS
|
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54-RNAP Per Se Can Contact Sequence Elements Located
Upstream the IHF Binding Site of Pu PromoterThe affinity of
54-RNAP for Pu promoter DNA lacking the sequence
region located upstream the position 79 is strongly reduced
(Fig. 1)
(26). These data suggested
that
54-RNAP could utilize per se additional DNA
affinity elements located far upstream of the 12/24 region
involved in the interaction with
54. Since
CTD-deleted derivatives of
54-RNAP showed reduced
promoter affinity (26), we
also suggested that
CTD could be directly involved in the recognition
of such upstream DNA, reminiscent, in this case, of the UP elements of
70 promoters. Furthermore, an
CTD/UP-like interaction
also seemed to be involved in the mechanism behind the stimulation of closed
complex formation between Pu DNA and
54-RNAP on IHF
binding and bending, which we referred to as IHF-mediated recruitment of
54-RNAP to the Pu promoter
(26). To map the contact sites
of
54-RNAP upstream 79 position and to reveal any
possible influence of IHF-induced bending on these upstream contacts, we
carried out either DNase I and hydroxyl radical footprinting assays on
end-labeled DNA fragments bearing the entire Pu mixed with increasing
amounts of
54-RNAP, both in the presence and in the absence
of sub-saturating concentrations of purified IHF. As shown in
Fig. 2A (lanes
13), the footprint of the
54-RNAP in the region
spanning approximately from 83 to 108 consisted in a generalized
hypersensitivity to DNase I. The addition of IHF
(Fig. 2A, lanes
4 and 5) caused a further slight increase of upstream DNA
reactivity to DNase I, with the exception of positions 90 and
91, appearing to be more protected from DNase I cleavage than when in
the presence of
54-RNAP alone. Similar conditions of labeled
Pu DNA/protein ratio were employed to perform hydroxyl radical
footprinting assays (Fig.
2B). The inspection of the Pu DNA sequence
upstream of the IHF binding site revealed both protected and hypersensitive
sites in a region spanning from 95 to 109. In particular, as
shown in Fig. 2B
(lanes 14), the hydroxyl radical footprint of
54-RNAP consisted of three hypersensitive positions,
103 to 105, which seemed to be flanked by short protected
regions. Apparently, the addition of IHF protein
(Fig. 2B, lanes
57) caused minimal changes to the pattern of hydroxyl radical
cutting. Examined together, these results strongly indicated that, even when
unassisted,
54-RNAP can establish contacts to DNA sites
located far upstream of the 12/24 region. In the case of the
hydroxyl radical footprint, the DNA region involved in interactions with
54-RNAP appeared to be limited to a short sequence in the
surroundings of position 104, which we named
UP-likePuI. In our previous work
(26), to explain the mechanism
behind the IHF-mediated recruitment of
54-RNAP, we suggested
that the distance between the 12/24 site and the UP element(s)
might disfavor either the formation or the maintenance of simultaneous binding
by
54-RNAP through
54 and
CTD,
respectively. Thus, the key recruiting action of IHF-induced bending would
have consisted of increasing
54-RNAP affinity for
Pu DNA by bringing the 12/24 site and the UP element(s)
into a closer proximity. Apparently, from the footprinting analysis presented
in Fig. 2, the ability of
54-RNAP to establish contacts with the Pu region
located upstream of the IHF binding site seemed to be enhanced to a limited
extent only by the addition of IHF. However, further analysis with free
radical-delivering
54-RNAPs (see below) showed more clearly
that IHF-induced bending can cause increased occupancy by
CTD of the
region upstream to the IHF binding site.
The Integrity of the UP-likePuI Site Is
Required Both for Full Promoter Affinity and IHF-mediated Recruitment of
54-RNAPThe footprinting experiments
presented above allowed us to identify an upstream DNA element,
UP-likePuI, which is contacted by
54-RNAP in the closed complex with the Pu promoter.
To evaluate the role of the contacts with
UP-likePuI in determining the affinity of
54-RNAP for the Pu promoter, we ran gel retardation
assays on the nucleoprotein closed complexes formed by
54-RNAP with DNA fragments bearing either the Pu
sequence up to position 126 (Pu-126;
Fig. 1) supposed to include the
whole UP-likePuI site or progressively shorter
Pu sequences extending up to 114, 105, and 85,
respectively (Pu-114, Pu-105, Pu-85;
Fig. 1). Side-by-side
comparison of the amounts of complex assembled by
54-RNAP
with either Pu-126 or Pu-114, respectively, showed no
significant difference (data not shown). On the contrary, the amounts of
complex formed by
54-RNAP with Pu-105 were strongly
reduced with respect to Pu-114
(Fig. 3, lanes 5 and
6). A more extended deletion up to position 85
(Pu-85) (Fig. 3,
lane 4) did not decrease further the amounts of nucleoprotein complex
with
54-RNAP.

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FIG. 3. Band-shift assay of the complexes formed between Pu segments
and 54-RNAP. Left, three sequences of
the Pu promoter spanning positions 114 to +45
(Pu-114), 105 to +45 (Pu-105), and 85 and +45
were end-labeled with 32P, mixed with 60 nM
54-RNAP in the absence and in the presence of 100
nM IHF, and run in a gel retardation assay as explained under
"Materials and Methods." The location of the different complexes
is indicated to the sides. Right, superimposed profiles from scans of
lanes 4 and 7 (Pu-85), 5 and 8
(Pu-105), 6 and 9 (Pu-114), respectively,
are shown. The vertical arrow indicates the position of the
54-RNAP-DNA complexes. The percentage of volume under each
peak with respect to the total volume of the lane profile was calculated. The
value close to the profiles of each promoter indicates the ratio between the
percentage volume of the peaks corresponding to the
54-RNAP/DNA complexes in the presence and in the absence of
IHF, respectively. For Pu-85, Pu-105, and Pu-114,
the absolute percentage values of peak volumes for
54-RNAP-DNA complexes in the absence of IHF resulted 8.5,
6.4, and 13.2%, respectively.
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To test the requirement of UP-likePuI
integrity for the IHF-mediated recruitment of
54-RNAP, we
added a sub-saturating concentration of purified IHF protein to the mixtures
of
54-RNAP with Pu-85, Pu-105, and
Pu-114, respectively. As shown in
Fig. 3 (lanes
49), while the binding of
54-RNAP to
Pu-114 could be stimulated by IHF as shown previously
(26), IHF failed to enhance
closed complex formation with Pu-105 and Pu-85,
respectively. Thus, these results strongly indicated that the interactions
established by
54-RNAP with
UP-likePuI site in the closed complex are
instrumental in determining promoter affinity. In addition, as the integrity
of UP-likePuI is also required to observe
IHF-mediated enhancement of
54-RNAP recruitment, we
speculated that the promoter architecture imposed by IHF binding contributes
to the interactions between
54-RNAP and the
UP-likePuI site.
The Scrambling of UP-likePuI DNA Region
Affects Pu Performance in VivoIn view of the previous results
in vitro, the disruption of the integrity of
UP-likePuI site was expected to affect
Pu activity. To address this issue, we aimed to monitor in
vivo the consequences of altering the sequence spanning the
UP-likePuI on Pu expression pattern.
To this end, we introduced progressive sequence scrambling into the DNA region
from 120 to 93 sites (Fig.
1) by replacement with synthetic double-stranded oligomers for the
wt DNA sequence located between the XhoI and ClaI
sites that were opportunely engineered within positions 126 and
121, and 84 and 79, respectively, in the Pu
variant, PuXhoCla (Fig.
1). From this procedure, we obtained two Pu derivatives,
PuScra1 and PuScra2 (Fig.
1), that, along with their parental PuXhoCla, were fused
to lacZ and recombined into the chromosome of P. putida
KT2442 hom.fg/xylRS as described previously
(36). As shown in
Fig. 4A, the comparison of
accumulation of
-galactosidase upon toluene induction of HFPu
(Pu::lacZ, xylR+) and KT2442PuXhoCla
(PuXhoCla::lacZ, xylR+), respectively, revealed
that the engineering of the XhoI and ClaI sites described
above did not affect Pu performance. On the contrary, as shown in
Fig. 4B, the
comparison of
-galactosidase accumulation upon toluene induction of
KT2442PuXhoCla, KT2442PuScra1 (PuScra1::lacZ,
xylR+) and KT2442PuScra2 (PuScra2::lacZ,
xylR+), respectively, revealed that the promoter activity of
PuScra2 was severely impaired. Since the sequence scrambling did not
introduce either phase alteration of key regulatory sites (UAS, IHF box and
12/24 sites) or a predictable drastic variation of promoter DNA
curvature (42), we inferred
that the reduction of promoter activity of PuScra2 was caused by the
destruction of upstream contacts between
54-RNAP and the
UP-likePuI site.

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FIG. 4. Involvement of UP-likePuI region
in Pu promoter activity in vivo. P. putida
KT2442 derivatives bearing the Pu-lacZ, PuXhoCla-lacZ, PuScra1-lacZ,
and PuScra2-lacZ transcriptional fusions recombined into the same
site of the chromosome, respectively, were tested for the performance of
expression of the lacZ reporter gene during the growth at 30 °C
in LB medium (38). Each strain
was grown until cultures had an absorbance of 0.5 at 600 nm. Toluene was then
administered and the incubation continued for the subsequent 3.5 h.
Accumulation of -galactosidase along the time and growth curves of each
strain after toluene addition are shown. A, comparison of the
performances of Pu-lacZ and PuXhoCla-lacZ in the presence
and in the absence of induction with toluene, respectively. B,
comparison of the performances of PuXhoCla-lacZ, PuScra1-lacZ, and
PuScra2-lacZ, respectively, upon toluene induction.
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Monitoring the Positioning of the Two
CTDs of
54-RNAP along Upstream Sequences of the Pu Promoter in
the Absence and in the Presence of IHFIn the
2
' core RNAP complex, the two identical
subunits can be distinguished by their arrangement with respect to
and
' subunits. In fact, one
,
I,
interacts with the
, whereas the other,
II, interacts
with
' (43,
40,
17). To provide a more precise
definition of the positioning of the
CTD of
I and
II (
CTDI and
CTDII,
respectively) along the Pu DNA region upstream the IHF site, we set
out to exploit the UP DNA cleavage capability caused by free radicals
originated from (p-bromoacetamidobenzyl)-EDTA·Fe
(Fe·BABE) attached to the UP contact surface of one or both
subunits assembled in the RNAP holoenzyme complex
(40,
17). We prepared free
radical-releasing
54-RNAP by adding a saturating amount of
54 both to oriented-
and non-oriented
(Fe·BABE)-labeled RNAP core complexes:
I(Fe)/
II and
I/
II(Fe), in which the (Fe·BABE)
moiety is bound to
CTDI and
CTDII,
respectively, and
I(Fe)/
II(Fe), in which
the (Fe·BABE) moiety is bound to both
CTDs. Such
(Fe·BABE)-labeled
54-RNAPs were incubated with
end-labeled Pu promoter DNA, and the pattern of Pu DNA
fragmentation was analyzed in sequencing gels as in the footprinting
experiments presented in Fig. 2 (A
and B). To test any differential influence of IHF on
upstream DNA occupancy by
CTDI or
CTDII, a
set of reactions was also incubated in the presence of IHF. As shown in
Fig. 5, in the absence of IHF,
both
I(Fe)/
II(Fe) and
I(Fe)/
II (lanes 2 and 3)
could produce in the UP-likePuI region a
fragmentation pattern that was very similar to the hydroxyl radical
hypersensitivity profile at positions 103, 104, and 105
displayed by the end-labeled Pu in the presence of unlabeled
54-RNAP (Fig.
2B, lanes 14). Furthermore, both
I(Fe)/
II(Fe) and
I(Fe)/
II could produce another discrete
pattern of fragmentation closer to the IHF binding site, involving positions
spanning from 74 to 79. We named this second
CTD-interacting region, which did not result so evident in the previous
footprinting experiments (Fig. 2,
A and B),
UP-likePuII. Unlike
I(Fe)/
II(Fe) and
I(Fe)/
II, in the absence of IHF the other
oriented-
54-RNAP,
I/
II(Fe), could not produce any
significant fragmentation of the end-labeled Pu DNA
(Fig. 5, lane 4). The
addition of IHF did not substantially modify the fragmentation pattern by
I(Fe)/
II(Fe) and
I(Fe)/
II. On the contrary, the presence of
IHF increased the fragmentation by
I/
II(Fe). In fact, under these conditions,
I/
II(Fe) was also able to generate a
fragmentation pattern (Fig. 5,
lane 8) identical to that of
I(Fe)/
II(Fe) and
I(Fe)/
II. Taken together, these results
clearly revealed that
CTD interaction with the Pu upstream
region can occur at two unusually distant sites,
UP-likePuI and
UP-likePuII, respectively. Both sites can be
bound interchangeably by
CTDI, both in the absence and in
the presence of IHF. Unlike
CTDI, and probably due to an
asymmetrical arrangement of the DNA upstream the IHF site,
CTDII can efficiently contact both
UP-likePuI and
UP-likePuII only in the presence of IHF
protein. These results strongly suggest that when the Pu promoter DNA
conformation is structured by the binding and bending of IHF, the two
CTD can distribute simultaneously and interchangeably among
UP-likePuI and
UP-likePuII.
 |
DISCUSSION
|
---|
The activity of the Pu promoter of P. putida is strongly
influenced by DNA architecture. Remarkably, the bending activity of IHF
strongly augments the probability of interaction between the activator XylR
and
54-RNAP
(44) and also positively
influences the docking of
54-RNAP on the promoter
(26). Our previous studies
showed the involvement of
CTD in the interaction with a Pu
region reminiscent of an UP element located upstream to the IHF binding site
(26). Furthermore, our results
suggested that the upstream interactions of
54-RNAP by
CTD could play an active role in its IHF-mediated recruitment. However,
the relationship between topology of the promoter DNA and topography of such
upstream interactions required further clarification. In fact, this might be
at the basis of the IHF-mediated enhancement mechanism of closed complex
formation. In this study, we investigated the nature and the role of the
upstream contacts that
54-RNAP is able to establish with the
Pu promoter both in the presence and in the absence of IHF protein.
The footprinting experiments presented in
Fig. 2 sustained more clearly
than our previous works (26)
the notion that
54-RNAP per se is able to establish
contacts with DNA sequences located upstream to the IHF binding site. In
addition, these experiments allowed us to identify a discrete upstream DNA
site surrounding position 104,
UP-likePuI
(Fig. 2B), engaged in
the interactions with
54-RNAP. Then, we addressed the issue
of the functional significance of the
54-RNAP/UP-likePuI contacts.
At least two lines of evidence indicated that the
54-RNAP
contacts with UP-likePuI are functional
interactions participating in Pu promoter activity. First,
alterations of the Pu sequence spanning from 114 to 85
positions (Pu-105 and Pu-85, respectively) affect promoter
affinity for
54-RNAP per se
(Fig. 3, lanes
46). Second, the rearrangement of the sequence surrounding
UP-likePuI in PuScra2 derivative
severely affects Pu activity in vivo. The experiment
presented in Fig. 3 also
indicated that UP-likePuI participates in the
IHF-mediated stimulation of closed complex formation by
54-RNAP even though its occupancy by
54-RNAP did not seem to change significantly upon the
addition of IHF (Fig.
2B). In our previous work
(26), to explain the
IHF-mediated recruitment of
54-RNAP, we considered the
possibility that IHF binding and bending could strengthen the upstream
contacts established by
CTD with Pu DNA. We figured that this
could be accomplished by one or the combination of the following two
mechanisms: (i) IHF-induced curvature centered at 68 would bring into
closer proximity the 12/24 region and an UP element located
upstream of the IHF binding site, thus favoring the simultaneous interaction
of
54-RNAP with both sites, and (ii) the IHF binding would
locally distort the double helix of DNA strengthening the
CTD/UP
interaction. However, the idea that IHF could reinforce upstream contacts by
CTD contrasted with the evidence presented in
Fig. 2 that IHF addition did
not substantially alter the pattern of the upstream footprint of the
54-RNAP. Furthermore, this had to be reconciled with the
fact that the integrity of UP-likePuI was
required both to determine promoter affinity and stimulate IHF-mediated closed
complex formation. The experiments with (Fe·BABE)-labeled
54-RNAPs showed that the hypothesis of the IHF-mediated
strengthening of the
CTD upstream contacts was correct. However, the
possible scenario (Fig. 6) can
be more complex than previously thought (Ref.
26 and see above). First,
CTD can bind to UP-likePuI and also to
another site, UP-likePuII, located downstream.
In the absence of IHF (Fig.
6A), only one
CTD,
CTDI
(i.e.
CTD of
that associates with
), can bind
interchangeably to both UP-likePuI and
UP-likePuII. These asymmetrical upstream
CTD interactions determine a first level of promoter affinity. The fact
that in the absence of IHF only
CTDI can interact
efficiently with upstream DNA could be explained by: (i) asymmetries in the
2
'
54 complex, (ii) the
axis of the upstream DNA is not in line with the body of
54-RNAP, and (iii) a combination of point i and ii. However,
the binding and bending of IHF makes the
CTD interactions more
symmetrical (Fig. 6B).
In fact, in this case, the other
CTD,
CTDII
(i.e.
CTD of
that associates with
'), can
also bind interchangeably to both UP-likePuI
and UP-likePuII. These symmetrical
CTD
interactions determine a higher second level of promoter affinity which would
underlie the IHF-mediated recruitment of
54-RNAP to the
Pu promoter. The topological shift from asymmetrical to symmetrical
CTD interactions can be attributed to the IHF-induced bending that
brings the 12/24 site and the upstream DNA into closer
proximity. In fact, this would favor the
54-RNAP contacts
with the upstream DNA also through the
CTDII domain. Despite
this IHF-mediated topological switch, the flexibility of the long unstructured
interdomain linker connecting
CTD to the rest of
(8,
22) might also account for the
ability of
54-RNAP to contact interchangeably the
UP-likePuI and
UP-likePuII sites located at a distance from
the 12/24 core promoter region.
Both UP-likePuI and
UP-likePuII sites may resemble the UP element
subsites, each of which constitutes a binding site for
CTD
(10). The certain assignment
of UP-likePuI and
UP-likePuII to one of the two classes, distal-
or proximal-type, of UP subsite
(10) cannot be deduced from
this study. Moreover, it remains to be clarified whether
UP-likePuI and
UP-likePuII are arranged adjacently as in the
UP element consensus sequence
(10) or are separated by turns
of DNA helix. Hypothetically, the A-tracts at positions 102 to
105 and 77 to 82 within
UP-likePuI and
UP-likePuII
(Fig. 1), respectively, could
constitute the core subsites
(7,
10,
45). In view of this, we
suggested that the distance between the core A-tracts is not consistent with a
side-by-side arrangement as for the UP subsites in the canonical UP element
(10), and there would be turns
of helix between UP-likePu subsites. Despite the distance
between UP-likePuI and
UP-likePuII, it would still be possible that
the binding of one
CTD to one UP-likePu subsite
cooperatively assists the second copy of
CTD to bind to the other
UP-likePu subsite. This could be accomplished by a
combination of protein-protein interaction between the two
CTDs and
local DNA flexibilization. The hypersensitivity to DNase I of the region
upstream to the IHF binding site (Fig.
2A), both in the absence and in the presence of IHF,
could trace the DNA bending of the UP-likePu arising from
the cooperative binding of the two
CTDs. We suggest that even in the
absence of IHF, the binding of
CTDI to one
UP-likePu subsite may recruit an otherwise distant
CTDII to the other UP-likePu subsite,
and this would originate bending of the UP-likePu. The
promoter bending introduced by IHF that renders the
UP-likePu region more accessible to
CTDII (see above) may result in mutual and stronger
cooperative binding of two the
CTDs. The slight increase in the general
DNase hypersensitivity and DNase I protection of positions 90 and
91 in the presence of IHF (Fig.
2A, lanes 3 and 4) might account for
the strengthening of the cooperative binding of
CTD to the
UP-likePu region. Since no evidence for the occupation of
other sites different to UP-likePuI and
UP-likePuII resulted from the assay with
I(Fe)/
II(Fe)
54-RNAP, we
also suggest that the cooperative occupancy of the upstream DNA region
involves only UP-likePuI and
UP-likePuII subsites.
In summary, the evidence presented in this work strongly supports the
notion of IHF-mediated topological switch that governs the occupancy of a
promoter by RNAP through the curvature-mediated modulation of
CTD
interactions with the upstream promoter region. It was shown previously that,
by protein-protein interaction, transcription factors can direct the
CTD positioning on the upstream promoter region
(17,
19). In a novel way, the
positioning of
CTD on the Pu promoter would be directed
predominantly by the DNA architecture.
 |
FOOTNOTES
|
---|
* This work was supported by QLK3-CT-2000-00170 (MIFRIEND) Contract of the
European Union. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Present address: Rockefeller University, 1230 York Ave., New York, NY
10021. 
||
To whom correspondence should be addressed: DGBM-Università degli Studi
di Milano, via Celoria 26, 20133 Milano, Italy. Tel.: 39-0250315027; Fax:
39-0250315044; E-mail:
giovanni.bertoni{at}unimi.it.
1 The abbreviations used are: RNAP, RNA polymerase holoenzyme;
CTD,
subunit carboxyl-terminal domains;
NTD,
amino-terminal
domain; IHF, integration host factor; UAS, upstream
activating sequences. 
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to S. Saini, I. Cases, E. Galli, and M. Valls for inspiring
discussions; to S. Goodman (University of Southern California, Los Angeles,
CA) for the kind donation of IHF protein; and to F. Vidal for helpful support
in band quantification.
 |
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