From the Department of Biology, Imperial College of
Science, Technology and Medicine, London SW7 2AZ, United Kingdom
and the § Departmento de Bioquímica,
Biología Molecular y Celular de Plantas, Estación
Experimental del Zaidín, Consejo Superior de Investigaciones
Científicas, Profesor Albareda 1, 18008 Granada, Spain
Received for publication, August 25, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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The Accessing the information in DNA often relies upon the action of
DNA-binding proteins that are able to generate non-canonical B-DNA
structures. Recombination, replication, methylation, repair, and
transcription are processes that proceed through intermediates in which
DNA is distorted. The process of RNA transcript formation by all RNA
polymerases must involve a DNA-melting event to reveal the template DNA
strand (1-3). Distortion of the DNA leading to the nucleation of
strand separation occurs within the closed complex formed between RNA
polymerases and the promoter. Following isomerization of the
polymerase, full stable DNA opening is evident, which is thought to
have spread from the initial nucleation site. Single-stranded
DNA-binding activities in the RNA polymerase are required for DNA
opening, and in the case of the bacterial RNA polymerase, the The closed complexes formed with the Here we use DNA footprinting to show that the DNA within the isomerized
DNA and Proteins--
The promoter fragments used in this work
were Escherichia coli glnHp2 from
The Klebsiella pneumoniae DNA Binding Assays--
End-labeled DNA (16-100 nM)
and 1 µM DNA Footprints--
Binding reactions were conducted as
described above; footprinting reagents were added; reactions were
terminated; and bound and unbound DNAs were separated on native gels as
described above. DNA was then excised, processed, and analyzed on a
denaturing 10% polyacrylamide gel. For DNase I footprints, 1.75 × 10 Previously, we showed that purified 54 subunit of the
bacterial RNA polymerase requires the action of specialized
enhancer-binding activators to initiate transcription. Here we show
that
54 is able to melt promoter DNA when it is bound to
a DNA structure representing the initial nucleation of DNA opening
found in closed complexes. Melting occurs in response to activator in a
nucleotide-hydrolyzing reaction and appears to spread downstream from
the nucleation point toward the transcription start site. We show that
54 contains some weak determinants for DNA melting that
are masked by the Region I sequences and some strong ones that require
Region I. It seems that
54 binds to DNA in a
self-inhibited state, and one function of the activator is therefore to
promote a conformational change in
54 to reveal its
DNA-melting activity. Results with the holoenzyme bound to early melted
DNA suggest an ordered series of events in which changes in core to
54 interactions and
54-DNA interactions
occur in response to activator to allow
54 isomerization
and the holoenzyme to progress from the closed complex to the open complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit plays an important role (1, 3-7). For the
70-type factor, binding to the core enzyme induces
conformational changes in a single-stranded DNA-binding region of the
protein. As a consequence of these conformational changes,
70 gains specificity for the non-template strand of the
melted region in the open complex (8, 9). For the
54-type factor, unrelated by sequence to
70, the determinants of single-stranded DNA binding are
less well described. Single-stranded DNA binding by
54
is evident, however (4, 10, 11). The sequences that
54
recognizes as single-stranded DNA are between the
12 promoter element
and the start site (4). Importantly, the activity of the
54 holoenzyme is tightly regulated at the DNA-melting
step, but promoter binding to form the initial
54
holoenzyme closed complex is not highly regulated (12).
54 holoenzyme are
silent for transcription unless acted upon by an enhancer-binding
activator protein (13-15). A network of protein and DNA interactions
involving
54 function to maintain a stable holoenzyme
conformation that rarely changes spontaneously to allow DNA melting and
transcript initiation (11, 16-22). The conformationally restricted
closed promoter complex isomerizes to an open promoter complex (in
which the DNA strands are melted out) in a reaction in which the
activator consumes ATP or another nucleoside triphosphate (14). As a
part of this reaction pathway,
54 contributes to the
creation of a local structural distortion within the closed complex
(23).
54 binds tightly to the distorted promoter DNA and
can be shown to isomerize independently of the core RNA polymerase in a
reaction that has all the remaining requirements for open complex
formation (4, 24). Isomerization is associated with an increased DNase I footprint of
54 on DNA, extending toward the
transcription start site (24).
54-DNA complex has melted and that some melting is
negatively regulated by Region I of
54. However,
extensive melting requires Region I. Additionally, changed interactions
between
54 and the nucleated DNA are evident in
complexes in which melting has occurred. We show that the presence of
core RNA polymerase inhibits those changes in
54-DNA
interactions that occur in response to activator, consistent with the
view that tight binding to the early melted DNA limits DNA opening (4).
The results provide clear evidence in favor of an activation mechanism
in which conformational changes in a basal
54-DNA
complex are brought about by the enhancer-binding activator. Activator-independent melting suggests that the activator does not
function exclusively as a site-specific DNA helicase for DNA opening (11, 18, 22).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60 to +28 in which T at
13 was replaced by G and the
11/
10 sequence was replaced with a
CA/TG mismatch to create a short unpaired DNA element next to a
consensus GC (
13/
12) promoter element or a mismatched sequence
between
11 and
6 (see Fig. 1). Sinorhizobium meliloti
nifH promoter fragments from
60 to +28 either containing a CA/TG
mismatch immediately adjacent to the consensus GC element or with the
A-12 top strand base missing (gapped duplex) were also used (see Fig.
1) (24). Synthetic DNA strands from
60 to +28 were annealed to create
the duplex, with either strand 5'-32P-end-labeled. The
unlabeled strand was at 2-fold molar excess.
54 protein, its
Region I-deleted derivative lacking the first 56 amino acids
(
I
54), and Region I (amino acids 1-56) were prepared
as described previously (11, 25). The activator was E. coli
PspF lacking a functional C-terminal DNA-binding domain (PspF
HTH)
(26). E. coli core RNA polymerase was from Epicentre
Technologies Corp.
54 or
I
54 in a
10-µl reaction in buffer containing 25 mM Tris acetate
(pH 8.0), 8 mM magnesium acetate, 10 mM KCl, 1 mM dithiothreitol, and 3.5% (w/v) polyethylene glycol 8000 were incubated for 5 min at 30 °C. Activator PspF
HTH (0.5-4
µM) and dGTP, GTP, or
GTP
S1 (4 mM)
were added for a further 10 min. Region I was at 0.5 µM. Where indicated, heparin (100 µg/ml) was added for 5 min prior to gel
loading. Free DNA was separated from
-bound DNA on 4.5% native
polyacrylamide gels run in 25 mM Tris and 200 mM glycine at room temperature.
3 units of enzyme (Amersham Pharmacia
Biotech) was added to a 10-µl binding reaction for 1 min, followed by
addition of 10 mM EDTA to stop cutting. For
KMnO4 footprinting, 4 mM fresh
KMnO4 was added for 30 s, followed by 50 mM
-mercaptoethanol to quench DNA oxidation.
Gel-isolated DNA was eluted into 0.1 mM EDTA (pH 8.0)
(DNase I footprints) or H2O (KMnO4 footprints)
overnight at 37 °C. KMnO4-oxidized DNA was cleaved with
10% (v/v) piperidine at 90 °C for 20 min. Recoveries of isolated
DNA were determined by dry Cerenkov counting, and equal numbers of
counts were loaded onto gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
54 bound to the
S. meliloti nifH promoter was able to isomerize if the DNA
template had an unpaired sequence downstream of the GC element of the
promoter (24). The isomerization also required activator and nucleoside triphosphate hydrolysis and was characterized as an extended
54-DNA interaction toward the transcription start site.
The unpaired DNA downstream of the GC promoter element was suggested to
mimic the nucleation of DNA melting (early melted DNA) seen in
54 closed complexes, which normally requires
54 and core RNA polymerase (23). Here we have used
variants of the
54-dependent E. coli
glnHp2 promoter (27) (Fig. 1) to
explore DNA melting by
54. We chose to generate a
nucleated glnHp2 promoter because the high AT content of the
sequence should facilitate the detection of unstacked T residues using
KMnO4 as a DNA footprinting reagent (28). Base unstacking
occurs when DNA melts, and the associated increased reactivity to
KMnO4 is readily detected.
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Fig. 1.
Sequence of the E. coli
glnHp2 and S. meliloti nifH derivatives used in
this work. Compared with the original glnHp2 m12
promoter sequence (27), heteroduplex promoter fragments contained
either unpaired DNA from 11 to
10 with A-10 (bottom strand)
replaced with G to form a pre-melted structure comparable to that for
the early melted S. meliloti nifH DNA (24) or from
11 to
6 (highlighted). In glnHp2 m12, the wild-type
T:A base pair at
13 is replaced by G:C to increase
binding.
S. meliloti nifH promoter fragments are as described
previously (24).
Isomerization of the 54-glnHp2
Complex--
Initially, we used a gel shift assay to show that
54 bound to the modified glnHp2 promoter with
unpaired DNA at
11/
10 and, in a reaction requiring hydrolyzable
nucleoside triphosphate (dGTP) and activator, produced a supershifted
heparin-resistant complex (ss
-DNA, Fig.
2A, lane 4). As
seen before, the same mobility-supershifted complex formed with
activators of different molecular weights, providing evidence that the
activator was not stably associated with the isomerized complex (Ref.
24 and data not shown). Formation of this complex required
54 Region I (Fig. 2A, compare lanes
4 and 8). Addition of Region I in trans to
I
54 resulted in a new species with similar mobility
to that of the supershifted complex, independent of activator and
nucleotide (Fig. 2A, lanes 9 and 10).
Using DNase I footprinting, we showed that the DNA within the
isomerized
54 complex was protected more than in the
complex formed with
54 in the absence of activator and
nucleotide (Fig. 2B). The downstream edge of the
54 footprint extended to about
5 (Fig. 2B,
lane 3). In the isomerized complex, the footprint extended
clearly to +2, but a partial footprint to +5 was also detected (Fig.
2B, lane 4). The upstream edge of the footprint
was not easily discernible due to background DNA fragments in the
undigested sample (Fig. 2B, lanes 1 and
5). Binding of
I
54 did not lead to the
extended DNase I footprint of
54 seen with the
activator-dependent isomerized complex, but weakly footprinted to about
7 (Fig. 2B, lanes 9 and
10). Addition of Region I in trans to
I
54 resulted in a DNase I footprint indistinguishable
from that of the non-isomerized
54-DNA complex (Fig.
2B, compare lanes 7 and 8 with the
lane 3).
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The overall results showed that 54-DNA interactions at
glnHp2 are changed by the action of activator in a
nucleotide-dependent manner. Non-hydrolyzable nucleotide
GTP
S did not substitute for dGTP to produce an extended footprint
(data not shown). Qualitatively, the results are similar to those
obtained with the S. meliloti nifH promoter (24) and clearly
indicate that activator-dependent
54
isomerization may be readily demonstrated with a number of different promoters.
Activator-dependent DNA Melting by
54--
We used KMnO4 to probe for DNA
melting within isomerized complexes. KMnO4 footprints at
30 °C using the S. meliloti nifH early melted DNA
(
12/
11) in the isomerized complex did not convincingly show extra
DNA melting, but the unpaired T residue at
12 of the heteroduplex
region was much less reactive in both
54-DNA complexes
(data not shown). These KMnO4 footprints were repeated at
37 °C and with a promoter derivative having a single base pair of
heteroduplex at
12 (top strand A replaced with C) (24). Results with
the
12/
11 heteroduplex showed no extra DNA melting at the elevated
temperature, but the unpaired T residue at
12 in the isomerized
complex was more reactive to KMnO4 (data not shown).
However, footprints using the C-12 heteroduplex DNA in the isomerized
complex showed that, at 37 °C, the template strand T at
9 had a
2-fold increase in KMnO4 reactivity, indicating some extra
DNA melting (data not shown). This contrasts with results obtained with
glnHp2 derivatives (see below and Fig.
3), where considerable extra DNA melting
in the isomerized complex was seen at 30 °C. It seems that melting
of the nifH promoter within the isomerized complex occurs
less frequently than with glnHp2 and may relate to
differences in the ease with which the DNA strands of the two promoters
can separate (see "Discussion").
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Footprints of the glnHp2 11/
10 promoter DNA show
convincingly that the isomerized complex has extra DNA melting. As
shown in Fig. 3A,
54 strongly protected the
template strand unpaired T residue at
11 from KMnO4
attack (compare lanes 2 and 3). In the isomerized complex, this protection was lost, and KMnO4 reactivity was
evident at the unpaired T residue at
11 as well as at the new
positions
9 and
7 (Fig. 3A, compare lanes 3 and 4). The bases at
11,
9, and
7 must be within an
altered DNA structure compared with the free DNA and the non-isomerized
54-DNA complex. It seems that activator brings about
some extra DNA melting as well as changing the interaction of
54 with the T residue at
11. The KMnO4
reactivity of the bands was quantified to enhance the reliability of
the interpretation (Table I). The
same patterns of enhanced reactivity and protection were seen in three
independent experiments. Controls in which either activator or
hydrolyzable NTP was omitted or a non-hydrolyzable NTP (GTP
S) was
used showed that the extra DNA melting at
9 and
7 and the changed
footprint at
11 required activator plus hydrolyzable nucleotide (data
not shown). In the absence of these components, the
54-DNA footprint remained unchanged, and the only
complex evident in the gel shift assay was the fast running
54-DNA complex (data not shown).
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KMnO4 was used to probe the non-template strand in
54-DNA and isomerized
54-DNA complexes
forming with the modified glnHp2 promoter. As shown in Fig.
3B (see also Table I), T residues at
8 and
6 were KMnO4-reactive in isomerized complexes that formed in
response to activator and hydrolyzable nucleotide (Fig. 3B,
lane 4). In the absence of activator and hydrolyzable
nucleotide, the same sequences were no more reactive to
KMnO4 than the naked DNA (Fig. 3B, compare
lanes 2 and 3; see also Table I). As seen for the template strand (Fig. 3A), isomerization of the
54-DNA complex was accompanied by some local DNA
melting. In both cases, melting seems to extend from the
11/
10
heteroduplex region by at least (interpretation is limited by the
placement of a potentially reactive T residue) an extra 4 base pairs
toward the transcription start site (summarized in Fig. 7). Melting at
these locations is seen in natural open promoter complexes forming with
the
54 holoenzyme (13-15, 29). The lack of reactivity
of non-template T at
4 suggests either that the transcription start
site sequence is not stably opened in the isomerized complex or that T
at
4 is protected by
54 from KMnO4 attack.
Weak Deregulated DNA Melting by 54 Lacking Region
I--
The amino-terminal 50 amino acids of
54 (Region
I) function to inhibit isomerization of the RNA polymerase holoenzyme
as well as to promote enhancer responsiveness (30-33). Activities of
Region I include contributions to the DNA-binding function of
54, particularly the recognition and creation of the
nucleated DNA near
12, and an interaction with core RNA polymerase
(4, 16, 23, 31, 34). Removal of Region I results in
activator-independent transcription if the DNA is transiently opened
and allows the holoenzyme to engage with pre-melted DNA (11, 18, 20,
25). Using KMnO4 to probe the template strand of the
glnHp2-
I
54 complex, we found some evidence
for weak activator-independent melting. As shown in Fig. 3A
(lane 8), the T residue at
9 showed some increased
KMnO4 reactivity in the
I
54 complex
compared with the zero protein control (lane 2). This region
of extra DNA melting appears to be a subset of that found within the
activator-dependent complex forming with full-length
54 (Fig. 3A, compare lanes 4 and
8). The restricted pattern of melting at
9 seen with
I
54 was independent of nucleotide and activator (Fig.
3A, compare lanes 7 and 8).
Quantitative treatment of the KMnO4 reactivity (Table I)
showed a constant increase in the reactivity of T at
9 when
I
54 was bound. The increase was seen in three
independent experiments. The T reside at
11 in the
I
54 complex was slightly more protected compared with
the isomerized
54 complex (Fig. 3A), but
significantly more reactive than
54 alone to
KMnO4 attack (compare lanes 3, 4, and
8; also see Table I). It seems that removal of Region I
partially deregulates
54 melting activity. Region I
supplied in trans did not result in a significant change in
KMnO4 reactivity to suggest a shift in the footprint toward
that of the non-isomerized
54-DNA complex (Fig.
3A, compare lanes 3 and 5; and Table
I). It seems that, although Region I binds to the
12/
11-
I
54 complex, this does not lead to large
changes in KMnO4 reactivity (24).
KMnO4 was used to probe the non-template strand in the
I
54-DNA complexes forming with the modified
glnHp2 promoter. As shown in Fig. 3B (lanes
5-8) and Table I, no sequences were significantly more
KMnO4-reactive than in the unbound DNA (Fig. 3B,
lane 2). For the
I
54-DNA complex, no
further changes in KMnO4 reactivity were detected in
response to activator and hydrolyzable nucleotide (Fig. 3B, lane 7). Region I supplied in trans to
I
54-DNA binding assays did not change non-template
strand KMnO4 reactivity (Fig. 3B, lanes
5 and 6).
Interactions of 54 with DNA Pre-opened from
11 to
6 and a Gapped Structure--
The extra DNA opening from
9 to
6
seen in the activator-dependent isomerized complex (Fig. 3)
could arise from activator functioning as a DNA helicase. To explore
this issue, we wished to learn if pre-opening the DNA from
11 to
6
would allow the
54 to bind promoter DNA in an isomerized
state without activator. The interaction of
54 with
glnHp2 promoter DNA mismatched from
11 to
6 (see Fig. 1)
to mimic the DNA opening seen in the activator-dependent
isomerized complexes was examined in the presence and absence of
activator. Gel shift assays showed that
54 bound the
11/
6 opened DNA to give an initial complex
(ss
*-DNA) with reduced mobility compared with
the
11/
10 opened DNA complex (Fig. 4,
compare lanes 2 and 5). The reduced mobility was
similar to that of the activator-dependent isomerized
complex (ss
-DNA) forming on the
11/
10
opened DNA (Fig. 4, compare lanes 3 and 5). The
mobility of the
54 complex with DNA opened from
11 to
6 was unchanged by activator and hydrolyzable nucleotide (Fig. 4,
compare lanes 4 and 5). To more fully understand
the properties of these slow running complexes, we probed them by DNase
I and KMnO4 footprinting. Using glnHp2 promoter
DNA mismatched from
11 to
6,
54 gave a short DNase I
footprint to
5 and no extra KMnO4 reactivity, and
footprints were insensitive to activator and nucleotide (data not
shown). We conclude that the reduced mobility of the
54
complex with DNA opened from
11 to
6 is due to the altered DNA
conformation and that pre-opening the DNA does not drive the change in
54 needed for the extended downstream DNase I footprint
to at least +2 seen in activator-dependent isomerized
complexes (Fig. 2B, lane 4) (24). Instead, a
change in
54 conformation driven by the activator seems
to be necessary for the extended footprint to +2. The insensitivity of
the
54 complex on the
11/
6 opened DNA to activator
suggests that the DNA from
9 to
6 should be in a double-stranded
form for activator to act on
54, consistent with prior
work with the S. meliloti nifH promoter (24).
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We previously observed with the S. meliloti nifH promoter
that removal of the top strand A-12 residue resulted in a
54-DNA complex that did not form a new slow running
species when incubated with activator and hydrolyzable or
non-hydrolyzable nucleoside triphosphate (Fig.
5A, compare lanes
2, 4, and 5 with lane 6) (24).
I
54 formed a slower running complex when Region I was
in trans (Fig. 5A, compare lanes 2,
6, and 7). To characterize these complexes and to
determine their relationship to the isomerized complex, we used DNase I
and KMnO4 footprinting. The S. meliloti nifH
promoter
12 gap (top strand) probe (see Fig. 1) gave a DNase I
footprint with a distinct region that was cut poorly compared with the
intact probe (data not shown). This region centered over the
12 gap and extended ~4 bases on either side, suggesting a locally altered DNA structure refractory to DNase I cutting. When
54
bound, extra cutting from
10 to
1 was also evident, suggesting that
54 stabilized a double-stranded DNA structure otherwise
absent from the unbound gapped DNA (data not shown). There was no
difference in the DNase I footprint under activating conditions (data
not shown). The effects of activating conditions were then gauged by
KMnO4 footprinting. When
54 bound the gapped
duplex, the single-stranded T residue at
12 (template strand) was
protected from attack by KMnO4, and a modestly increased
reactivity to KMnO4 was seen at T-9 indicative of some activator- and nucleotide-independent DNA melting (Fig. 5B,
compare lanes 2 and 3). The same pattern was seen
in the presence of activator and nucleotide (Fig. 5B,
compare lane 3 with lanes 5-7) without evidence
for extra activation-dependent melting. This suggests that
a mismatch at
12 is needed for activator response. The slow running
I
54 complex with Region I in trans
footprinted like wild-type
54 (Fig. 5B,
compare lanes 3 and 8), whereas the
I
54 complex showed less KMnO4 reactivity
at T
9 (compare lanes 8 and 9). This suggests
that Region I stabilizes the melted DNA at
9. The DNA melting seen
with the gapped DNA when bound by
54 (Fig.
5B) is consistent with the proposed role of the
12
nucleotide in restricting melting prior to activation (4). The gapped DNA allows melting within the
54-DNA complex that is not
evident with homoduplex DNA (Fig. 5B and data not
shown).
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Interactions of the 54 Holoenzyme with Early Melted
DNA--
The early melted DNA structure just downstream of the GC
promoter that enables
54 isomerization is believed to
exist in closed promoter complexes, but is apparently absent in the
activator-dependent open complex (23, 24). The chemical
reactivities at
12/
11 seen in closed complexes are not evident in
open complexes; rather, new melting is evident nearer the transcription
start site (23). To explore the activator responsiveness of the
54 holoenzyme on the S. meliloti nifH and
E. coli glnHp2 early melted DNAs, we conducted gel shift and
footprinting assays. As noted above, the
54 holoenzyme
bound to the early melted DNA assumes a complex with greater resistance
to heparin than does the holoenzyme complex on homoduplex DNA (31).
This has been suggested to involve a changed interaction between
54 and core RNA polymerase since the binding of
54 to core RNA polymerase in the absence of early melted
DNA is heparin-sensitive (31, 34). Gel shift assays with either the S. meliloti nifH (Fig.
6A) or the E. coli
glnHp2 (data not shown) early melted DNA in the presence of the
54 holoenzyme showed, that under activating conditions,
no new slow running holoenzyme-DNA complex was detected (compare
lanes 6 and 7). A reduction in
54
holoenzyme concentration led to increased formation of the supershifted
54-DNA complex (ss
-DNA, Fig.
6A, compare lanes 2-6) in the presence of
activator and nucleotide, reflecting free
54.
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We next used DNase I and KMnO4 footprinting to characterize
the 54 holoenzyme-DNA complexes on the early melted DNA
and to learn if they had isomerized. Results showed that, under
non-activating conditions, the isolated
54 holoenzyme
complex gave a short DNase I footprint to
5 and no extra
KMnO4 reactivity (data not shown). The footprints were
essentially as for
54, except that some extra protection
from DNase I by the
54 holoenzyme upstream of
34 was
observed with the E. coli glnHp2 promoter (data not shown).
We next footprinted the
54 holoenzyme-DNA complexes
under activating conditions. The isolated holoenzyme complexes gave
footprints indistinguishable from those obtained under non-activating
conditions, suggesting that core-bound
54 was not able
to isomerize efficiently (data not shown). Further experiments with the
use of initiating nucleotide (GTP) to potentially stabilize complexes
on opened DNA through allowing initiation or with the omission of the
heparin challenge to help preserve unstable complexes failed to produce
54 holoenzyme footprints in which
activator-dependent changes were evident (data not shown).
We conclude that
54 does not isomerize efficiently when
bound to core RNA polymerase in assays using early melted DNA probes.
We confirmed (data not shown), using S. meliloti nifH 60
to +28 homoduplex DNA, that the heparin-resistant open promoter complexes formed by the action of activator and GTP had extended DNase
I footprints to at least +13, definition of an exact end point being
limited by the resolution of the gel and fragment size to +28 (14, 20,
35). However, the efficiency of activator-dependent stable
complex formation was low in this assay. To address the issue that the
54 holoenzyme bound to the early melted DNA might form
new activator-dependent complexes (not distinguished from
the activator-independent complexes because of the common property of
heparin resistance) but with low efficiency, we also used
KMnO4 as a probe of isomerization events. Here
isomerization would be evident as increased reactivity to
KMnO4 rather than protection from DNase I (see Fig.
3A, lane 4). Unlike the activator- and
nucleotide-dependent complexes forming with
54 and the early melted glnHp2 promoter DNA
(Fig. 3A), the
54 holoenzyme did not respond
to activator to yield detectable extra melting or any associated loss
of KMnO4 reactivity of the glnHp2 heteroduplex
sequence (data not shown). The overall results show that the holoenzyme
complex, in contrast to
54, is poorly (if at all)
responsive to activation conditions when bound to the early melted DNA.
As discussed below, this may relate to unusually stable complex
formation between the early melted DNA and the
54 holoenzyme.
Core RNA Polymerase Binding to Isomerized
54-DNA--
Having shown that
54 bound
to early melted DNA forms an isomerized complex (Ref. 24 and this
work), but that
54 holoenzyme does so inefficiently if
at all (see above), we conducted an experiment to determine whether the
conformation of the isomerized
54-DNA complex allowed
core RNA polymerase binding. In this assay, the isomerized complex was
formed using an end-labeled
35 to +6 S. meliloti nifH
12/
11 DNA fragment (24) in excess of
54 to diminish
the amount of free
54 and to ensure that core RNA
polymerase interactions were potentially largely with DNA-bound
54. Isomerization reactions were carried out and stopped
by adding GDP (Fig. 6B, lane 2) (24). Increasing
amounts of core RNA polymerase (E) were then added to bind
54-DNA complexes. As shown in Fig. 6B,
addition of increasing amounts of core RNA polymerase (lanes
3-8) depleted the
54-DNA complex; and in parallel,
an increasing amount of the
54 holoenzyme bound to DNA
(E
-DNA) was detected. The amount of isomerized
54-DNA complex (ss
-DNA) remained
relatively constant throughout the titration with core RNA polymerase.
It seems that core RNA polymerase preferentially binds the
non-isomerized
54-DNA complex. At high core RNA
polymerase concentrations (in excess of 0.6 µM) (Fig.
6B, lanes 7 and 8), some core bound to
DNA was detected, and this contributed to the apparent increased amount of
54 holoenzyme bound (graphed in Fig. 6C)
since core-DNA and holoenzyme-DNA complexes were not fully resolved. We
infer that weak binding of the isomerized
54-DNA complex
by core RNA polymerase is because the interface between core and
54 and DNA has changed upon isomerization. This leads to
the suggestion that movements in
54 and DNA are
concerted with some in core RNA polymerase for forming the natural open
promoter complex and is discussed below.
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DISCUSSION |
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DNA Melting by
54--
Activator-dependent isomerization
of
54 results in the spreading of DNA melting away from
the nucleated DNA structure and toward the transcription start site
(Fig. 7). A structure melted over at
least 6 base pairs is generated. Interactions with the nucleated DNA
located at
11/
10 are lessened in the isomerized complex compared
with the initial
54-DNA complex. Although removal of
Region I sequences allows some weak DNA melting by
, this is not as
extensive as activator-driven isomerization. A comparison of results
obtained with
I
54 and intact
54 shows
that the full DNA-melting activity of
54 has some
determinants outside of regulatory Region I (for weak melting at
9)
and some that depend upon Region I (for extra melting from
8 to
6).
|
Isomerized 54-nifH promoter complexes failed
to show KMnO4 reactivity as extensively as
glnHp2 (Fig. 3 and data not shown). If this reflects less
melting (as opposed to shielding from KMnO4), differences
in intrinsic DNA opening rates between the two promoters in combination
with the sequence-independent single-stranded DNA-binding activity in
54 (36) could contribute. DNA opening at
9 seen in
natural nifH open complexes but weakly detected in
isomerized complexes with
54 might therefore reflect a
stabilizing contribution from the core enzyme (37). Clearly, changes in
the
54-DNA relationship seen in DNase I footprints of
isomerized complexes in which melting may not have occurred is
consistent with the idea that activator changes
54
structure and that pre-opening the DNA does not drive this change (22,
24).
Comparisons of the closed and activator-dependent open
54 holoenzyme-promoter complexes using KMnO4
and ortho-copper phenanthroline footprinting suggest that
the structure of the DNA immediately downstream of the GC element
differs between these complexes and that contact with G-13 is also
altered (14, 15, 23). These observations are fully consistent with the
lessened
interaction at
11 detected by the KMnO4
footprints of isomerized complexes reported here (Fig. 3). Based on the
known interaction between Region I and core polymerase (34, 38), we
suggested that the silencing interactions associated with the
13 to
11
54-DNA contact (4) can contribute significantly to
inhibiting RNA polymerase isomerization through restricting
conformational changes within the core subunits (16). Changes in
promoter DNA and
54 holoenzyme conformation are probably
coupled through
54 Region I to maintain either the
closed or open state of the promoter. Open promoter complexes that form
in deregulated transcription by the
54 holoenzyme are
unstable compared with activator-dependent open complexes
(18, 19, 25). Their instability may be related to incomplete DNA
melting beyond
9 as a consequence of the mutations in Region I of
54 and a need for Region I to stabilize melted DNA (Fig.
5B).
Role of Activator--
Results with DNA mismatched from 11 to
6 across the sequences melted in the isomerized
54-DNA
complex support the view that activator drives a conformational change
in
54 rather than generating isomerization by solely
creating an opened DNA structure for
54 binding. For
both transcription and
54 isomerization, pre-opening of
the DNA through heteroduplex formation does not by pass activator
requirements (22), unless the structure recognized by
54
at
11 is destroyed (36). Rather, activator-dependent
conformational changes in
54 and the holoenzyme seem
necessary for open complex formation and
54
isomerization (24, 36). DNase I footprinting shows that
54 clearly binds to double-stranded DNA ahead of the
locally melted DNA or the fork junction that forms next to the GC
promoter element in closed complexes (this work and Refs. 4 and 24).
This suggests that the further melting of the double-stranded DNA
within the
54-DNA complex is an active process in the
sense that it is not a primary result of a domain of
54
translocating along the duplex and trapping DNA strands at a fraying
fork junction. Energy for duplex destabilization may come from the
tight binding of
54 to the initially locally melted DNA.
An activator-driven change in
54-DNA interaction might
release
54 from binding to the double-stranded DNA
downstream of the
12 GC and switch
54 to a
conformation that then allows it to bind single-stranded DNA. Combined
with the single-stranded DNA-binding activities in
54
(4, 36) and the core RNA polymerase (39),
54
isomerization would lead to formation of the stable open promoter complex.
Stable Holoenzyme Binding--
When bound to core RNA polymerase,
activator-dependent isomerization of 54 was
not evident, in marked contrast to its efficient isomerization without
core. It seems that the short sequence of heteroduplex downstream of GC
inhibits isomerization of
54 within the holoenzyme
since, on linear DNA, activator-dependent isomerization of
the holoenzyme is evident (11, 23, 39). The
activator-dependent movement of
54 across
the sequence opened downstream of GC implied by the results of our
KMnO4 footprints may be inhibited when
54 is
bound by core RNA polymerase. This suggests that isomerization of
54 within the holoenzyme may normally require that the
local DNA opening next to GC does not strongly persist. In the
homoduplex, the DNA can base pair again, but not in the heteroduplex.
Consistent with this view is the observation that the local DNA
distortions downstream of GC and present in the closed complex are
apparently changed in the open complex, as judged by the reduced
sensitivity of the DNA to two chemical probes of DNA structure (23).
The spread of melting observed in the
isomerization assays would then normally be associated with a changing of the DNA structure believed to be locally melted in the closed complex, achieved through a
breaking of DNA contacts and a rebinding of
54 to DNA.
These considerations suggest an ordered series of events in which
changes in core RNA polymerase to
54 interactions and
54-DNA interactions occur in response to activator to
allow
54 isomerization and the holoenzyme to progress
from the closed complex to the open complex. This view is consistent
with activator interacting with both core RNA polymerase and
54 (24, 40) to achieve isomerization of the holoenzyme
and with the view that the promoter sequences around GC contribute to
preventing the holoenzyme from isomerizing prior to activation and to
set the target of the activator (4, 24).
Core- Interactions and DNA Melting--
Results of core RNA
polymerase binding with the isomerized and non-isomerized
54-DNA complexes strongly suggest that some points of
interaction between
54 and core are changed upon
isomerization of the
54-DNA complex. The poorer core
binding of the isomerized complex likely correlates with the changes in
protease sensitivity of
54 in the isomerized complex
(24). Changed DNA structure within the isomerized
54-DNA
complex may also contribute to poor binding by core RNA polymerase. The
strong reduction in isomerization of
54 resulting from
core RNA polymerase binding prior to exposure to activating conditions
(Fig. 6A) and the weak binding of isomerized
54 to core RNA polymerase (Fig. 6, B and
C) is striking. It seems that normally for efficient
54 holoenzyme isomerization and open complex formation,
activator-dependent changes in
54 structure
would occur in concert with a changed binding of parts of
54 to core RNA polymerase; but on the early melted DNA,
this is not occurring properly. As indicated above, when using the
early melted DNA as template, the failure to reconfigure interactions with DNA next to the GC promoter may simply strongly stabilize a
54 conformation that is unfavorable for core RNA
polymerase binding. We therefore suggest that, in closed complexes,
activator drives a conformational change in
54 that
results in altered contacts with the early melted DNA (as detected in
our KMnO4 footprinting; see above), allowing binding of
54 and core RNA polymerase to permit full
54 isomerization and the associated isomerization of the
closed complex to the open complex. It seems that Region I of
54 greatly contributes to these events through (i) its
requirement for creating the early melted DNA when the holoenzyme binds
homoduplex DNA, (ii) directing
54 binding to the early
melted DNA in heteroduplexes and associated fork junction structures,
(iii) a binding interaction with core RNA polymerase, and (iv) the
changing Region I structure in isomerized
54 and the
activated
54 holoenzyme (reviewed in Ref. 12). The
recent demonstration that Region I sequences localize over the
12
promoter region is fully consistent with these observations and points
to a central role of the protein and DNA elements that localize there
in establishing new interactions that allow DNA melting and a changing
binding relationship between core subunits and
54 (41).
We also note that the refractory behavior of the
54
holoenzyme bound to the early melted DNA and the poor core binding of
isomerized
54 are fully consistent with the view that
interactions
54 makes with the
12 promoter region, in
particular sequences just downstream of GC, are key in limiting
spontaneous activator-independent open complex formation (4).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jörg Schumacher and Susan Jones for useful comments on this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a Wellcome Trust grant (to M. B.) and by a Biotechnology Marie Curie fellowship (to M.-T. G.). Work was conducted in the Imperial College Center for Structural Biology.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 should be addressed: Dept. of Biology, Sir Alexander Fleming Bldg., Imperial College of Science Technology and Medicine, London SW7 2AZ, UK. Tel.: 207-594-5442; Fax: 207-594-5419; E-mail: m.buck@ic.ac.uk.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M007779200
![]() |
ABBREVIATIONS |
---|
The abbreviation used is:
GTPS, guanosine 5'-O-(3- thiotriphosphate).
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