From the Department of Biological Sciences, Imperial College of
Science, Technology and Medicine, Sir Alexander Fleming Building,
Imperial College Road, London SW7 2AZ, United Kingdom and the
Waksman Institute and Department of Genetics, Rutgers,
The State University, Piscataway, New Jersey 08904
Received for publication, September 16, 2002, and in revised form, October 17, 2002
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ABSTRACT |
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Recent determinations of the structures of the
bacterial RNA polymerase (RNAP) and promoter complex thereof establish
that RNAP functions as a complex molecular machine that contains
distinct structural modules that undergo major conformational changes
during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core
RNAP ( Multisubunit DNA-dependent RNA polymerases
(RNAP)1 are complex molecular
machines that synthesize a RNA copy from a DNA template. In
Escherichia coli, five subunits ( Sequence comparisons reveal two unrelated families of RNAP Binding of a E Proteins and Promoter DNA Probes
Klebsiella pneumoniae Wild-type and mutant E. coli RNAP core enzymes, which
contained a COOH-terminal hexahistidine tag on the plasmid-borne Core RNAP Binding Assays
Native Gel Assembly Assay--
Increasing amounts of core RNAP
(10-400 nM) was added to 10-µl reactions containing 100 nM 32P end-labeled RNAP Holoenzyme Dissociation Assays--
For core
RNAP- Promoter Probe Binding Assays
These were conducted essentially as described by Wigneshweraraj
et al. (10) in STA buffer (25 mM Tris acetate,
pH 8.0, 8 mM magnesium acetate, 10 mM KCl, 1 mM dithiothreitol, and 3.5% (w/v) PEG 8000) using 100 nM Activator Binding Assays
Activator binding assays were conducted as described by Chaney
et al. (18). Holoenzymes were formed by incubating 50 nM 32P-labeled DNA Footprinting Assays
DNase I Footprinting--
DNase I footprinting of closed, open,
and initiated promoter complexes formed on the S. meliloti
nifH homoduplex (Fig. 4a) or early melted (Fig.
8c) promoter probes (32P end-labeled template
strand) were conducted essentially as described by Wigneshweraraj
et al. (10). The 10-µl binding reactions were conducted in
STA buffer; 1.75 × 10 KMnO4 Footprinting--
Binding reactions were
conducted as described above for DNase I footprinting in STA buffer
without dithiothreitol. 4 mM fresh KMnO4 was
added for 30 s, followed by 50 mM Ortho-Copper Phenanthroline (Ortho-CuOP)
Footprinting--
Binding reactions were conducted essentially as
described for KMnO4 footprinting. The reactions were
treated with 0.5 µl of a solution of 4 mM
ortho-phenanthroline and 0.92 mM
CuSO4 followed by 0.5 µl of 0.116 M
mercaptopropionic acid for 2 min. The reaction was terminated by the
addition of 1 µl of 28 mM
2,9-dimethyl-1,10-phenanthroline and loaded onto a 4.5% native gel.
Unbound and RNAP bound DNA was then excised from the gels. Gel-isolated
DNA was eluted into H2O overnight at 37 °C and processed
and analyzed on a 10% denaturing gel as described above.
In Vitro Transcription Assays
These were performed essentially as described by Wigneshweraraj
et al. (10). For the single round transcription assay,
supercoiled plasmid (pMKC28 or pSLE1) containing the S. meliloti
nifH or the E. coli pspA promoter,
respectively, was used. Plasmids pMKC28 and pSLE1 contain a T7 early
transcriptional terminator sequence downstream of the multiple cloning
site. The promoter fragment is inserted into the multiple cloning site
in such a way to direct transcription to generate a discrete transcript
of ~470 (for pMKC28) and ~510 (for pSLE1) bases. 10-µl reactions
contained 20 nM template, 100 nM RNAP
holoenzyme (formed with 1:6 ratio of core RNAP to FeBABE Cleavage of Promoter Complexes
Single-cysteine forms of Core RNAP Lacking the Previously, we have demonstrated that deletion of the 2
'
; E) associates with a sigma
(
) subunit to form the holoenzyme (E
). A mutation removing the
subunit flap domain renders the Escherichia coli
70 RNAP holoenzyme unable to recognize promoters.
54 is the major variant
subunit that utilizes
enhancer-dependent promoters. Here, we determined the
effects of
flap removal on
54-dependent
transcription. Our analysis shows that the role of the
flap in
54-dependent and
70-dependent transcription is different.
Removal of the
flap does not prevent the recognition of
54-dependent promoters, but causes multiple
defects in
54-dependent transcription. Most
importantly, the
flap appears to orchestrate the proper formation
of the E
54 regulatory center at the start site proximal
promoter element where activator binds and DNA melting originates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
'
)
form the RNAP catalytic core (E) that associates with a sigma (
)
subunit to form the holoenzyme (E
). The
subunit imparts on the
core RNAP the ability to specifically recognize and initiate
transcription from promoters. Biochemical and structural studies
indicate that the protein-protein contacts at the interface between
core RNAP and
, an extensive and functionally specialized sets of
surfaces, govern the conformational changes that allow efficient
promoter recognition and transcription initiation (1-3).
factors.
Members of the major family of
factors form RNAP holoenzymes that
recognize promoters and form transcriptionally competent promoter
complexes in the absence of other factors or energy
sources. This family, which includes most bacterial
factors is
named after the prototypical housekeeping
of E. coli,
70. Members of the second, minor family of
factors,
the
54 family, form RNAP holoenzymes that recognize
promoters but require additional protein factors and a source of energy
in the form of ATP or GTP hydrolysis for formation of transcriptionally
competent promoter complexes (4-6). Despite the differences in
pathways that lead to transcription-competent open promoter complexes, both classes of
factors occupy similar positions within their respective RNAP holoenzymes and appear to utilize some common RNAP
surfaces for transcription initiation (7-10).
70 family factor induces
conformational changes within the core RNAP (2, 11, 12). Structural
modules of the core RNAP, designated as the
' clamp, the
flap,
and the
lobes, interact with a
70 family
subunit
(
A) in the structures of Thermus aquaticus
and Thermus thermophilus RNAP holoenzymes and undergo
conformational changes, which orientate and position
70
DNA-binding domains within the RNAP holoenzyme to allow promoter recognition (2, 3). The importance of these conformational changes is
underlined by our recent observation that removal of the E. coli RNAP
flap domain abolished the ability of the mutant E
70 to recognize promoters of the
10/
35 class
(13).
54 recognizes and binds promoters
containing conserved consensus elements centered around
24 and
12
nucleotides upstream of the transcription start site at +1. These
promoter elements can be considered as functional analogues of the
35/
10 consensus promoter elements recognized by E
70
class holoenzymes. The activity of
54 promoters is
strictly regulated at the DNA melting step and is dependent upon the
presence of an enhancer DNA-bound activator. The maintenance of the
transcriptionally silent state of the activator-independent E
54 closed complex depends on the integrity of (i) the
amino-terminal 56 amino acids of
54 (known as Region I;
Fig. 1a) and (ii) promoter
sequences at
12 (14-17). In the closed complex, Region I localizes
close to the
12 promoter element where DNA melting originates. This
protein-DNA arrangement, which we called the "regulatory center,"
constitutes a target for mechanochemical action of the activator (18,
19). Very little is known about the contribution of the core RNAP
mobile modules to promoter binding and transcription initiation by
E
54. Here, we studied the properties of E. coli E
54 reconstituted from mutant core RNAP
harboring the
flap deletion,
885-914 (hereafter called
flapE), to gain insights into the contribution of a core
RNAP structural module, which is critical for transcription initiation
by E
70, to enhancer-dependent transcription
by E
54 (Fig. 1b). Our results demonstrate
that the
flap domain of the RNAP has multiple roles in
transcription by the E
54. Most importantly, it appears
to orchestrate the formation and organization of the regulatory center
at the start site proximal promoter element at
12.
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Fig. 1.
a, domain organization of
K. pneumoniae 54. b, domain
organization of the E. coli
subunit (top) and
structure of the T. aquaticus core RNAP (bottom).
In the E. coli
subunit, the gray shaded
regions indicate evolutionarily conserved segments
(A-I). Dispensable regions are shown in white.
Evolutionarily conserved segment G is expanded and aligned with
corresponding segments from T. aquaticus (Taq)
and yeast RNAP II (YP2). Dots and
hyphens indicate identical or missing amino acids,
respectively. The secondary structure of the
flap from
T. aquaticus is also given. The deletion mutation
(
885-914) characterized in this work is shown above the
E. coli sequence. In the T. aquaticus core
RNAP structure, the
,
',
2, and
are shown in
cyan, pink, green, and
white, respectively. The active center Mg2+ is
shown in blue. The portion of the
flap deleted in this
work is shown in yellow. c, S. meliloti nifH 88-mer homo- and heteroduplex promoter probes
used in this work. The consensus GG and GC elements of
54-dependent promoters are in
bold and their positions with respect to the transcription
start site at +1 is given. Boxed are the sequences that are
mismatched to generate the early and late melted heteroduplex
probes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
54, mutant
variants thereof (
R1
54, Ala24-26,
R336A
54, Cys20
54, and
Cys46
54), E. coli full-length
PspF and PspF-(1-275) were purified as amino-terminal
His6-tagged fusion proteins essentially as described in
Refs. 16, 18, 20, and 21.
subunit, were purified as follows. Plasmid pRL706 expressing wild-type rpoB, or the corresponding plasmid expressing mutant
rpoB with the flap deletion were transformed in the E. coli XL1-Blue cells and cells were grown in 4 liters of LB
containing 200 µg/ml ampicillin. Cells were grown to
A600 of ~2 without induction. Cells (~14 g) were collected and lysed, by sonication, in 70 ml of grinding buffer
(40 mM Tris-HCl, pH 7.9, 10 mM EDTA, 15 mM
-mercaptoethanol, and 0.2 mM
phenylmethylsulfonyl fluoride) containing 200 mM
NaCl. The supernatant after low speed centrifugation was made 0.8%
with Polymin P (Sigma), pH 8.0. The Polymin P pellet was washed twice with grinding buffer containing 500 mM NaCl, and RNAP was
eluted twice with 15 ml of grinding buffer containing 1000 mM NaCl. The combined 1000 mM extract was
precipitated with ammonium sulfate (0.3 g/ml), the pellet was
collected, dissolved in 40 ml of TG buffer (40 mM Tris-HCl,
pH 7.9, 5% glycerol), and loaded onto a 5-ml Heparin HiTrap column
(Amersham Biosciences) equilibrated in TG buffer containing 100 mM NaCl. The column was washed with TG buffer containing
300 mM NaCl, and RNAP was eluted in TG buffer containing
600 mM NaCl. The 600 mM NaCl fraction was
loaded onto a 5-ml HiTrap chelating column (Amersham Biosciences)
loaded with Ni2+ using the manufacturer's instructions and
equilibrated in a buffer containing 25 mM HEPES, pH 8.0, 500 mM NaCl, 5% glycerol. The column was step-eluted with
the same buffer containing 10, 20, 50, and 200 mM
imidazole, pH 8.0. RNAP containing plasmid-borne, hexahistidine-tagged
mutant
eluted at 50 mM imidazole in the buffer and did
not contain
70 subunit as judged by visual inspection of
Coomassie-stained SDS gels and abortive initiation assays on a strong
70-dependent promoter. RNAP was precipitated
by ammonium sulfate, the pellet was dissolved in 250 µl of TG buffer
containing 1 mM EDTA and loaded onto a Superose-6 column
(Amersham Biosciences) equilibrated in TG buffer containing 1 mM EDTA and 200 mM NaCl. The column was
developed isocratically with the same buffer, RNAP-containing fractions
were collected, concentrated on Centricon-100 (Amicon) to ~0.3 mg/ml,
glycerol was added to a final concentration of 10%, the enzyme was
aliquoted and stored at
80 °C. For native PAGE and
footprinting assays, 32P end-labeled 88-mer (from
60 to
+26) homoduplex and heteroduplex Sinorhizobium meliloti nifH
promoter probes were prepared essentially as described by Cannon
et al. (21).
54 in core
binding buffer (40 mM Tris-Cl, pH 8, 10% (v/v) glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl) at 37 °C. The reactions were incubated for 5 min and complexes were resolved on a 4.5% native polyacrylamide gel.
RNAP holoenzyme complexes were visualized and quantified by
PhosphorImager analysis of the dried gel.
54 dissociation assays, 50 nM
holoenzyme complexes (at a molar ratio of 1:6 core RNAP to unlabeled
54) were pre-formed in core binding buffer containing
200 µg/ml
-lactoalbumin in a total volume of 50 µl and incubated
for 5 min at 37 °C. After incubation, 300 nM
32P-labeled
54 was added for 0-60 min and
samples were taken and loaded on a 4.5% native polyacrylamide gel that
was run for 2 h at 50 V. PhosphorImager analysis was used to
quantify 32P-labeled
54·RNAP complexes and
the free
54 in each sample.
54-RNAP (formed with 1:6 ratio of core
RNAP to
54), 16 nM 32P
end-labeled homoduplex or heteroduplex (as indicated in figures and
legends thereof) S. meliloti nifH promoter probes, 4 mM ATP or GTP (where indicated), and 4 µM
PspF-(1-275) (where indicated) at 37 °C. When required heparin (100 µg/ml) was added for 5 min prior to analysis of the complexes by
native PAGE. Gels were dried and promoter complexes were quantified by
PhosphorImager analysis.
54 with 100 nM core RNAP at 37 °C in STA buffer for 5 min. 10 µM PspF
HTH, 0.2 mM ADP, and 5 mM sodium fluoride were added to the reaction for a further
5 min; 0.2 mM aluminum chloride was then added and the
reactions were further incubated for 10 min at 37 °C prior to
separation of complexes by native PAGE. Dried gels were visualized
using a PhosphorImager.
3 units of DNase I (Amersham
Biosciences) was added (for 1 min), reactions terminated, and bound and
unbound DNAs were separated by native PAGE. Unbound and RNAP-bound DNA
was then excised from the gels. Gel-isolated DNA was eluted into 0.1 mM EDTA overnight at 37 °C and recoveries of the
isolated DNA were determined by dry Cherenkov counting. Equal numbers
of counts were loaded onto a 10% denaturing gel. Dried gels were
visualized and quantified by using a PhosphorImager.
-mercaptoethanol to quench DNA oxidation. The reactions were phenol:chloroform:isoamyl alcohol extracted, ethanol precipitated, and stored overnight at
80 °C. The DNA was then pelleted and washed with 80% (v/v) ethanol. The dried DNA pellet was resuspended in 50 µl of TE buffer (10 mM Tris-Cl, pH 7.0, and 0.1 mM EDTA) to
which 1 µl of 0.4% (w/v) SDS and 500 µl of a 25 mg/ml stock of
proteinase K were added and incubated for 30 min at 37 °C. The
reaction was stopped by phenol:chloroform:isoamyl alcohol extraction
and the DNA was precipitated and pelleted as before. The DNA pellet was
then resuspended in 30 µl of H2O and the oxidized DNA was
cleaved with 10% (v/v) piperidine at 90 °C for 20 min. The cleavage
reaction was stopped by flash-freezing. The reaction was then dried
using a speed-vacuum drier. The recoveries of DNA was quantified and
analyzed by denaturing PAGE as described above for DNase I footprinting.
54).
Where indicated 4 mM ATP or GTP and 4 µM
PspF-(1-275) were added for open complex formation. The elongation
mixture contained 100 µg/ml heparin, 0.1 mM ATP, CTP, and
GTP, and 1.5 µCi of [
-32P]UTP. Reactions were done
at 37 °C and stopped with 4 µl of formamide dye mixture. 7 µl of
the samples were run on a 6% denaturing gel and the dried gel was
quantified and analyzed by PhosphorImager analysis. Run-off
transcription assays were performed using the late melted promoter
probe essentially as described above. However, reactions were stopped
by phenol:chloroform:isoamyl alcohol extraction and ethanol
precipitation. Recoveries of transcripts were determined by dry
Cherenkov counting and equal numbers of counts were loaded onto 15%
denaturing gels. Dried gels were visualized and quantified by using
a PhosphorImager.
54,
20
54, and 46
54
were modified with the FeBABE reagent
((p-bromoacetamidobenzyl)-EDTA Fe; Dojindo Chemicals) and
conjugation yield determined essentially as described by Wigneshweraraj et al. (9). DNA cleavage assays were performed as described by Wigneshweraraj et al. (19). Briefly, promoter complexes
were formed (in 10-µl reactions) using 20 nM S. meliloti nifH early melted promoter probe (template strand
32P end-labeled), 50 nM core RNAP, and 300 nM
54 in cleavage buffer (40 mM
HEPES, pH 8.0, 10 mM MgCl2, 5% (v/v) glycerol,
0.1 mM KCl, and 0.1 mM EDTA) at 37 °C. DNA
cleavage was initiated by the rapid sequential addition of 2 mM sodium ascorbate, pH 7.0, and 1 mM hydrogen
peroxide. Reactions were allowed to proceed for 10 min and quenched
with 50 µl of stop buffer (0.1 M thiourea and 100 µg/ml
sonicated salmon sperm DNA). The stopped reactions were
phenol:chloroform:isoamyl alcohol extracted and precipitated with
ethanol. Recoveries of DNA were determined by dry Cherenkov counting
and equal numbers of counts were loaded onto 10% denaturing gels.
Dried gels were visualized and analyzed using a PhosphorImager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Flap Forms an Unstable
54
Holoenzyme
flap
does not significantly affect the ability of mutant core RNAP to bind
70 (13). We tested the ability of
flapE
to bind
54. For this purpose, a fixed amount of
32P-labeled
54
(32P-
54) was combined with various amounts
of
flapE or control wild-type core RNAP and the mixtures
were separated on a native polyacrylamide gel (Fig.
2a, (i)) and
quantified using a PhosphorImager (Fig. 2a (ii))
(10, 20). As shown in Fig. 2a, with the wild-type RNAP most
of the 32P-
54 was in the holoenzyme form
(E
54) when the molar ratio of core RNAP to
54 reached 1:1. In contrast, a 4-fold excess of
flapE was required to convert
32P-
54 to
flapE
54. Thus, it appears that
flapE has a reduced ability to bind
54,
suggesting that the
flap contributes to the
54
binding site.
View larger version (38K):
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Fig. 2.
Binding of flap core
RNAP to
54. a:
i, native gel showing the binding of
32P-
54 to wild-type and
flap core RNAP.
The migration positions of 32P-
54 and RNAP
holoenzyme are indicated. ii, quantification of the data
from i using a PhosphorImager. Saturation curves for
flapE
54 (
) and E
54
(
) formation are shown. Indicated on the x axis is the
molar ratio of core RNAP to 32P-
54 included
in the reaction and on the y axis the percentage of RNAP
holoenzyme formation. b, stability of
flapE
54 and E
54 over time
(1-60 min; shown on the top of the gel). Lanes
1, 3, 5, 7, 9, and
11 contain the wild-type RNAP; lanes 2,
4, 6, 8, 10, and
12 contain the
flap RNAP.
To estimate the stability of the flapE
54
complex, we conducted experiments in which unlabeled
54
was mixed with core RNAP at a ratio of 6:1 to allow complete conversion
of mutant core RNAP into the holoenzyme form, and then added
32P-
54 to capture free core RNAP arising
from dissociation of the preformed holoenzyme complex. As shown in Fig.
2b, little formation of the 32P-labeled RNAP
holoenzyme was detected 1 min after the addition of
32P-
54 to the wild-type RNAP holoenzyme
(lane 1), indicating that the wild-type E
54
complex had not yet dissociated. Strikingly, ~6-fold increased formation of 32P-labeled RNAP holoenzyme complex was
observed in the case of
flapE
54 (Fig.
2b, lane 2), indicating that the mutant
holoenzyme dissociated much faster than did the wild-type
E
54. The presence of promoter DNA did not markedly
improve the stability of
flapE
54 (data
not shown). Overall, the data suggests that the
flap contributes to
the binding of
54 to RNAP core and the stability of the
resulting RNAP holoenzyme. This observation is consistent with previous
results in which a derivative of
54 harboring a FeBABE
at residue Cys198 within the major core RNAP binding
surface of
54 (residues 120-215; Fig. 1a)
cleaved the RNAP
subunit between residues 850 and 890 (9, 20),
close to or within the
flap domain (residues 885-914).
The Flap Contributes to Heparin-stable Promoter Complex
Formation by E
54
In E70,
70 region 4 (which
recognizes the
35 promoter consensus element) interacts with the
flap (2, 13). This interaction is functionally important and is
required for correct positioning of the
70 region 4 to
allow promoter complex formation on promoters of the
35/
10 class
(2, 13). To investigate the contribution of the
flap domain to
promoter binding by E
54, we analyzed promoter complexes
formed by
flapE
54 on the S. meliloti nifH promoter and its derivatives (Fig. 1c) under activating and nonactivating conditions.
Experiments with S. meliloti nifH Homoduplex Promoter
Probe--
Initially, flapE
54 binding
activity was investigated on a fully double-stranded S. meliloti
nifH promoter probe (homoduplex probe; Fig. 1c).
Complex formation was investigated under three conditions that allowed
the monitoring of different promoter complexes. Under nonactivating
conditions, when RNAP alone or with added ATP and GTP was present in
the reaction, only closed promoter complexes were expected to form.
Under activating conditions, when RNAP, E. coli
54-dependent activator PspF-(1-275) (a form
of PspF that lacks the enhancer DNA binding
domain)2 and ATP were
present, open promoter complexes were expected to form. Under
initiating conditions, when RNAP, PspF-(1-275), and GTP were present,
RNAP could use GTP to synthesize an RNA trimer.
Wild-type or mutant E54 were used to form promoter
complexes that were then resolved by native gel electrophoresis, or
were first challenged with heparin, followed by electrophoretic
separation of promoter complex from free promoter probe. Acquisition of
heparin stability by E
54 promoter complex on the
homoduplex promoter probe is a hallmark of either open or
initiated E
54 promoter complexes. The results were
quantified and are presented in Table I.
Under nonactivating conditions
flapE
54
bound the homoduplex probe with similar activity as E
54,
and the complexes were sensitive to heparin, as expected. Promoter complex formed by the wild-type E
54 became
heparin-stable under activating or initiating conditions, as expected.
In contrast, neither activation nor initiation conditions led to the
acquisition of heparin stability by
flapE
54 on the homoduplex promoter (Table
I). Because in the absence of heparin
flapE
54 and E
54 bound the
homoduplex probe equally well, we conclude that (i) the
flap
contributes to DNA opening and/or engagement of single-stranded DNA by
E
54 to acquire heparin stability, or (ii) deletion of
the
flap compromises the ability of the mutant RNAP to respond to
activation conditions.
|
Experiments with S. meliloti nifH Heteroduplex Promoter
Probes--
To distinguish between the above possibilities, we
performed binding and heparin stability assays on a
heteroduplex promoter probe. The late-melted promoter probe contains a
mismatched segment from positions 10 to
1 relative to the
transcription start point and is believed to represent the conformation
of promoter DNA in E
54 open complexes (21, 22) (Fig.
1c). E
54 efficiently forms heparin-stable
promoter complexes on the late-melted promoter probe only under
activating conditions. Initial binding assays with
flapE
54 in the absence of heparin showed
that
flapE
54 bound the late-melted probe
with wild-type activity under all the conditions tested (Table I). Like
E
54,
flapE
54 was unable to
form heparin-stable complexes in the absence of activation (Table I).
Activation (+PspF-(1-275) and ATP) increased the heparin stability of
wild-type E
54. In contrast, activated
flapE
54 late melted promoter DNA
complexes were heparin-sensitive (Table I). However, under activating
conditions that permitted initiation (+PspF-(1-275) and GTP) a
significant number of heparin-stable
flapE
54 promoter complexes was detected
(Table I), suggesting that (i)
flapE
54
was able to respond to activation when the template strand was available as single-stranded DNA, and (ii) initiation of RNA synthesis stabilized
flapE
54 on the late melted
promoter probe.
Acquisition of heparin stability on late melted promoter probes
independent of activation is a property of deregulated forms of
E54, for example, those lacking the
54
regulatory Region I (
RI
54) (21) (Fig. 1a).
To further assess the ability of
flapE
54
to engage pre-melted promoter DNA in a heparin-stable manner we
conducted experiments with double mutant holoenzyme lacking the
flap and
54 regulatory Region I. Initially, we tested
whether
RI
54 can form holoenzyme complexes with
flapE. The results showed that a 1:8 ratio of
flapE to
RI
54 was needed to form
stable
flapE·
RI
54 complexes,
consistent with the
54 binding defects of
flapE (data not shown and Fig. 2). In the absence of
heparin,
flapE·
RI
54 bound the late
melted promoter probe with an activity similar to that of
E
·RI
54 (data not shown). Strikingly and in marked
contrast to E
·RI
54 complexes, very little (<2%)
of the
flapE·
RI
54 late melted
promoter complexes survived heparin challenge (Fig. 3, compare lane 2 with
5). However, conditions that permitted activator-independent
initiation by E
·RI
54 (i.e. the presence
of GTP) detectably stabilized
flapE·
RI
54-late melted promoter
complexes (Fig. 3, compare lane 5 with 6). In
contrast, the presence of noninitiating nucleotides, ATP or dGTP, did
not lead to heparin resistance of
flapE·
RI
54 promoter complexes (data
not shown). We note that increased heparin resistance of
flapE·
RI
54 promoter complexes was
not dependent on the presence of activator (Fig. 3, compare lanes
6 and 7), suggesting that the deregulated properties of
RI
54 have not been altered in the absence of the
flap. Similar binding patterns were obtained with
flapE
in the context of other deregulated mutants of
54, such
as F318A (23), R336A (24), and Ala24-26
54 (16), further
confirming that initiation of RNA synthesis stabilizes late melted
promoter complexes in the context of
flap RNAP (data not shown).
|
Overall, several conclusions can be derived from the binding properties
of flapE
54 on the late melted promoter
probe. First, DNA interactions with melted DNA made by
E
54 are destabilized in the absence of the
flap.
Second,
flapE
54 closed complexes respond
to activator and engage pre-melted DNA to form a heparin-stable
complex, albeit less efficiently than the wild-type E
54.
This result suggests that the
flap is not essential to the RNAP
site that interacts with the activator. Third, the deletion of the
flap does not deregulate E
54, at least in the context of
the late melted promoter probe complex.
DNase I Footprinting Reveals Altered Interactions of
flapE
54 with the S. meliloti nifH
Promoter
To further characterize the marked differences in promoter complex
stability caused by deletion of the flap we conducted DNase I
footprinting experiments. Promoter complexes on homoduplex DNA were
formed in the absence of heparin because of the instability of mutant
open and initiated complexes (Table I), footprinted, and complexes were
separated from free DNA by native PAGE. Both E
54 and
flapE
54 formed equal amounts of promoter
complexes under the conditions tested (data not shown). Promoter DNA
from promoter complexes was recovered and analyzed by denaturing PAGE
(see "Experimental Procedures"). The DNase I footprint of the
E
54 on the S. meliloti nifH homoduplex probe
was typical of E
54 closed promoter complexes. Promoter
DNA was protected from DNase I cleavage between positions
34 and
5
with respect to the transcription start site at +1 (Fig.
4a
(i), lane 3). The
flapE
54 closed promoter complex footprint
was similar to that of wild-type E
54 between positions
34 and
4 (Fig. 4a (i), lanes 3 and
4, and (ii)). PhosphorImager analysis of the
wild-type and mutant closed promoter complex footprints revealed that
several sites on the DNA outside of the closed complex-specific
protection region (
34 to
4) were also protected from DNase I
cleavage in the mutant closed promoter complexes (Fig. 4a
(ii), indicated by arrows). This result is
suggestive of an overall inhibition of DNase I cleavage, perhaps
because of (i) altered conformation of the
flapE
54 closed promoter complex or (ii)
nonspecific binding of the
flapE
54. In
promoter complexes formed with the wild-type E
54, open
promoter complex formation or the initiation of RNA synthesis results
in the extension of the DNase I footprint in the downstream direction
(Fig. 4a (i), compare lane 3 with
lanes 6 and 8, respectively). No further
increased protection of downstream DNA was detected in open complexes
formed with the
flapE
54 (Fig.
4a (i), compare lanes 4 and
5). Strikingly, conditions that allowed initiation from
flapE
54 promoter complexes resulted in
increased DNase I cleavage between
34 and
5 and shortening of the
footprint in the upstream direction (Fig. 4a (i),
lane 7). Analysis of mutant-initiated complexes by native
PAGE revealed that the
flapE
54 formed an
equal number of initiated promoter complexes as E
54,
suggesting that the increased cleavage of DNA between the
34 and
5
positions was not because of simple dissociation of promoter complexes
(data not shown and Table I). Overall, the DNase I footprinting data
suggests that
flapE
54 promoter complexes
differ significantly from promoter complexes formed by
E
54. The failure to observe a fully extended downstream
footprint in mutant complexes under activating conditions, as well as
the apparent hypersensitivity of mutant initiated complexes to DNase I
possibly indicates an altered response of the
flapE
54 closed complexes to activation
and altered conformations of
flapE
54
compared E
54 under closed complex conditions.
|
flapE
54 Is Defective for Promoter DNA
Melting
We used DNA melting within open or initiated promoter complexes as
an indicator of activator responsiveness and analyzed promoter complexes formed by flapE
54 and the
wild-type E
54 by KMnO4 probing. Reactions
were set up essentially as described above for DNase I footprinting. As
shown in Fig. 4b, in the absence of activation, DNA melting
was barely detectable within closed promoter complexes formed by the
flapE
54. However, very little deregulated
DNA melting was observed within the wild-type closed promoter complex.
Activation of E
54 promoter complexes resulted in
increased KMnO4 reactivity of template strand thymines at
positions
10 and
8 indicating promoter opening in response to
activation (Fig. 4b, (ii)). In contrast, the
KMnO4 reactivity of the
10 and
8 thymines was greatly
reduced within activated mutant promoter complexes. Under initiating
conditions, a 2-fold further increase in KMnO4 reactivity
of thymine at position
8 was seen in wild-type promoter complexes,
consistent with the expectation that initiation stabilizes DNA opening
(Fig. 4b, compare (ii) and (iii)). In
contrast, no significant increase in KMnO4 reactivity was
detected in
flapE
54-initiated promoter
complexes. Overall, KMnO4 probing data are consistent with
DNase I footprinting results and strongly suggests that
flapE
54 promoter complexes are defective
for (i) DNA melting and/or (ii) activator responsiveness.
flapE
54 Forms a Stable Complex with
the
54 Activator PspF-(1-275)
To determine whether the absence of the flap affects the
ability of E
54 to form a stable binary complex with
54 activator PspF-(1-275), we conducted binding assays
in the presence of ADP-AlFx, a nonhydrolysable analogue of ATP
that allows stable activator-E
54 complexes to form (18).
E
54 or
flapE
54 were
combined with PspF-(1-275) in the presence of ADP-AlFx and
protein complexes were analyzed by native PAGE. Fig.
5 shows that both holoenzymes formed
similar amounts of stable, ADP-AlFx-dependent complexes with PspF-(1-275) (Fig. 5, compare lane 3 with
6). Similar results were obtained in the presence of
promoter DNA (data not shown). Thus, it appears that the greatly
reduced DNA melting in
flapE
54 promoter
complexes formed under activating or initiating conditions is not
because of a major defect in activator interaction. Furthermore, these
activator binding assays also suggest that the
flap is not part of
the putative
subunit activator interaction site previously detected
by chemical cross-linking (25).
|
flapE
54 Is Defective for
Activator-dependent in Vitro Transcription from Supercoiled
Templates
To assess the consequence of 54-binding and
promoter DNA-interaction defects of
flapE on
transcription activity, we conducted single-round in vitro transcription assays using the supercoiled plasmid pMKC28, which contains the S. meliloti nifH promoter (24). When ATP and
PspF-(1-275) were used to form open complexes prior to the addition of
heparin, [
-32P]UTP, and the remaining nucleotides,
flapE
54 was only about 15% as active as
the wild-type E
54 for transcription (Fig.
6, compare lanes 1 and
2). This result is very consistent with the results of the
late melted promoter complex stability assay where only about 14% of
the initial
flapE
54 promoter complexes
survived heparin challenge (Table I). When GTP and PspF-(1-275) were
used to form initiated promoter complexes prior to the addition of
heparin and remaining nucleotides,
flapE
54 showed less than 5% of the
wild-type E
54 activity, indicating a destabilization of
complexes under initiating conditions. This result is striking as the
presence of GTP-stabilized
flapE
54 and
flapE
R1
54 complexes on late melted
promoter (Table I and Fig. 3, respectively). However, the result is
consistent with the DNase I and KMnO4 footprinting data,
where initiation resulted in an altered DNase I footprint for
flapE
54 (Fig. 4a
(i), lane 7) and greatly reduced DNA melting was
observed in mutant complexes at initiating conditions (Fig.
4b, (iii)). Additional in vitro
transcription assays using supercoiled pSLE1 plasmid, which harbors the
pspA promoter and the DNA binding site for PspF, and
full-length PspF (26), gave transcription patterns similar to those
obtained with PspF-(1-275) (Fig. 6b, compare lanes
1 and 2 with 3 and 4), suggesting
that the presence of enhancer-bound activator did not detectably affect
transcription activity of the
flapE
54.
Overall, the in vitro transcription data from supercoiled
templates show that
flapE
54 is
transcriptionally active, but to a lesser degree than
E
54. A likely explanation for this could be the weakened
54 and promoter DNA interactions made by
E
54 in the absence of the
flap.
|
flapE
54 Is Active for
Activator-dependent in Vitro Transcription from
Heteroduplex Promoter Probes
To investigate whether by-passing the DNA melting step would
permit efficient transcription by
flapE
54, in vitro
transcription from the S. meliloti nifH late melted promoter
probe was performed. After closed promoter complex formation, PspF-(1-275) and either ATP or GTP were added to induce open or initiated promoter complex formation, respectively. Next, a mixture of
heparin, [
-32P]UTP, and remaining nucleotides was
added to allow a single round of transcription to occur. When ATP and
PspF-(1-275) were used for activation, the
flapE
54 was 40% as active as the
wild-type E
54 (Fig. 6c, compare lanes
3 and 4). This run-off transcript was of the expected
size (~28 bp) and its production was
54 and
activator-dependent (Fig. 6c, lanes
1, 2 and 5, 6, respectively). In
the presence of GTP and PspF-(1-275), transcription by the
flapE
54 was poor, resulting in ~12% of
wild-type activity.
flapE
54 was inactive
on the S. meliloti nifH homoduplex promoter probe (data not
shown). Thus, it appears that some of the in vitro
transcription defects of the E
54 associated with the
flap mutation can be overcome by using pre-melted promoter
templates. Overall, the single-round and run-off transcription data,
together with the KMnO4 probing of mutant promoter
complexes, strongly implies that the
flapE
54 is defective for stable DNA
melting in response to activation.
flapE
54 Is Inactive for in Vitro
Transcription with Deregulated
54 Mutants
In deregulated forms of E54 DNA opening occurs in
the absence of activator, but the resultant open complexes are unstable
unless initiated. As a result, transcription by deregulated
E
54 proceeds via an unstable open complex that was
proposed to correspond to an intermediate promoter complex prior to
fully formed stable open complex formation (27). Therefore,
transcription by deregulated E
54 measures the formation
of unstable intermediate open complexes. We performed
activator-independent transcription assays with three different
deregulated mutants of
54 (
RI
54,
Ala24-26
54 and R336A
54 in the context of the
flap RNAP holoenzyme. The activator-independent transcription assay
was performed using the S. meliloti nifH promoter in the
pMKC28 plasmid (24). To allow activator-independent initiation, RNAP
holoenzymes, template DNA, and GTP were preincubated, followed by the
addition of heparin to destroy residual unstable complexes. [
-32P]UTP and remaining nucleotides were next added to
initiate a round of transcription. As expected, RNAP holoenzymes
reconstituted from wild-type core RNAP and either of the three
deregulated
54 mutants were active in both
activator-dependent (Fig. 7,
lanes 2-4) and activator-independent (Fig. 7, lanes
6-8) transcription. Strikingly, the double mutant holoenzymes
reconstituted from
flapE and either of the deregulated
54 mutants were transcriptionally inactive either in the
presence (Fig. 7, lanes 10-12) or absence (Fig. 7,
lanes 14-16) of activator. This result contrasts the late
melted promoter probe stability data in which the deregulated forms of
flapE
54 formed heparin-stable complexes
in the presence of GTP independent of activation (Fig. 3 and data not
shown). We therefore considered a possibility that deregulated forms of
flapE
54 were defective for promoter DNA
binding. However, DNase I footprints of closed complexes formed with
deregulated forms of
flapE
54 indicated
that this was not the case (data not shown). Overall, it appears that
removal of the
flap while only partially affecting the ability of
wild-type
54 RNAP holoenzyme to transcribe from
supercoiled templates, completely abolishes transcription by RNAP
holoenzymes reconstituted with deregulated forms of
54.
This suggests that the
flap is required for unstable open promoter
complex formation and/or that the
flap is needed for transcription
initiation from the unstable promoter complexes.
|
flapE
54 Makes Altered DNA
Interactions within Closed Promoter Complexes
The apparently contradicting properties exhibited by deregulated
forms of flapE
54 in the late melted
promoter probe binding experiments and transcription assays prompted us
to investigate DNA interactions made by
flapE
54 early during open promoter
complex formation. The transcriptionally silent state of the
E
54 closed promoter complexes strictly depends on the
integrity of promoter sequences immediately downstream of the
12
promoter consensus element (the GC-element; Fig. 1c). In
wild-type E
54 closed promoter complexes, the base pair
immediately downstream of the GC element shows increased reactivity
toward KMnO4, diethylpyrocarbonate, and
ortho-CuOP and therefore appears to be melted (28). This limited DNA opening within closed promoter complexes is termed "early
DNA melting" and is one hallmark of regulated E
54
transcription. In stable activator-dependent,
transcriptionally competent open complexes, early DNA melting is not
evident, suggesting that early DNA melting is a transient feature en
route to regulated transcription by E
54 (28). To
investigate early DNA melting within closed promoter complexes formed
by
flapE
54, ortho-CuOP
footprinting was performed. As expected, DNA cleavage by
ortho-CuOP was seen at position
12 (immediately downstream the GC-element; Fig. 1c) in closed complexes formed with the
wild-type E
54 on the S. meliloti nifH
homoduplex promoter probe (Fig.
8a, lane 3).
Activation of the wild-type closed complexes resulted in ~5-fold reduction of the
12 signal, indicating open complex formation (Fig.
8a, compare lanes 3 and 5). In
contrast, cleavage at
12 was absent in closed complexes formed by
flapE
54 (Fig. 8a, lane
4), suggesting that the mutant enzyme is either defective for
early DNA melting and/or is unable to make stable interactions with
early melted DNA. To test the latter possibility, we conducted
ortho-CuOP footprinting assays on promoter complexes formed
on a heteroduplex promoter probe in which 2 base pairs immediately
downstream of the GC element were mismatched, thus mimicking early
promoter melting (Fig. 1c, early melted promoter probe).
Ortho-CuOP treatment of early melted DNA in the absence of
RNAP revealed a hypersensitive site around the mismatched region, as
expected (Fig. 8b, compare lanes 1 and
2), and E
54 protected the mismatched DNA
region from ortho-CuOP cleavage, indicating the binding of
E
54 to the promoter probe (Fig. 8b, compare
lanes 1 and 3). A clear protection of the
hypersensitive site at
12 was also seen in reactions containing
flapE
54 indicating complex formation by
flapE
54 on the early melted promoter
probe (Fig. 8b, lane 4). DNase I footprinting
experiments also showed protection between DNA positions
34 to
5 in
the presence of both E
54 and
flapE
54 (Fig. 8c, compare
lanes 3 and 4), thus confirming efficient complex formation on early melted promoter probe by both enzymes.
|
To gain further insights into flapE
54
interaction with early melted DNA, we tested heparin stability of
nifH promoter early melted complexes. As summarized in Table
I,
flapE
54 bound the early melted
promoter probe as efficiently as wild-type E
54 under all
conditions tested. As reported previously (29, 30), and in contrast to
the situation on homoduplex promoter probes, conditions that permitted
activation (+PspF-(1-275) and ATP) or initiation (+PspF-(1-275) and
GTP) did not markedly increase the heparin stability of wild-type
E
54 complexes on the early melted promoter probe (Table
I). This property of wild-type E
54 complexes is
attributed to tight binding of
54 to the heteroduplex
region of the early melted promoter probe (15, 29). Significantly, the
flapE
54-early melted promoter probe
complex was unstable and did not survive heparin challenge under any of
the conditions tested (Table I), suggesting that
E
54-early melted DNA interactions are altered in the
absence of the
flap and do not lead to stable promoter complex
formation. Alternatively, it is also possible that the mutant
holoenzyme dissociates to core RNAP and free
in the presence of
heparin. However, band shift analysis of holoenzymes on native gels
similar to the ones shown in Fig. 2 showed that
flapE
54 was as sensitive to disruption by
heparin as the wild-type E
54 (data not shown). Thus, the
failure of the
flapE
54 to form
heparin-stable complexes on the early melted promoter probe seems to be
because of a failure of the
flap core RNAP to bring about a
DNA-dependent change(s) in the core RNAP-
54
interaction that results in the formation of heparin-stable promoter complexes. In other words, we suggest that
flapE
54 is not defective in the binding
to the early melted DNA structure, but is defective in the formation
and maintenance of the early melted structure.
Organization of the Regulatory Center within
E54 Closed Complexes Is Dependent on the
Flap
Regulated transcription by E54 relies on the
integrity of
54 Region I (Fig. 1a), which
localizes proximal to promoter DNA around the GC promoter element
(14-16, 19, 31-34) (Fig. 1c). Within the RNAP holoenzyme,
54 Region I localizes proximal to the
and
'
subunit residues that contribute to the formation of the RNAP active
center (9). Region I is directly contacted by activators of
E
54 transcription (18). We have termed the
54 Region I-DNA-core RNAP arrangement within closed
E
54 promoter complexes the regulatory center
(19). Experiments presented above suggest that some of the properties
of
flapE
54 could be accounted for by
defects in the organization of the regulatory center. To investigate
whether the
flap contributes to proper positioning of
54 Region I and hence to the organization of the
regulatory center, we conducted localized hydroxyl radical cleavage
experiments using FeBABE derivatives of
54. RNAP
holoenzymes were reconstituted with the wild-type and mutant RNAP core
enzymes and
54 containing FeBABE conjugated to
Cys20 and Cys46
(20*
54 and
46*
54)3
in Region I4 and were used to
form complexes on early melted promoter probes. Preliminary experiments
demonstrated that the mutant and the wild-type holoenzymes
reconstituted with 20*
54 and
46*
54 formed promoter complexes on the early
melted promoter probe as efficiently as holoenzymes reconstituted with
unmodified
54 (data not shown). After complex formation,
hydroxyl radicals were generated and the pattern of cleavage within the
early melted promoter probe is presented in Fig.
9. All resulting cleavage of promoter DNA
was dependent on the FeBABE reagent being coupled to unique cysteine
residues in Region I because no cleavage was observed in control
complexes formed with RNAP holoenzymes reconstituted with cysteine-free
54 (Cys-free
54) that was
subjected to FeBABE conjugation conditions as a control (Fig. 9,
lanes 1, 2, 7, and 8). In
contrast, strong cleavage of promoter DNA template strands between
positions
11 and
16 was seen within promoter complexes formed by
E20*
54 (Fig. 9, lanes 3 and
4), suggesting that
54 Region I residue 20 is
proximal to the regulatory center. Strikingly, no DNA cleavage was
detected in complexes formed by
flapE20*
54 (Fig. 9, compare
lanes 3 and 4 with 9 and
10), suggesting that in the absence of the
flap, residue
20 in
54 Region I is unable to occupy its correct
position in the regulatory center. In E46*
54
complexes, cleavages in the template strand of promoter DNA between positions
6 and
2 were obtained (Fig. 9, compare lanes 5 and 6), indicating that residue 46 in
54
Region I is localized outside of the regulatory center. Interestingly,
flapE46*
54 complexes
also cleaved promoter DNA at these positions (Fig. 9,
compare lanes 5 and 6 with 11 and
12), suggesting that the positioning of
54
Region I residues that do not contribute to the regulatory center is
not affected by the
flap mutation, at least in the context of this
assay. Thus, this suggests that the
flap directly and selectively
contributes to the organization of the E
54 regulatory
center.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcription from enhancer-dependent promoters relies
upon the RNAP containing the 54 factor. Unlike the
enhancer-independent promoters recognized by the
70
containing RNAP, the conversion of closed
enhancer-dependent promoter complexes to
transcription-competent open complexes requires the mechanochemical
action of ATPases belonging to the AAA+ protein family (4, 35). The
contribution of the RNAP mobile structural modules (
flap,
'
clamp,
downstream and upstream lobes) to enhancer-dependent and enhancer-independent transcription
by E
54 and E
70, respectively, is not well
understood at a molecular level. By using a mutant RNAP harboring a
deletion of the
flap (
885-914;
flap), we have investigated
the contribution of this RNAP structural module to E
54
functioning. The data establish that the
flap domain has multiple roles in enhancer-dependent transcription. Strikingly, the
flap domain appears to have different functionalities in
enhancer-dependent and enhancer-independent transcription.
54 Binding--
Even though the
enhancer-dependent
54 and the
enhancer-independent
70 bind the common RNAP with
similar affinities and occupy similar positions within the RNAP
holoenzyme (7, 9, 36), the
flap mutation markedly reduces the
activity of the RNAP to bind
54 and the stability of the
resulting holoenzyme. In contrast,
70 binding and the
stability of E
70 were not greatly affected by the
flap mutation (13). Holoenzyme formation is accompanied by
large-scale conformational changes of all the structural modules of the
and
' subunits that serve to orientate and position the
factor for promoter-specific transcription initiation. Upon binding
70, the
flap re-positions region 4 of
70 to facilitate recognition of the
35 promoter
element (2, 3, 13). The contribution of the
flap to
E
54 formation appears to be 2-fold. First, the
instability of the
flapE
54 complexes
suggests that the
flap contributes to the anchoring of
54 to the core RNAP. Second, differences in the DNA
cleavage patterns by FeBABE-modified
54 in the context
of the wild-type and
flap RNAP imply a role for the
flap in the
proper positioning of
54 domains within the holoenzyme
that is important for E
54 functioning.
Promoter Complex Formation--
Promoter complex formation by
E70 induces major conformational changes in two of the
mobile modules of the RNAP, notably also including the
flap domain
(3). Consequently, E
70 with the
flap mutation is
unable to efficiently bind and utilize the
35/
10 class of bacterial
promoters (13). In the context of the E
54, the
flap
mutation has little effect on initial promoter complex formation as
judged by gel shift and DNase I footprinting assays. In contrast to
E
54,
flapE
54 promoter
complexes formed under activating conditions were sensitive to heparin
and KMnO4 probing showed greatly reduced DNA opening within
such mutant promoter complexes. Consequently, in vitro transcription activity of the
flapE
54
from supercoiled templates is severely affected. By-passing the DNA
melting step by using heteroduplex promoter probes marginally improves
the transcription activity of the
flapE
54. In contrast, DNA opening was
fully normal in promoter complexes formed with the
flapE
70, but mutant promoter complexes
were less stable than the wild-type ones to heparin
challenge.5
The isomerization of closed promoter complexes to open complexes
proceeds via several heparin-sensitive intermediates involving several
protein and DNA conformational changes and alterations in the
interfaces between the RNAP and DNA (37). It is proposed that the entry
of DNA into the active-cleft of the RNAP triggers a slow protein
conformational change that nucleates DNA melting and the formation of
the heparin-resistant open promoter complex (38). We propose that the
low levels of open complex formation by the
flapE
54 proceed via an altered pathway
that neither results in efficient DNA melting nor acquisition of
heparin stability. The limitation of the
flapE
54 to only function with
the wild-type
54 for transcription, but not with
deregulated
54 mutants that transcribe via unstable and
heparin-sensitive open complexes, supports this view. Thus, we propose
that the
flap contributes to the formation of an intermediate,
heparin-sensitive promoter complex state or states en route to heparin
stable E
54 open complex formation.
Acquisition of Heparin Stability--
On the late melted promoter
probe, activated E54 promoter complexes are resistant to
heparin, suggesting that the E
54 must undergo
conformational changes in response to activation to acquire stability
(21, 22). The
flapE
54 does not
efficiently form heparin-stable complexes on the late-melted promoter
under activating conditions, suggesting that, in the absence of the
flap, the E
54 does not undergo the
same set of conformational changes that leads to heparin stability.
Furthermore, unlike the wild-type E
54, the
flapE
54 also forms heparin-sensitive
complexes on the early melted promoter probe under nonactivating
conditions. Thus, we propose that the
flap makes a significant
contribution to the acquisition of heparin stability by the
E
54. Previously, we have reported that either the
absence of
54 Region I or mutations affecting the
subunit downstream lobe functionality reduces the ability of the
E
54 to form heparin-stable promoter complexes (10, 29).
We envisage a functional co-operation between Region I of
54 and two mobile structural modules, the
flap and
the
downstream lobe, which leads to heparin stable-promoter complex
formation during enhancer-dependent transcription.
Organization of the E54 Regulatory Center--
In
enhancer-dependent closed complexes formed by the
E
54, Region I of
54, and the catalytic
center of the RNAP co-localize over the
12 position (GC-element) of
the promoter where DNA melting originates. This nucleoprotein complex
is defined as the E
54 regulatory center and constitutes
the target for the activator protein (9, 18, 19). Comparison of FeBABE
cleavage profiles of closed promoter complexes formed with the
wild-type and
flap RNAP clearly indicates that residues in Region I
of
54 that contribute to the regulatory center are not
proximal to the regulatory center in the absence of the
flap
domain. This suggests that the
flap may directly contribute to the
formation of the regulatory center by positioning Region I over the
GC-element. Furthermore, the FeBABE data also serves to help explain
the absence of the Region I-dependent DNA distortion within
closed complexes formed with the
flapE
54
in ortho-CuOP footprinting experiments.
Overview--
Comparison of multisubunit RNAP structures of
bacterial and yeast RNAPs and promoter complexes thereof reveals
conserved structural mobile modules and conformational changes that are
important for the functioning of the RNAP as a complex molecular
machine (39). Factors that target these mobile modules act to modulate
and regulate the activity of RNAPs. In bacteria, factors make
extensive interactions with the structural mobile modules (
' clamp,
flap, and
downstream and upstream lobes) of the bacterial RNAP
during the process of transcription initiation (1, 2, 40). Unlike the
downstream lobe module, which is commonly used by
E
54 and E
70 during open complex formation
(8, 10), the
flap module makes markedly different contributions to
enhancer-dependent and -independent transcription by
E
54 and E
70, respectively. For
E
70, the
flap plays a crucial role in the proper
positioning of
70 region 4 within the holoenzyme for
promoter-specific transcription initiation (13). Thus, it appears that
for enhancer-independent transcription by E
70, the
flap contributes to RNAP-promoter DNA interactions at the start site
distal promoter element. For E
54, the
flap has multiple roles in modulating E
54 activity and
most important of these appears to be the positioning of Region I at
the start site proximal promoter element. It remains to be
determined whether it is
54 Region I or other parts of
54 that interact with the
flap en route to
enhancer-dependent transcription initiation. However, the
data presented here highlights the fact that the extensive interface
between
factor and RNAP is crucial for the co-ordinated
reconfiguration of both partners for efficient transcription initiation.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Wendy Cannon for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a Biotechnology and Biological Sciences Research Council project grant (to M. B.) and National Institutes of Health RO1 Grant GM64530 (to K. S.).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.
§ To whom correspondence may be addressed. Tel.: 732-445-6095; Fax: 732-445-573; E-mail: severik@waksman.rutgers.edu.
¶ To whom correspondence may be addressed. Tel.: 44-207-594-5442; Fax: 44-207-594-5419; E-mail: m.buck@ic.ac.uk.
On leave from Limnological Institute of the Russian Academy of
Sciences, Irkutsk, Russia.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M209442200
2 E. coli PspF-(1-275) lacks the helix-turn-helix enhancer DNA binding domain and essentially represents the central catalytic domain of the protein.
3 Superscript asterisk indicates conjugation to FeBABE.
4 S. R. Wigneshweraraj, P. Burrows, K. Severinov, A. Ishihama, and M. Buck, manuscript in preparation.
5 K. Severinov and K. Kuznedelov, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RNAP, RNA polymerase; CuOP, ortho-copper phenanthroline.
![]() |
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