Nucleic Acids Research, 2003, Vol. 31, No. 11 2745±2750
DOI: 10.1093/nar/gkg400
Effect of DNA bases and backbone on s70
holoenzyme binding and isomerization using fork
junction probes
Mike S. Fenton and Jay D. Gralla*
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles,
PO Box 951569, Los Angeles, CA 90095-1569, USA
Received March 6, 2003; Revised and Accepted April 16, 2003
ABSTRACT
Abasic substitutions in the non-template strand and
promoter sequence changes were made to assess
the roles of various promoter features in s70
holoenzyme interactions with fork junction probes.
Removal of ±10 element non-template single strand
bases, leaving the phosphodiester backbone intact,
did not interfere with binding. In contrast these
abasic probes were de®cient in promoting holoenzyme isomerization to the heparin resistant conformation. Thus, it appears that the melted ±10
region interaction has two components, an initial
enzyme binding primarily to the phosphodiester
backbone and a base dependent isomerization of
the bound enzyme. In contrast various upstream
elements cooperate primarily to stimulate binding.
Features and positions most important for these
effects are identi®ed.
INTRODUCTION
s70 dependent bacterial promoters (1±3) contain two moderately conserved hexamers separated by an optimal spacer
length of 17 bp. These two recognition elements are near
positions ±10 and ±35 relative to the start site at +1. The
elements stabilize polymerase binding to DNA, direct
polymerase dependent DNA melting within the ±10 element
and promote isomerization of the polymerase to its functional
heparin-resistant form. Naturally occurring promoters deviate
from the consensus resulting in a diverse array of promoter
activities (4,5). DNA sequences outside these elements also
have a very important in¯uence (6,7).
Recent studies have emphasized that the ±10 and ±35
elements may have signi®cantly different functions. The ±35
region acts through duplex DNA and makes stabilizing
contacts to region 4 of the polymerase s subunit (8). This
can help anchor the enzyme to the DNA throughout the
preinitiation pathway (9,10). The role of the ±10 region is
more complex because it is converted to the single stranded
form during open complex formation. Electrophoretic mobility shift assay (EMSA) studies using either duplex or fork
junction probes (where the ±10 region is presented as a
non-template single strand) show effects of base substitutions
(11). When fork junction probes lacking the ±35 region are
tested using energy transfer the effects are larger (12).
However, when fork junction probes (with ±35) are tested
by EMSA in the presence of heparin, nucleotide substitutions
lead to large defects (11,13). This led to the idea that a primary
function of the ±10 region is to assist in isomerizing
polymerase to the heparin resistant form via interactions
with melted DNA (11). The comparisons also point out the
importance of DNA context in evaluating the function of
the ±10 region.
Several studies have suggested that the A on the nontemplate strand at ±11 is of paramount importance for the
function of the ±10 region DNA element in single stranded
form (12±15). The extension of an upstream duplex to include
this as a single melted nucleotide confers stability in EMSA
assays using fork junction probes (13). The effect is greater
when assays measure the ability of the isomerized enzyme to
resist heparin challenge. It has been suggested that that this
nucleotide performs a `gating' (13) or `master' (16) function
during the melting of the promoter DNA. This may be related
to its unpairing during the nucleation of the melting process.
The unpairing at ±11 to create a fork junction is likely to be a
seminal event, although how the structure contributes to
subsequent events is still unclear. Substitutions at this position
can have wide-ranging effects on binding, isomerization and
ultimately transcription.
Understanding the roles of sequence-speci®c recognition of
the ±10 region nucleotides has been hampered by the lack of a
high-resolution structure and by the lack of a uniform context
for diverse biochemical experiments. It is clear that many
contacts come from the s subunit (17,18), but it is not clear as
to how the effects of these contacts partition before and after
the isomerization of the enzyme. It is also not clear how the
upstream DNA elements, and the fork junction itself,
contribute to ±10 function. This is because different experiments have used probes with and without upstream sequences,
and in the presence and absence of heparin.
In this report we investigate the effect of upstream
sequences and the nature of sequence-speci®c interactions
with the ±10 element, with and without heparin challenge. The
primary approach uses EMSA on fork junction probes. In one
set of experiments various upstream elements are combined to
learn their contributions to probe binding. Other experiments
*To whom correspondence should be addressed. Tel: +1 310 825 1620; Fax: +1 310 267 2302; Email: [email protected]
Nucleic Acids Research, Vol. 31 No. 11 ã Oxford University Press 2003; all rights reserved
2746
Nucleic Acids Research, 2003, Vol. 31, No. 11
use probes in which bases have been removed individually but
the backbone remains intact. These allow the assessment of
the base-speci®c interactions in the duplex ±35 region and in
the single stranded ±10 region of the non-template strand. The
results show that probes with an intact non-template strand
backbone but lacking single bases can be bound quite well,
with signi®cant reductions seen only in the presence of
heparin. In this circumstance the binding is reduced somewhat
more when the abasic site receives a non-consensus base.
Overall, the results support a signi®cant role for the ±10 region
backbone in polymerase binding with a major role for the
bases in enzyme isomerization.
MATERIALS AND METHODS
Proteins and DNA
The plasmid pQE30-rpoD was over-expressed and s70 was
puri®ed as described (19). Escherichia coli RNA polymerase
core is a commercial product from Epicenter Technologies.
Oligonucleotides and probes were prepared as described (20).
Brie¯y, the bottom strand of each probe was labeled with
[g-32P]ATP. The 40-ml mixture, containing 4 pmol of kinased
DNA and 6 pmol complementary strand in 20 mM Tris±HCl,
pH 7.5/50 mM NaCl, was annealed by rapid heating to 95°C
and slow cooling to room temperature in a PCR thermocycler (MJ Research). The resulting annealed probes were
diluted in Tris±EDTA buffer containing 50 mM NaCl to the
desired concentration. Proper annealing was monitored by
electrophoresis.
Electrophoretic mobility shift assay
Mobility shift assays with and without heparin were as
described (11). Brie¯y, 20 nM core was mixed with 50 nM
s70 in a 10 ml reaction mixture with 13 buffer A [30 mM
Tris±HCl pH 7.9, 100 mM KCl, 3 mM MgCl2, 0.1 mM EDTA,
1 mM dithiothreitol (DTT), 100 mg/ml bovine serum albumin
(BSA), 3.25% glycerol] with 6 ng/ml (dI-dC) and 1 nM
annealed probe, and incubated for 20 min on ice. For heparin
challenge experiments, 0.25 ml of 2 mg/ml heparin was added
into samples for an additional 5 min (11). Samples were run on
5% PAGE with 13 TBE buffer, packed in ice.
RESULTS
Upstream elements
A collection of seven probes is used to assess the importance
of the upstream elements in fork probes (Fig. 1). Each probe
contains a ±35 element and spacer but within the ±10 element
only base pair ±12 and non-template strand nucleotide ±11 are
present. Previously we found that such a short fork probe
modeled after the PR¢ promoter can be bound well by RNA
polymerase (13) but an analogous UV5 fork probe is bound
poorly (11). Differences in PR¢ and UV5 fork probes include
the spacer and the ±35 element. To learn the source of the
difference in binding we altered the UV5 probe to include
features of PR¢. These include changing position ±34T in the
±35 region to a consensus G, altering the spacer length of 18 bp
to an optimal 17 bp, and swapping the upstream PR¢ sequence
from ±14 to ±20 into UV5. Pairs of changes were also made in
Figure 1. Parental and hybrid T11/B12 fork junction probes. The parent PR¢
(lowercase DNA sequence) and UV5 (uppercase DNA sequence) probes are
at the top with the ±35 element boxed. DB refers to a PR¢ sequence speci®ed
as the downstream block (±14 to ±20) placed in UV5; S refers to a spacer
length change that shortens the UV5 spacer by 1 bp (position ±30 deleted,
as indicated by an underscore); 35 refers to the indicated change in the UV5
±35 region.
two probes. The collection of ®ve hybrid probes and the two
parents are shown in Figure 1.
EMSA experiments were done to assess binding on these
altered UV5 fork junction probes. The data show that only
probes containing pairs of changes formed tightly bound
complexes with polymerase (Fig. 2A, lanes 3 and 4). Each of
the individual changes also increased binding somewhat
(Fig. 2A, lanes 5±7). The ±14 to ±20 region substitution was
slightly better than the spacer length change which in turn was
slightly better than the ±35 base substitution. It appears that
spacer length, non-conserved spacer sequence and the ±35
element work together to stabilize binding to fork junction
probes.
In Figure 2B the experiments were repeated in the presence
of heparin. Because the DNA is pre-melted the comparison
with Figure 2A bypasses DNA melting and assesses only the
ability of the polymerase itself to isomerize to a heparin
resistant form. The binding was less but the overall patterns
were preserved (compare Fig. 2A with B). Thus no upstream
feature appears to play a unique role either in DNA binding or
in isomerization, but all features are important.
The ±35 region is known to be involved in base-speci®c
recognition (21,22). We wished to evaluate the properties of
the ±35 region bases in a fork junction probe context. The nontemplate strand was altered in order to provide a direct
comparison with studies of the ±10 region. Individual bases
were deleted, leaving the backbone fully intact, leading to
abasic positions in the non-template strand. One of the stronger
binding hybrid fork junction probes (35-S) was used in order to
reveal partial defects that might occur. The collection of
probes contained abasic sites on the non-template strand of the
±35 hexamer at each of the six consensus positions (±30 to ±35
on the 17 bp spacer 35-S promoter). As a comparison the nonconserved base at position ±39 was also made abasic. EMSA
experiments were done with and without heparin.
Nucleic Acids Research, 2003, Vol. 31, No. 11
Figure 2. EMSA of holoenzyme with fork junction probes shown in
Figure 1. Bound complexes are indicated by an arrow. (A) Without heparin:
PR¢, S-DB, and 35-S bound from 80±95%; S, DB and 35 bound from
20±35%; UV5 did not bind detectably. The lower band in the UV5 lane is
representative of core binding. (B) With heparin: PR¢ and S-DB, bound from
60±75%; 35-S at ~30%; DB, S and 35 at 2±10%.
Figure 3A shows that the removal of any one of four out of
the six ±35 element bases leads to a reduction in binding to
fork junction probes (X30A, X32A, X33G and X35T).
Removal of either of the other two consensus bases, ±31C
and ±34T, leads to only a slight reduction compared to either
the parent or the control probe outside the ±35 region (X39).
The pattern is similar with and without heparin except that the
±30 abasic is somewhat more deleterious in a heparin
challenge protocol (compare Fig. 3A with B). The data
show that some bases are important for fork probe binding but,
rather surprisingly, some have a limited importance despite
their conservation and the contacts made by their template
strand partners to s (8). Apparently the template strand
contacts to ±31 and ±34 can be maintained in the absence of
the non-template base partner.
Removal of the overhanging ±11A base from a short
fork junction probe
These short fork junction probes contain a single unpaired
nucleotide, a consensus A at non-template strand position ±11.
This ±11 nucleotide overhang at the optimal fork junction is
required for strong binding (13). The nucleotide must be the
consensus A to obtain ef®cient enzyme isomerization (13).
2747
Figure 3. EMSA using the 35-S T11/B12 fork junction probes with abasic
sites. Abasic sites are at individual positions in the ±35 element nontemplate strand (TTGACA from positions ±35 to ±30) with a preceding X
indicating removal of the speci®ed base. The control used outside the ±35
element was at position ±39. (A) Without heparin: abasic probes 31, 34 and
39 bound at wild type levels (50% in lane 2), probe 30 bound at 30%,
probes 32 and 33 at 15% and probe 35 did not bind detectably. (B) With
heparin: probe 39 bound at wild type levels (20% in lane 2), probes 31 and
34 bound at 15%, and binding of probes 30, 32, 33 and 35 was barely
detectable.
The next experiment tests the contribution of the base and
backbone to these properties. A hybrid fork junction probe
with a 3¢ phosphate on the non-template strand (35-S) was
used to obtain the strongest parental signal. The test probe was
constructed to be abasic at ±11 with the backbone remaining
intact.
In the absence of heparin, removal of the base led to a 2- or
3-fold reduction in binding (Fig. 4A, compare lane 3 with 4).
In the presence of heparin, the ±11 abasic fork junction probe
was incapable of enzyme isomerization (Fig. 4B, compare
lane 7 with 8). We conclude that the consensus base A at ±11,
and not just the backbone, is helpful for DNA binding by
polymerase. However, the base itself is necessary for
polymerase isomerization.
Effects of abasic sites within the melted ±10 region
Abasic sites were also introduced into a probe containing a
duplex ±10 region in order to assess effects on double strand
2748
Nucleic Acids Research, 2003, Vol. 31, No. 11
Figure 4. EMSA using 35-S fork junction probes. The 35-S-P has a phosphate at the 3¢ end of the non-template strand and X11 is also abasic at ±11.
(A) Without heparin: probes 35-S and 35-S-P bound ~50% and probe X11P bound ~20%. (B) With heparin: probes 35-S and 35-S-P bound ~15% and
X11-P did not bind detectably. The core lanes used the 35-S fork probe.
DNA binding. However, binding to all duplex probes was
stimulated by the removal of a base (not shown), likely due to
the creation of easily melted DNA around the abasic positions.
To investigate the roles of bases in single stranded DNA a fork
junction probe with a longer single strand tail was used. In
such probes the melted non-template strand extends to
position ±6. The probes with a longer single strand tail bind
better than the short fork probes with a one nucleotide
overhang under non-saturated binding conditions. When bases
and backbones together are progressively removed from such
probes binding is strongly decreased (11). Nonetheless, the
effects of base substitution are modest on binding; they remain
substantial on enzyme isomerization (11).
In order to evaluate the contributions of the consensus
bases, six abasic probes were created by removal of individual
bases from ±12 to ±7 (Fig. 5 top). The backbone remains intact
in all cases. EMSA was done in the absence and presence of
heparin. The data was tabulated and is presented in Figure 5.
For the sake of comparison, data from prior EMSA experiments (11) where G was substituted at each position is also
presented in Figure 5.
Results in the absence of heparin show that individual
abasic substitutions have little or no effect on probe binding
(Fig. 5A). For example removal of the ±7 base had little effect
whereas it was shown previously that removal of both the base
and backbone led to a large decrease in binding. The results
indicate that the backbone is a critical determinant for binding.
In comparison, G substitutions show effects at positions ±11
and ±7 (see Fig. 5A). Because the consensus base can be
removed without detectable effect, the comparison suggests
that inclusion of a non-consensus base can actively inhibit
binding. Strong effects are seen with a probe abasic at both
±11 and ±7 (data not shown). Thus, although the main binding
interaction is with the backbone, the bases present at positions
±11 and ±7 have an in¯uence on binding.
Experiments that evaluate enzyme isomerization via
heparin challenge gave a different picture of the effects of
removing bases (Fig. 5B). In this circumstance ®ve of the six
Figure 5. EMSA on T6/B12 fork + tail probes with abasics in the single strand ±10 element. The left column indicates the abasic position (X7 is a probe
with an abasic at ±7 and so forth). (A) Without heparin and (B) with heparin. The independent data for G substitutions at each positon is reproduced from
prior EMSA experiments (11).
Nucleic Acids Research, 2003, Vol. 31, No. 11
probes showed reductions (the abasic at position ±10 is the
exception). The remaining abasics led to an average 3-fold
reduced binding. When compared with data obtained from G
substitutions, the results indicate that substitution of a nonconsensus base can lead to a weakened signal compared with
simple removal of a consensus base. As was the case for
binding without heparin challenge this is most clear at
positions ±11 and ±7 (see Fig. 5). Overall, the results suggest
that the bases themselves, not just the backbone, are centrally
involved in enzyme isomerization.
In order to place these results in the context of intact DNA,
±11 and ±7 cytosine substitutions were studied on supercoiled
plasmid DNA (pTH8-UV5). Occupancy of the mutant
promoters was measured via DNase I protection (data not
shown). Both mutants (±7C and ±11C) led to a partial
reduction in protection against DNase I digestion. The
reductions in occupancy were re¯ected in diminished melting
as assessed by permanganate reactivity and diminished
transcription (data not shown) and the ±11C mutation was
slightly more defective in all assays. The transcription results
with the ±11 mutant were consistent with the defects observed
in transcription from the PR¢ promoter (23). The ±7C mutant
on lacUV5 was more defective in transcription than on the PR¢
promoter, consistent with binding assays that demonstrate the
PR¢ promoter has more optimal upstream elements (Fig. 2 and
data not shown). Overall, the data indicate that binding studies
on these probes re¯ect properties of transcription complexes.
DISCUSSION
DNA binding
The data show that a variety of DNA elements and structures
cooperate to promote holoenzyme binding prior to polymerase
isomerization. These include the ±35 element, the spacer
length, the spacer sequence (independent of length), the fork
junction structure and the phosphodiester backbone of the
non-template strand when the ±10 region is melted. No feature
appears to be truly unique in that any can be altered and
binding still occurs when an optimal combination of the others
is present. Some aspects of how they contribute to binding are
unusual.
Prior data showed that when the nucleotides of the melted
±10 region are removed progressively from position ±7,
binding is very strongly diminished (11). However, the current
data show that when individual bases are removed, leaving the
phosphodiester backbone intact, binding levels are not
signi®cantly altered (Fig. 5A). Effects can be seen only
when multiple bases are removed (data not shown), or when
using a weaker-binding truncated probe in which only the ±11
nucleotide is melted (Fig. 4). This implies that a large
component of the initial binding of holoenzyme to melted
DNA involves the DNA backbone. The effects of removing
the base at either ±11 or ±7 are less than when a non-consensus
base is substituted [(11) and see Fig. 5B]. This suggests that
non-consensuses bases in the ±10 regions of natural promoters
may interfere with binding by hindering the holoenzyme from
properly engaging the melted DNA backbone.
These results are consistent with prior experiments using
fork junction probes in which substitutions had modest effects
on DNA binding in the absence of heparin (11). Other studies
2749
detected more signi®cant reductions in binding upon substitution of non-consensus nucleotides (12,14). These studies
used probes that did not contain upstream stabilizing features
and thus would allow smaller differences to be detected. The
larger effects on enzyme isomerization (see below) were not
explored in those investigations.
Removal of non-template stand bases in the duplex ±35
promoter region, also leaving the backbone intact, led to
variable effects on holoenzyme binding. Signi®cant reductions were seen in four of the six positions (underlined in
TTGACA) (Fig. 3). Only the initial ±35T appears to be
contacted directly in the structure of s region 4 with short
duplex DNA (8). Consistent with this, the removal of the
contacted ±35T led to a more severe binding defect than at the
other three affected positions. It is possible that the other base
removals disrupt the DNA helical structure and thus indirectly
interfere with polymerase contacts on the template strand.
However, removal of either ±34T or ±31C is without effect so
this view is dif®cult to support except in a strongly localized
context. It should be noted that non-consensus bases at these
two positions (±35T and ±32C at the lacUV5 promoter) lead to
a reduction in transcription (24). It may be that the source of
these various effects would be more apparent if the overall
kinetic pathway of recognition were understood.
Enzyme isomerization
The ability of holoenzyme to isomerize to its functional
conformation was assessed using a heparin challenge assay.
Because the DNA probes were pre-melted the need to melt
DNA is not a part of this assay. Thus, comparing EMSA
results on various probes with and without heparin reveals
how features of the probes contribute to driving the enzyme
into the heparin-resistant conformation.
All of the elements tested were important in obtaining
maximal amounts of heparin-resistant binding. There were
two experiments in which isomerization was much more
affected than binding. When individual bases were removed
from the melted ±10 region, leaving the backbone intact, large
effects were only seen in the heparin challenge protocol
(compare Fig. 5A with B). With probes containing only the
±11 overhanging nucleotide, the reduction caused by removing the base was signi®cantly greater in the presence of
heparin than in the absence (Fig. 4, lane 4 versus lane 8). The
comparisons indicate that within the melted ±10 region DNA
the bases themselves, as distinct from the backbone, are
important for holoenzyme isomerization. The exception is
position ±10, which in the fork probe context shows no
importance in either binding or isomerization.
The overall data suggest two stages in the use of the melted
±10 region DNA during formation of functional open complexes. During the initial stages of opening the holoenzyme
could make its most important contacts with the DNA
backbone in this region, as suggested by the overall binding
data (Fig. 5) (11). The data indicate that the most important
contributions at this stage would come from exposure of
position ±11 (Figs 4 and 5), which would help to create the
fork junction. Subsequently, establishment of holoenzyme
contacts with most or all of the ±10 element bases, along with
stabilization provided by upstream interactions, would help to
stabilize the isomerized form of the enzyme (Figs 2 and 5).
As promoters typically do not have fully consensus ±10
2750
Nucleic Acids Research, 2003, Vol. 31, No. 11
sequences or optimal upstream elements they would vary in
how easily the isomerization phase would be promoted. This is
consistent with prior data on the lacUV5 promoter (25), which
was the parent for these studies. In general the degree of
binding and the rate of isomerization, set by the combination
of these and other promoter features along with activators,
would determine the initiation rate for each promoter.
ACKNOWLEDGEMENT
This work was supported by NIH grant GM35754.
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