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INTRODUCTION |
To initiate transcription, RNA polymerases
(RNAP)1 must locate and bind
to a promoter region, separate the two DNA stands near the start site,
and position the template strand in the active center. Understanding
how this process occurs is important to our knowledge of the
transcription process. As is the situation with other RNAPs, promoters
for T7 RNA polymerase have a bipartite structure that consists of an
upstream binding region that is recognized in a sequence-specific
manner and an initiation region that must be melted open prior to
initiation (1). The binding region (
17 to
5) appears to
function independently of the initiation region, and a
double-stranded (ds) DNA fragment that contains only this region of the
promoter is tightly bound by the RNAP (2). The binding region is
recognized only in the form of duplex DNA, and removal of either the
template (T) or non-template (NT) strands in this region prevents
polymerase binding (3). In contrast, the NT strand in the initiation
region is not required for initiation, and a partially single-stranded
(pss) promoter in which this strand has been removed downstream of
4
(essentially a "premelted promoter") allows efficient and accurate
initiation (3).
A variety of lines of evidence suggests that the binding
and initiation regions function independently and that it may be possible to physically separate the two while retaining promoter function. In the crystal structure of a T7 RNAP-promoter
complex, as well as in an initiation complex in which the first 3 bases in the template strand have been transcribed, the two promoter domains
are observed to interact with separate regions of the RNAP (4, 5).
Furthermore, a number of structural changes that disrupt the linkage
between the binding and initiation regions do not prevent initiation.
These include the introduction of non-nucleosidic linkers into the T
strand between these two regions, as well as interruption or removal of
a portion of the T strand that lies between the binding region and the
start site of transcription (nicked or gapped promoters) (6, 7).
The question arises, then, as to whether the binding region serves
merely to recruit the RNAP to a (melted) region of the template that is
suitable for initiation or whether its interaction with the RNAP is
required for other functions during initiation and isomerization to a
stable elongation complex. To answer this question, we took
advantage of a Gal4:T7 RNAP fusion protein in which the yeast Gal4
binding domain is fused to the amino terminus of T7 RNAP, thereby
conferring an independent binding capacity upon the RNAP (8). We found
that recruitment of the Gal4:T7 RNAP protein to a ss promoter via a
Gal4 binding site is not sufficient to activate transcription, but that
transcription could be activated by the addition, in trans,
of a ds hairpin loop that contained only the binding region.
Strikingly, the same results were obtained with wild type (WT) enzyme,
and in the absence of the Gal4 DNA binding site. Gel-shift experiments
indicate that exposure of the RNAP to the isolated promoter binding
region facilitates recruitment of the ss template and that the binding
region is displaced prior to initiation.
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EXPERIMENTAL PROCEDURES |
RNA Polymerase and Templates--
His-tagged wild type T7 RNAP
was purified as described previously (9, 10). The Gal4:T7 RNAP fusion
protein (8) was purified as described for unmodified WT RNAP (9) except
that the chromatography step on DEAE-cellulose was omitted.
Oligonucleotides were purchased from Macromolecular Resources and
purified by reversed phase chromatography. The sequences of all
oligonucleotides are presented in Table
I. Where indicated, synthetic oligomers
were labeled with 32P utilizing T4 polynucleotide kinase
(11) and purified from free label by chromatography on QuickSpin
columns (Qiagen). To assemble synthetic templates, DNA oligomers were
taken up in transcription buffer (see below) to a final concentration
of 0.5 µM each, heated to 70 °C for 10 min, and
allowed to cool slowly to room temperature (2-3 h).
Transcription Assays--
Unless otherwise indicated, reactions
were carried out in a volume of 10 µl containing: transcription
buffer (20 mM Tris acetate, pH 7.9, 10 mM
magnesium acetate, 0.05% Tween 20; 5 mM
-mercaptoethanol, 0.1 mM EDTA), 0.5 mM ATP,
GTP, CTP, and UTP, 2 µCi of [
-32P]GTP (NEN, 6,000 mCi/mmol), 50 nM template, and 20 ng of RNA polymerase for
15 or 30 min at 37 °C. Reactions were terminated by the addition of
an equal volume of 90% formamide, 50 mM EDTA, 0.02%
bromphenol blue, 0.02% xylenecyanol, heated to 100 °C for 2 min,
and analyzed by electrophoresis at 85 watts for 1.5 h in 20%
polyacrylamide gels cast in TBE buffer (11) containing 7 M
urea. The gels were analyzed by exposure to a
PhosphorImagerTM screen.
Gel-shift Experiments--
Reactions (10 µl) contained
transcription buffer, 5% glycerol, and RNA polymerase and labeled
probe as indicated. Samples were incubated at 37 °C for 15 min and
resolved by electrophoresis at room temperature in 5% polyacrylamide
gels cast in TAE buffer (11) containing 10 mM magnesium
acetate. Prior to loading, the gel was pre-electrophoresed at 100 volts
for 30 min, and samples were run into the gel at 35 mA for 2.5-3.5 h.
Gels were dried and analyzed by PhosphorImagerTM screen
analysis (Amersham Biosciences).
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RESULTS |
Recruitment of T7 RNAP to a Single-stranded Template via an
Auxiliary DNA Binding Motif Is Not Sufficient to Activate
Transcription--
The Gal4:T7 RNAP fusion protein binds to the Gal4
site with an affinity that is greater than that of T7 RNAP for its
promoter, yet it retains catalytic activity and is able to initiate
transcription specifically at a T7 promoter (2, 8, 12). To recruit the fusion protein to a ss promoter template, we tethered a ds Gal4 recognition site to the template via a 5' "tail" that is
complementary to a region located 43 nt upstream from the promoter
(Fig. 1, A and C,
template b). Gel-shift experiments demonstrate assembly of
the composite template and binding by Gal4:T7 RNAP (Fig.
1B). At the same time, we examined a similar construct in
which the upstream binding region of the T7 promoter was tethered to
the template in the same manner (Fig. 1, A and C,
template c).

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Fig. 1.
Recruitment of T7 RNAP to a ss template does
not activate transcription. A, the single-stranded template
MJ1 contains the consensus promoter sequence from 17 to +6 and
extends upstream to position 77 and downstream to +10. Annealing of
oligomers MR5 and MR4 results in the formation of a 24-bp region that
contains the Gal4 recognition sequence (CGGAGGACAGTACTCCG) flanked by 3 and 4 base pairs upstream and downstream, respectively. A 17 nt
tail at the 5' end of MR5 is complementary to 17 nt at the 3'
end of the ss template; this region of complementarity is separated
from the promoter region by 43 nt of intervening ss DNA. AK33 contains
a ds stem loop that includes the binding region of the T7 promoter from
17 to 5 (shaded box) and has the same 5' tail as MR5.
B, templates were assembled by annealing together
32P-labeled MR5 (asterisk) with other oligomers,
as indicated. The resulting constructs were analyzed by electrophoresis
in non-denaturing polyacrylamide gels in the presence or absence of the
Gal4:T7 RNAP fusion protein, as indicated. C, templates were
assembled by annealing together the oligomers indicated. Note that
MR5/MR4 provides the Gal4 binding site, AK33 provides the promoter
binding region, and that hybridization of AK22 to MJ1 renders the
promoter region double-stranded from 24 to +10. D, the
templates shown in C were transcribed by T7 RNAP, and the
products were resolved by electrophoresis in 20% polyacrylamide gels
in the presence of 7 M urea. The position of the 10-nt
run-off product is indicated to the right.
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As shown in Fig. 1, C and D, recruitment of the
RNAP to the ss template via the Gal4 binding motif (template
b) was not sufficient to activate transcription. In contrast, the
presence of a ds promoter binding region tethered upstream
(template c) resulted in efficient transcription, giving
rise to a spectrum of products that was nearly identical to that
observed from a control template in which the target promoter was
rendered double-stranded by annealing of the complementary NT strand
(template d).
A potential explanation for our failure to observe efficient
transcription from the ss template using the Gal4 binding site is that
the geometry of the Gal4 site relative to the initiation region of the
promoter is not appropriate and prevents correct placement of the
initiation region into the active site. This explanation is diminished,
however, by the finding that the Gal4 binding site did not interfere
with initiation by the fusion protein when the promoter was rendered
double-stranded by annealing of a complementary NT strand
(template e). The presence of an additional promoter binding
region upstream of the ds promoter also had little effect on initiation
at the ds promoter (template f).
Exposure of T7 RNAP to the Isolated Binding Region of the Promoter
Activates the Enzyme to Transcribe a Single-stranded Template--
The
observation that the tethered promoter binding region activated
transcription from the ss promoter, whereas the tethered Gal4 binding
site did not, suggests that the promoter binding region provides other
functions in addition to its ability to recruit the RNAP to a nearby
initiation site. To examine this, we asked whether the addition of the
isolated promoter binding region in trans (as an untethered
ds hairpin loop) could activate transcription by an RNAP that was bound
to the ss template via the Gal4 binding site (Fig.
2). Two stem-loop structures were employed in these studies. RC12 contains the upstream binding region of
the T7 promoter from
5 to
23; RC13 has a similar stability and
structure but involves a sequence that is unrelated to the T7
promoter.

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Fig. 2.
Addition of the isolated binding region of
the promoter activates T7 RNAP to transcribe a single-stranded
template. Left panel, the RC12 oligomer forms a stem-loop
structure that contains the T7 consensus binding region from 17 to
5 flanked by an additional 6 base pairs upstream; RC13 has a similar
structure and stability but contains an unrelated sequence.
Template a consists of the ss template MJ1 bound to the
tethered Gal4 binding region (see Fig. 1C, template
b); template b consists of only the ss template.
Right panel, templates were transcribed either by wild type
T7 RNAP (WT) or by the Gal4:T7 RNAP fusion protein
(F) in the presence of RC12 or RC13, as indicated. Products
were resolved by electrophoresis as in Fig. 1D.
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Strikingly, addition of the isolated binding region to the tethered
Gal4:T7 RNAP activated transcription from the ss template (Fig. 2,
lane 2 versus lane 4). Even more
striking, however, were the observations that stimulation also occurred
with the WT enzyme (lane 1 versus lane
3) and in the absence of the Gal4 binding site (template
b, lanes 7 and 8). The addition of RC13 (the
control stem loop structure) did not lead to activation (lanes
9 and 10) nor did the addition of another stem-loop
structure that deviates from the consensus binding site only at
position
8 (which prevents promoter function (13, 14)) (data not shown).
The Entire Promoter Sequence Must Be Present in the Single-stranded
Template for Activation to Occur--
In the templates described
above, the target promoter in the ss template contained the entire
consensus sequence from
17 to +6. To explore what features of the
target promoter are required to allow activation (e.g. is
the presence of the binding region required, or is the presence of a ss
initiation region sufficient?), we constructed templates in which
various portions of the binding region in the target promoter were
deleted or replaced (Fig. 3). Truncation
of the original template (MJ1) to 24 nt upstream from the start site
(AK18) did not prevent activation by RC12 nor did substitution with a
different sequence upstream of
17 (AK24). However, commencing at
15, substitution of the binding region with an unrelated sequence
prevented activation. While necessary, the binding region is not
sufficient, as additional flanking sequences upstream are also required
(compare templates c and f).

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Fig. 3.
The entire promoter sequence must be present
in the ss template for activation to occur. Left panel, the
ds control template (template a) was formed by annealing
together oligomers AK22 and AK18, as in Fig. 1. Single-stranded
templates (b-i) contained the same sequence in the T strand
(template b) or had substitutions (templates
c-e) or deletions (templates f-i) in the region
upstream of the promoter, as indicated. Right panel, the
templates indicated were transcribed by WT T7 RNAP in the presence or
absence of RC12, as noted, and the products were resolved by
electrophoresis as in Fig. 1D. The position of the 10-nt
run-off product is indicated.
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A Model for Activation of the RNAP by Exposure to the Isolated
Binding Region of the Promoter--
The affinity of T7 RNAP for a
promoter that lacks a ds binding region is very low (Refs. 2, 3, and 5
and see Fig. 5). Activation of the RNAP to transcribe such a template
must therefore result (at least in part) from an increased ability to
bind to and/or retain the template. A possible explanation for the
observations reported here is that exposure of the RNAP to the isolated
binding region reorganizes the enzyme, allowing binding of the ss
template and its entry into the active site. While in principle this
could involve only transient exposure of the RNAP to the binding
region, the affinity of the polymerase for the isolated ds binding
region is even greater than that of the enzyme for a complete promoter
(2, 16, 17), suggesting that once formed, the binary complex may be
quite stable.
For these reasons, and for reasons that will be explained below, we
suggest that binding of RC12 results in the formation of a binary
complex that subsequently binds the single-stranded template. Three
alternative pathways that could account for continued retention of the
ss template and activation of transcription are presented in Fig.
4. First, formation of a stable ternary
complex (consisting of RNAP, RC12, and the ss template) might be
sufficient to allow initiation through cooperative interactions between
the binding and initiation regions. However, this does not appear to be
consistent with the observation that the ss template must also contain
the sequence of the upstream binding region for activation to occur
(see Fig. 3). Alternatively, the binding region present in RC12 may be
denatured within the complex. Annealing of the T strand portion of the
binding region to its complement in the ss template would then result
in the formation of a ds binding region in the target promoter
(i.e. a pss promoter; annealing model). In a third
alternative, the ss template might displace the ds binding region,
allowing it to interact with both the promoter binding region of the
RNAP and with the active site (displacement model). The latter complex
might mimic some of the features of an elongation complex in which
promoter clearance has occurred.

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Fig. 4.
Potential models for activation of T7 RNAP by
exposure to the isolated binding region of the promoter. Exposure
of T7 RNAP (ellipse) to the isolated binding region of the
promoter (RC12) allows binding of the ss template
(T). Once formed the ternary complex may be able to support
transcription through cooperative interactions. Alternatively,
denaturing of the ds binding region in RC12 might allow reannealing
with the upstream binding region in the ss template, leading to the
formation of a pss template (Annealing model). Last, binding
of the ss template may result in displacement of the ds binding region
(Displacement model).
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The 23-bp ds stem portion of RC12 is expected to be quite stable
(predicted
G =
34.6 kcal/mol) and to rapidly
self-anneal even if transiently denatured. Heating and annealing of
RC12 to the ss target sequence in solution did not result in
significant hybridization of the binding region to the ss template,
even when the template was present at a 10-fold molar excess (data not
shown). We therefore conclude that spontaneous annealing of RC12 to MJ1 cannot account for the observed activation. (However, on the basis of
this experiment alone we cannot rule out the possibility that the
polymerase might facilitate separation and annealing of the strands of
RC12 within the ternary complex.)
To investigate the alternative pathways shown in Fig. 4 more directly,
we used a gel-shift assay to examine binding of RC12 and the ss
template (Fig. 5). All three of the
models predict that RC12 will enhance binding and retention of the ss
template. As expected, adding increasing amounts of RC12 to reactions
that contained a fixed concentration of RNAP and labeled ss template resulted in enhanced binding of the ss template (Fig. 5, A
and B).

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Fig. 5.
Binding of the ss template results in
displacement of the ds binding region. A and B,
32P-labeled MJ1 (50 nM) and T7 RNAP (100 nM) were incubated with increasing concentrations of RC12,
as indicated, and the complexes were analyzed by electrophoresis in 5%
polyacrylamide gels under non-denaturing conditions. The fraction of
MJ1 bound (open squares) as a function of increasing RC12
concentration is presented in the bottom panel (B).
C and D, as above, except that the concentration
of RC12 was 50 nM in all samples, and the concentration of
MJ1 increased from 0 to 100 nM (as indicated). The fraction
of RC12 bound (open squares) as a function of increasing MJ1
concentration is presented in the bottom panel (D).
Transcription activity in the samples (filled circles)
was determined by measuring the production of the 10-nt run-off product
as in Fig. 3 and is plotted as a function of MJ1 concentration.
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While the annealing model and the cooperative interaction model predict
that adding increasing amounts of the ss template to complexes that
have bound RC12 should not result in displacement of the ds binding
region (and might even enhance its retention in the case of the
annealing model), the displacement model predicts that addition of
increasing amounts of the ss template should result in loss of the ds
binding region from the complexes. As shown in Fig. 5, C and
D, the latter result is observed. Here, binding of RC12 was
observed in the absence of the ss template, but as the concentration of
the ss template was increased, RC12 was displaced. Moreover, the
transcription activity of the complexes increased with MJ1
concentration and remained at a high level even when nearly complete
displacement of RC12 was observed. These observations are in direct
contradiction of the annealing and cooperative interaction models, but
are in agreement with the displacement model.
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DISCUSSION |
In this work, we have shown that recruitment of T7 RNAP to a ss
template that contains the consensus promoter sequence is not
sufficient to permit initiation of transcription. Strikingly, the
presence of the ds binding region, either tethered to the ss template
some distance away from the promoter or added in trans as a
stem loop structure, activates the RNAP to initiate transcription on
such a template. Gel-shift assays demonstrate that exposure of the RNAP
to the isolated binding region enables the RNAP to bind to the ss
template, but that the binding region is displaced prior to initiation.
From these experiments, we conclude that while T7 RNAP is ordinarily
unable to bind a ss promoter template, exposure of the RNAP to the ds
binding region of the promoter alters the enzyme in such a way as to
enable it to bind and initiate transcription on the ss template. The
observation that the ss template must contain the upstream binding
sequence to be activated (Fig. 3) indicates that the ss template must
replace some of the contacts made by the ds binding region,
not just displace them. Most of the base-specific contacts
made between the RNAP and the binding region of the promoter involve
the template strand (4, 18, 19). Perhaps these interactions are
important in the displacement process or in activation.
Why is the RNAP unable to bind to the ss template unless it is first
exposed to the binding region? Some insight into this question may come
from studies with bacterial RNAPs, which also have promoters with a
bipartite structure comprising an upstream binding region and a
downstream initiation region. The transition to an open complex
involves multiple stages, in which promoter bound as duplex DNA (a
closed complex) is locally melted, and the T strand is led down into
the active site (to form an open complex). Recent structural analyses
indicate that in the open complex the T strand of the initiation region
is enclosed in a channel that is surrounded on all sides by protein and
that movement of region 1.1 of the transcription initiation factor
is required to permit entry of the T strand and of
downstream DNA into their respective binding sites (20-22). In view of
the observations reported here, it would be of interest to determine
whether exposure of the multisubunit RNAPs to the isolated binding
regions of their promoter might induce conformational changes that are
associated with open promoter formation.
The formation of an open complex by T7 RNAP also involves multiple
steps. The initial closed complex (EDC) isomerizes to an intermediate form (EDO1) in which the promoter is melted
from
4 to
1 and subsequently to a fully open complex
(EDO2) that is melted to +2 (23). Kinetic studies indicate
that the enzyme may exist in two conformations, which differ in their
rates of promoter binding and open promoter formation, with the fast
form predominating in most RNAP preparations (24). It has not yet been
determined whether exposure of the enzyme preparation to the isolated
binding region alters the distribution of these two forms.
Based upon these observations, we suggest that unliganded T7 RNAP is
unable to bind to and/or insert the T strand in the initiation region
into the active site and that coordinated changes in the structure and
organization of the transcription complex in response to base-specific
contacts in the binding region are required to capture the T strand or
otherwise stabilize the open conformation. A number of structural
elements in T7 RNAP are known to be involved in promoter binding and
melting. The specificity loop (amino acid residues 740-769) interacts
in a sequence-specific manner with bps in the binding region from
11
to
7 (4, 13, 14, 25). In addition, an AT-rich recognition loop
(residues 93-101) interacts with the promoter 13-17 nt upstream from
the start, and a
-hairpin intercalating loop (residues 245-250)
interacts with the promoter between
5 and
4 and is thought to be
important for promoter melting and directing the T strand into the
active site) (4, 5). Whereas the positions of the AT-rich recognition
loop and the specificity loop remain largely unchanged before and after promoter binding and during the early stages of initiation, the
-hairpin intercalating loop is disordered and becomes visible in the
crystal structure only after promoter binding (4, 26, 27). Speculating
that the unformed
-hairpin loop might occlude the binding site or
otherwise inhibit insertion of the T strand, we examined whether mutant
RNAPs in which this element had been disrupted might be able to
initiate transcription from a ss template. Neither of the mutant RNAPs
that we tested (DelbH, in which residues 236-240 are deleted (28), or
231-241, in which residues 231-241 are deleted (29)) provided this
effect (data not shown); perhaps more substantial changes in this
region are required. Other regions of the RNAP whose organization
changes after promoter binding include the tip of the thumb domain, and
a portion of the fingers domain, which forms part of the downstream DNA
binding site in the elongation complex (4, 26, 27, 30). We have not
tested the effects of mutations in the latter regions.
The templates used in the experiments described here extend only 10 nt
downstream from the start site for transcription. While useful for
studies of transcript initiation, the lengths of these templates are
not sufficient to allow isomerization to a stable elongation complex
(which is not complete until the transcription complexes have extended
10-14 nt downstream). In earlier studies, we had determined that the
presence of the NT strand in the initiation region is important to
isomerization and that transcription on promoters that lack the NT
strand downstream of
4 (pss promoters) results in enhanced release of
products in this size range (7, 31). To examine transcription
from ss templates in more detail, we utilized templates that extend 20 nt downstream and found that the pattern of transcription was nearly
identical to that of the pss promoters (data not shown).