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INTRODUCTION |
Eukaryotic messenger RNA synthesis is a complex biochemical
process that depends on RNA polymerase II and a variety of general and
gene-specific transcription factors. Much information about the
function of the RNA polymerase II transcription complex has been
obtained in reconstituted, in vitro transcription systems, in which the contributions of individual cofactors to rate-limiting steps can be specifically evaluated (1, 2). Stable binding of RNA
polymerase II to the promoter requires minimally the presence of the
general transcription factors
TFIID1 (or TBP), TFIIB, and
TFIIF. Before transcription can begin, the double-stranded structure of
the DNA template surrounding the initiation site on the promoter needs
to be melted into single-stranded DNA in a process that is referred to
as open complex formation (3). Open complex formation and transcription
initiation (i.e. the formation of the first phosphodiester
bond (Refs. 4-6)) depend on the presence of two additional general
transcription factors, TFIIE and TFIIH, and are catalyzed by an
ATP(dATP)-dependent DNA helicase activity associated with
TFIIH (6-9).
Transcription initiation is followed by a short phase that is referred
to as promoter escape. This phase primarily includes the formation of
the first 10-15 phosphodiester bonds of nascent RNA transcripts and is
characterized by functional instability of the RNA polymerase II
transcription complex (10). In the absence of either TFIIH or an
ATP(dATP) cofactor, early RNA polymerase II elongation intermediates
are prone to premature arrest at ~10 to ~14 base pairs downstream
of the transcriptional start site (11, 12). In contrast, further
transcript elongation by very early RNA polymerase II elongation
intermediates that have successfully synthesized transcripts 14 or 15 nucleotides long requires neither TFIIH nor an ATP(dATP) cofactor.
Finally, completion of promoter escape depends on the presence of an
extended region of downstream DNA; digestion of a duplex template
containing the AdML promoter with a restriction enzyme that cuts the
template 35/39 nucleotides downstream of the DNA template has no effect
on initiation but results in arrest by RNA polymerase II at a position
10-15 nucleotides downstream of the transcriptional start site.
However, once polymerase has successfully synthesized ~14 nucleotide
transcripts, digestion of the template with the same restriction enzyme
has no effect on further elongation by RNA polymerase II (13). Based on
these characteristics, we operationally define early RNA polymerase II
elongation complexes that have synthesized transcripts ~15 nucleotides or longer as those that have successfully escaped the promoter.
During the transcription cycle, promoter escape follows immediately
after initiation. There are a number of notable similarities between
these two very early stages of transcription; both share a requirement
for an ATP(dATP) cofactor and depend on the presence of TFIIE and TFIIH
(6, 11, 12, 14), and both are suppressed by mutations in the
same DNA helicase subunit of TFIIH, encoded by the
Xeroderma pigmentosum complementation group B
(XPB) gene (8, 9, 15, 16), arguing that promoter
melting is required for both transcription initiation and promoter
escape and is likely catalyzed by the same mechanism in both steps.
Finally, initiation, like promoter escape, depends on the presence of
an extended downstream DNA region, which has been shown to extend to
somewhere between 23 and 35 nucleotides downstream of the
transcriptional start site (13).
Although previous studies have provided strong support for the model
that interactions between component(s) of the transcription complex and
downstream DNA are important for both initiation and promoter escape, a
variety of important questions regarding the structure and function of
downstream DNA remain unanswered. These include the following. (i) What
are the precise boundaries of the regions required to support
transcription initiation and promoter escape? (ii) Does the requirement
for downstream DNA in promoter escape depend on downstream DNA
sequence? (iii) Is the completion of promoter escape accompanied by a
major rearrangement of the transcription complex? The experiments
presented here provide evidence that the downstream DNA requirements do
not change significantly through the synthesis of the first 14-15
bonds of nascent transcripts, that downstream DNA operates in a
sequence-independent manner, and that a major conformational change in
the transcription complex likely happens at the completion of promoter escape.
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MATERIALS AND METHODS |
DNA Templates--
The primary DNA template used in this study
is M13mp19-AdML, which contains original AdML promoter sequences from
50 to 10 (12). AdML promoter mutants were prepared by the
uracil-containing DNA method for site-directed mutagenesis (17), using
the Bio-Rad Mutagen 2 system. Resulting clones were verified by
sequencing. For transcription, a 444-base pair fragment was produced by
PCR from M13mp19-AdML. The primers were 5'-GACGGCCAGTGAATTCGA and 5'-CCAGCGTGGACCGCTTGC. The resulting DNA fragment, which contains sequences that extend 77 bp upstream and 367 bp downstream of the
transcriptional start site, was gel-purified by agarose gel electrophoresis prior to use in transcription reactions.
RNA Markers--
Hybrid templates were prepared containing the
core T7 promoter fused to AdML promoter sequences with downstream
HaeIIII restriction sites. The T7 promoter sequence was
introduced to the AdML templates by performing PCR using M13mp19-AdML
derivatives as templates. We used an upstream PCR primer consisting
of the T7 promoter: 5'(
25)-ATGGTACCTAATACGACTCACTATAGGGAGAACTCTCTTCCTCTAGAGTCG.
The underscored sequence is a 20-base sequence complimentary to the AdML template strand at promoter positions 1-20. At the 5' end of the
T7 promoter, a six-base "clamp" sequence has been added (18). The
downstream primer was (5'-CCAGCGTGGACCGCTTGC), identical to that used
in the AdML template amplification reactions. The M13mp19-AdML
templates used had HaeIII sites positioned at 34, 40, and 44 relative to the AdML start site. Because T7 begins transcription at
6
relative to the AdML insertion, after digestion of templates with
HaeIII the resulting transcripts are 40, 46, and 50 nt long.
T7-AdML template amplification was carried out in 100 µl with 5 units
of Taq DNA polymerase, 20 mM Tris-HCl, pH 8.0, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM each of dNTPs (dATP, dCTP, dTTP, dGTP), 50.0 pmol of
each primer, and 830 fmol of M13mp19-AdML template DNA. The length of
the PCR product is 398 bp. PCR products were gel-purified by agarose
gel electrophoresis.
RNA Polymerase II and Transcription Factors--
RNA polymerase
II (19) and TFIIH (20) were purified from rat liver nuclear extracts as
described. Recombinant yeast TBP (21, 22) and TFIIB (23) were expressed
in Escherichia coli and purified as described. Recombinant
TFIIE was prepared as described (24), except that the 56-kDa subunit
was expressed in E. coli BL21(DE3)-pLysS. Recombinant TFIIF
was purified as described (25) from E. coli JM109(DE3)
coinfected with M13mpET-RAP30 and M13mpET-RAP74.
Transcription Experiments--
Transcription experiments are
performed in vitro using a reconstituted transcription
system that includes RNA polymerase II, the five general transcription
factors (TBP, TFIIB, TFIIE, TFIIF, and TFIIH), and gel-purified
promoter-DNA. Promoter DNA and proteins are combined in a final volume
of 30 µl to form preinitiation complexes during a 30 °C, 45-min
incubation (11). The incubation buffer contains (final concentrations
are given) 0.3 mM HEPES-NaOH, pH 7.9, 25 mM
Tris-HCl, pH 7.9, 25 mM KCl, 4 mM
MgCl2, 0.2 mM EDTA, 1 mM
dithiothreitol, 0.5 mg/ml bovine serum albumin, 2% (w/v) polyvinyl
alcohol, and 6% (v/v) glycerol. Each reaction included 20 ng of AdML
DNA fragment, 50 ng of recombinant yeast TBP, 10 ng of recombinant
TFIIB, 20 ng of recombinant TFIIF, 20 ng of recombinant TFIIE, 150 ng
of highly purified TFIIH, and 0.01 units of RNA polymerase II.
Digestion of template DNA by PstI or HaeIII
endonuclease was included in various phases of the transcription
experiment by adding 2 µl of a solution containing 0.1-0.5 units of
the respective enzymes. Digestions were carried at 30 °C for the
times indicated, mostly 20 min. Separate experiments were carried to
verify that these conditions allowed for complete digestions.
Transcription was initiated by a labeling mix containing
[
-32P]CTP (3,000 Ci/mmol), a CpU dinucleotide primer,
ATP or dATP cofactor, and other ribonucleoside triphosphates as
described in the figure legends. The final volume of reaction mixes was 35 µl. Transcription was carried at 30 °C for the time indicated in the figure legends. Transcription was stopped by addition of 15 µl
of stop solution containing 100 mM EDTA. A 55-µl loading dye containing 10.0 M urea, 0.025% bromphenol blue, and
0.025% xylene cyanole was added to each sample. Samples were then
heated to 90 °C for 3 min, briefly centrifuged to remove insoluble
particles, and separated on a 25% acrylamide, 3% bisacrylamide, 6.0 M urea gel as described (6) and visualized by
autoradiography. For run-off transcription experiments, reactions were
stopped by addition of 0.05 M EDTA, 0.1 M NaCl,
and 0.5% SDS to the reaction mixture. Precipitation is accomplished by
ethanol followed by a 70% ethanol wash. Dried pellets were resuspended
in 27 µl of solution of 10 M urea, 0.025% bromphenol
blue, and 0.025% xylene cyanol FF, heated at 90 °C for 5 min, then
loaded on urea-containing acrylamide gels for electrophoretic
separation of transcripts.
T7-AdML templates obtained by PCR above were digested with
HaeIII prior to transcription by combining T7-AdML PCR
product, 1 unit of HaeIII, and 1× enzyme buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2,
and 50 mM NaCl) in a 10-µl reaction incubated for 15 min
at 37 °C. The RNA markers were synthesized in vitro as follows; reactions of 50 µl included 50 units of T7 RNA polymerase, 1× enzyme buffer (40 mM Tris-HCl, pH 7.9, 6 mM
MgCl2, 2 mM spermidine, 10 mM
dithiothreitol), 0.1 mg/ml bovine serum albumin, 100 µM ATP, 100 µM UTP, 100 µM GTP, 5 µM cold CTP, 67 nM
[
-32P]CTP, and pre-digested T7-AdML DNA template.
Reactions were incubated for 20 min at 37 °C. Reactions were stopped
by using the same solutions, and protocols as are used to stop RNA
polymerase II transcription reactions by the run-off protocol.
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RESULTS |
Experimental Strategy and Assays for Initiation and Promoter
Escape--
To map precisely the extent of downstream DNA required at
specific stages of early transcription, we generated a series of AdML
promoter-containing constructs that contain restriction sites for the
endonuclease HaeIII at various distances downstream of the
transcription initiation site. The locations of the newly inserted
cleavage sites correspond to promoter positions 26-50 from the start
site in the AdML promoter (Fig. 1). When
treated with HaeIII, these DNA templates acquire new,
shorter ends that differ by 2-nucleotide increments from each other. In
addition, following cleavage by HaeIII, templates are left
with blunt ends, and the 3' end of the templates differs from the
sequence of the original, parental plasmid by at most 2 nucleotides.

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Fig. 1.
AdML promoter mutants with inserted
HaeIII restriction sites in the downstream promoter
region. Promoter sequences begin from the in vivo
transcription initiation site (+1). The sequence titled
AdML is a portion of the AdML promoter region from the
M13mp19-AdML DNA (12). Mutant promoters carrying HaeIII
restriction sites are shown above with the insert locations
indicated at the left (expressed as the distance from the
in vivo initiation site to the HaeIII cleavage
site).
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In our studies of early transcription by RNA polymerase II, we utilized
a transcription system reconstituted with recombinant TBP, TFIIB,
TFIIE, and TFIIF and purified polymerase and TFIIH from rat liver.
Promoter-specific initiation was assayed by measuring synthesis of
abortive, dinucleotide-primed trinucleotide transcripts. As shown
previously, transcription initiation by RNA polymerase II from the AdML
promoter can be primed by a variety of dinucleotides. These
dinucleotides must be complementary to template DNA surrounding the
transcriptional start site (26). We assayed the synthesis of the first
phosphodiester bond of nascent transcripts by measuring synthesis of
trinucleotide transcripts in reactions containing the initiating
dinucleotide CpU and [
-32P]CTP. These nucleotides
support synthesis by polymerase of radioactively labeled CpUpC
transcripts initiated at a position 3 base pairs upstream of the normal
AdML transcriptional start site.
To measure promoter escape, we monitor the synthesis of short
transcripts in reactions containing the initiating dinucleotide CpU,
ATP, UTP, [
-32P]CTP, as well as the RNA
chain-terminating nucleotide 3'-O-methylguanosine triphosphate (3'-O-MeGTP). The maximal transcript length
under these conditions is 18 nucleotides, determined by the insertion of 3'-O-MeGTP at the first G downstream of the initiation
site. Because early RNA polymerase II elongation intermediates that have synthesized transcripts of ~15 nucleotides or longer are considered to have successfully escaped the promoter, formation of the
3'-O-MeGTP terminated transcripts is a useful assay for the
completion of promoter escape (11-13).
The Region of Downstream DNA Required for Initiation Extends to 34 Base Pairs Downstream of the Transcriptional Start Site--
Previous
experiments (13) have shown that initiation is strongly inhibited by
cleaving an AdML promoter-containing template (pDN-AdML) with
PstI, which cuts at position 23/27 and to a lesser degree by
cleaving with SphI or HindIII, which cut at
positions 29/33 and 35/39, respectively. However, these results only
roughly defined the downstream border of the DNA required for
initiation. In addition, because HindIII leaves a 5'
overhang and PstI leaves a 3' overhang, the results of these
earlier experiments could have been affected by the difference in DNA
ends left by the different restriction enzymes used.
In the experiments presented here, we used the abortive initiation
assay described above to compare the activities of the Ad+26 through
Ad+32 templates, with or without prior cleavage by HaeIII.
As shown in Fig. 2, the promoters on all
templates were capable of supporting abortive initiation when not
cleaved with HaeIII, although several (Ad+30 and Ad+34)
appeared somewhat less efficient. Following HaeIII cleavage,
however, very little initiation occurred on the Ad+26 and Ad+28
templates. An intermediate level of initiation was observed following
HaeIII cleavage of the Ad+30, Ad+32, and Ad+34 templates,
whereas HaeIII cleavage had little or no effect on
initiation from the Ad+36 and Ad+40 templates. Thus, DNA downstream of
position 34 is largely dispensable for initiation from the AdML
promoter, and the region of downstream DNA most critical for initiation
extends to 28 base pairs downstream of the transcriptional start
site.

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Fig. 2.
Downstream DNA up to position 34 is necessary
for transcription initiation. Dinucleotide-primed, trinucleotide
formation assay for analysis of the requirements for first
phosphodiester bond formation. Preincubation mixtures were prepared
with twice the regular amounts of all ingredients as described under
"Materials and Methods," except for TFIIE and TFIIH. Following a
30-min preincubation, reactions were divided into two equal portions.
One portion was treated for 15 min with the HaeIII
endonuclease, and the other served as a "no HaeIII"
control. At the end of the 15 min, TFIIE and TFIIH were added to both
reactions for an additional incubation of 15 min. To begin
transcription, a nucleotide mix was added containing (final
concentrations are indicated): 200 µM CpU, 0.5 µM [ -32P]CTP, and 5 µM
dATP. Reactions were stopped after 20 min. The single RNA band shown
corresponds to the CpUpC trinucleotide product.
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During Promoter Escape, Efficient Elongation by RNA Polymerase II
Requires the Presence of DNA Extending ~28 Base Pairs Downstream of
the Transcript 3' End--
To determine the extent of downstream DNA
required for efficient promoter escape, templates that can be cleaved
with HaeIII at positions from 34 to 46 base pairs downstream
of the site of transcription initiation were tested for their abilities
to support promoter escape, with and without prior treatment with
HaeIII. As shown in Fig. 3,
both transcription initiation and promoter escape were supported on all
templates not treated with HaeIII. Cleavage of the Ad+42,
Ad+44, and Ad+46 templates with HaeIII prior to
transcription reactions had very little or no effect on the efficiency
of promoter escape, as measured by synthesis of 18 nucleotide,
3'-O-MeG-terminated transcripts. Thus, DNA downstream of 42 is dispensable for promoter escape. In contrast, the efficiency of
promoter escape was substantially reduced when the Ad+34, Ad+36, Ad+38,
and Ad+40 templates were treated with HaeIII prior to the reactions, indicating that DNA extending to 40 is needed for efficient promoter escape. Notably, the length of the longest major transcripts synthesized following HaeIII cleavage depended on the
position of the HaeIII cleavage sites. Thus, the lengths of
the longest major transcripts were 8, 11, and 13-14 nt following
cleavage with HaeIII at 34, 36, and 38, respectively,
whereas cleavage at 40 allowed synthesis of a reduced level of 18-nt,
3'-O-MeG-terminated transcript.

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Fig. 3.
Downstream DNA up to position 40 is necessary
for completion of promoter escape. Preincubation mixtures were
prepared with twice the regular amounts of all reagents including
template DNA, RNA polymerase II and the five general transcription
factors. After a 45-min incubation at 30 °C to allow the formation
of preinitiation complexes, reaction mixtures were divided into two
equal portions. HaeIII endonuclease (10 units) was added to
one set of reaction mixtures, whereas the other set served as a "no
cleavage" control. After a 15-min incubation at the same temperature,
transcription was initiated by adding the following nucleotides (final
concentration are indicated): 200 µM CpU, 5 µM ATP, 0.5 µM [ -32P]CTP,
5 µM UTP, and 150 µM 3'-O-MeGTP.
Transcription was stopped 10 min later. The numbers
above the lanes indicate the HaeIII
cleavage site in the DNA template. RNA transcript length is indicated
by the arrows to the left.
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In Fig. 3, HaeIII digestion is performed before
transcription initiation. To examine the effect of downstream DNA on
promoter escape alone, we utilized a two-step, pulse-chase
transcription protocol. In the first stage of the reaction, RNA
synthesis was carried out in the presence of limiting
concentrations of radioactive nucleotides, resulting in the formation
of "pre-escaped" transcription complexes containing
3-9-nucleotide-long RNA transcripts. This initiation stage was
followed by a chase phase, in which a large excess of unlabeled
nucleotides was added, allowing further extension of transcripts and
completion of promoter escape. Reaction conditions can be
selectively changed between the two stages, allowing for the
assessment of specific template and cofactor requirements for promoter escape.
In the experiment of Fig. 4, we included
a 15-min incubation with HaeIII to allow cleavage of
downstream DNA between the pulse and chase stages of the reactions.
This experiment was performed using promoters with restriction sites at
positions 36, 38, 40, 42, and 44. Four transcription reactions were
performed with each of the mutant templates; transcription in two of
the reactions included only the labeling step, whereas in the other two
it also included the cold chase phase. In each pair, one reaction was incubated with the HaeIII endonuclease at the end of the
labeling stage. The other reaction served as a "no
HaeIII" control. Transcripts at the end of the labeling
stage of the reaction were 3-9 nucleotides long. The 3-nucleotide-long
product is abortive, and the remaining bands corresponded primarily to
5- and 7-nucleotide-long transcripts. Addition of HaeIII to
the "pulse-only" reactions did not significantly affect the length
or amount of transcripts detected.

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Fig. 4.
Efficient elongation of short, pre-escaped
transcripts requires downstream DNA up to position 40/42.
Preincubation mixtures including the various promoter mutants were
prepared with four times the regular amounts of all reagents. After a
45-min incubation at 30 °C to allow the formation of preinitiation
complexes, initiating nucleotides were added (final concentrations are
indicated): 200 µM CpU, 0.5 µM
[ -32P]CTP, 5 µM ATP, and 0.5 nM UTP. Twenty minutes later, reaction mixtures were
divided into four equal portions. HaeIII endonuclease (10 units) was added to two of the mixtures, whereas the other two served
as "no cleavage" controls. At the end of a 15-min incubation with
the endonuclease, transcription in one HaeIII-treated
reaction and one control reaction was stopped. A cold chase nucleotide
mixture containing (final concentrations are indicated): 200 µM CTP, 100 µM ATP, 100 µM
UTP, and 150 µM 3'-O-MeGTP was added to the
remaining two treatments. These reactions were stopped after 20 min of
additional incubation. The numbers above the
lanes indicate the HaeIII cleavage site in the
DNA template. RNA transcript length is indicated by the
arrows to the left.
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The other two lanes shown for each template are reactions in which a
cold nucleotide chase phase was added following the HaeIII treatment. Chase reactions were performed in the presence of ATP. As in
the first two lanes, one reaction included a HaeIII
treatment and the other was a "no HaeIII" control. In
all the templates in the control lanes, promoter escape was supported
to a similar level, as judged by the level of 18-nucleotide-long,
3'-O-MeGTP-terminated transcripts. In reactions that
included a HaeIII treatment, significant differences
appeared between the various templates. On templates with the
HaeIII restriction sites at positions 36 and 38, promoter escape was substantially suppressed. The 40 promoter showed an intermediate level of inhibition, and the 42 promoter showed only slight inhibition. Promoter escape on the template with a
HaeIII site at 44 was not affected by the endonuclease.
Therefore, in the two-step promoter escape experiment, DNA up to
position 40/42 from the transcription initiation site appears critical
for efficient promoter escape. This result is in full agreement with
the result obtained in the single-step promoter escape experiment (Fig.
3).
The experiment presented in Fig. 4 provides additional information
regarding transcription complexes that became arrested at
promoter-proximal positions. The reduction in the level of promoter
escape is accompanied by an increase in the level of promoter proximal
arrest. This is evident by a new pattern of arrested RNA transcripts
11-15 nucleotides in length formed in reactions containing templates
cut at positions 36 through 42. Small, but highly reproducible
differences in the size distribution of arrested transcripts formed on
the various templates can be recognized. Arrested transcripts were
primarily 11 and 12 nucleotides in length on the 36 promoter, 12 and 13 nucleotides for the 38 template, and 13 and 14 nucleotides for the 40 template. On the 42 template, which is only slightly affected by
HaeIII treatment, arrested transcripts were primarily 14 and
15 nucleotides in length.
Because transcription is primed with CpU at position
3 relative to
the in vivo initiation site, the observed RNA length less 3 nucleotides corresponds to the precise location of the RNA polymerase catalytic site at the time when its progress was stopped. The distance
between the end of the downstream DNA and the size of paused
transcripts, as seen in Fig. 4, is ~28 ± 2 nucleotides for all
templates where escape was suppressed by HaeIII treatment. These results suggest that, during the individual steps of the promoter
escape stage, the precise position of the polymerase is in phase with
the distal end of the required downstream DNA.
Downstream DNA Functions in a Sequence-independent
Manner--
During promoter escape, the downstream DNA region does not
function as a template for transcription as it is at a distance of more
than 20 nucleotides downstream from the catalytic site of the
polymerase. Instead, it is likely that the downstream region forms
critical contacts with component(s) of the transcription complex. These
interactions might directly involve TFIIH, based on evidence from
UV-induced protein-DNA cross-linking studies (27, 28) and functional
studies (29). To begin to address the question whether downstream DNA
functions in a sequence-dependent or sequence-independent
manner, we introduced a series of sequence mutations in the downstream
region of M13mp19-AdML, substituting every base pair in promoter
positions 25 through 42 (Fig.
5A). To increase the odds of
perturbing sequence-specific interactions, we have consistently
substituted purines for pyrimidines and vice versa.

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Fig. 5.
Sequence mutations in the downstream DNA
region have little effect on ATP-dependent promoter
escape. A, sequence mutations inserted in the
downstream region of the AdML. M13mp19-AdML DNA was used as the
template for mutagenesis (labeled AdML, top). The
mutated promoters are labeled by the position of the mutation relative
to the transcription initiation site (+1). The substituted
nucleotides in each derivative are highlighted in bold.
B, transcription experiment using the AdML sequence mutants.
Transcription was performed according to the two-step protocol (see
"Materials and Methods" and Fig. 3). After formation of
preinitiation complexes and short RNA chains, 100 mM UTP,
CTP, 150 mM 3'-O-methyl-GTP, and 100 mM either ATP or ATP S were added to each reaction to
allow RNA extension and promoter escape.
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To determine whether ATP-dependent promoter escape is
sensitive to downstream DNA sequence, we utilized the two-step promoter escape protocol described in Fig. 4. Transcription complexes were first
assembled on the various promoters (three identical reactions for a
single promoter mutant). Following the formation of
5-9-nucleotide-long transcripts, the nucleotides were added to extend
the RNA transcripts to a maximal length of 18 nucleotides, limited by
including 3'-O-MeGTP in the assay. Then, one reaction from
each set was stopped, and the other two were treated with cold chase
nucleotide mixtures: one that contained ATP and the other that
contained ATP
S.
As can be seen in Fig. 5B, formation of 5-9-nucleotide
transcripts was quite comparable in all templates. Comparison of the 18-nucleotide-long, 3'-O-methyl-GTP-terminated transcripts
in the extension reaction shows that, in the presence of ATP, all templates supported promoter escape efficiently. When the
ATP-dependent step was suppressed by adding high levels of
ATP
S, formation of the full-length, 18-nucleotide transcripts was
inhibited to a similar extent in reactions performed with all
promoters. The appearance of promoter-proximal arrested transcripts in
the size range of 11-14 nt was very comparable in all templates. These results indicate that ATP-dependent formation of
pre-escaped (5-9 nt long) and fully escaped transcripts (18 nt long)
transcripts, as well as ATP
S inhibition of promoter escape do not
depend on downstream DNA sequence, providing strong evidence that
downstream DNA does not operate in a sequence-specific manner during
promoter escape.
Evidence for a Conformational Change in the Early Elongation
Complex at the Completion of Promoter Escape--
As mentioned above,
published functional (29) and structural (27, 28) data suggest that
TFIIH physically occupies DNA downstream of the active site of the
polymerase. An explanation for our observation that RNA polymerase II
that has not yet escaped the promoter tends to arrest transcription
~28 nt proximally to the 3' end of the available template may be that
TFIIH bound to the downstream DNA blocks further elongation by the
enzyme. If this is the case, we should be able to determine when TFIIH
(or other template-associated components of the transcription complex) are released by determining when RNA polymerase II gains the capacity to synthesize transcripts that extend to the end of the DNA template.
The downstream length of template was manipulated by the use of
HaeIII endonuclease restriction sites inserted incrementally between positions 40 and 50 downstream of the AdML promoter (Fig. 1).
As shown earlier in Figs. 3 and 4, the removal of downstream DNA
proximally to 40 causes arrest of early transcription, whereas those
cut downstream of 40 have little or no effect on the formation of 18-nt
transcripts. Therefore, constructs with restriction sites in the 40-50
range would be suitable to determine whether the polymerase can utilize
the entire downstream region as a template.
In the experiment shown in Fig. 6, DNA
templates with HaeIII restriction sites at 34, 44, 46, and
50 were digested with HaeIII, and incubated with the general
transcription factors and RNA polymerase to allow pre-initiation
complexes to form. Transcription was initiated by the addition of a
nucleotide mixture containing CpA, ATP, UTP, GTP, and
[
-32P]CTP, allowing for the formation of a full-length
runoff transcripts.

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Fig. 6.
Lack of requirement for a 28-nt downstream
spacing in elongation following the completion of promoter escape.
The ability of transcription complexes to synthesize full run-off
transcripts after the completion of promoter escape was examined. AdML
DNA templates with the indicated restriction sites (see Fig. 1 for
complete sequences) were incubated with HaeIII followed by
incubation with basal factors to allow pre-initiation complex
formation. Nucleotides were added in excess, as described under the
"Run-off Transcription Assays" under "Materials and Methods."
RNA markers of defined lengths were synthesized using T7 RNA polymerase
as described under "Materials and Methods."
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To accurately determine whether transcripts produced under the
experimental conditions correspond to the full runoff length of the
template, we prepared a series of sequence-specific RNA size markers.
These markers were created by using a bacterial T7 RNA polymerase
promoter fitted with AdML sequence downstream of 1, including the
HaeIII restriction sites as in the M13mp19-AdML series.
Lanes 1-3 show the run-off products generated by
bacterial T7 RNA polymerase and the size indicated by
arrows, corresponding to 40, 46, and 50 nucleotides in
length. Lanes 4-7 show transcripts synthesized
by RNA polymerase II from AdML promoters that were cleaved at 50, 46, 44, and 34, respectively. In each of lanes 4,
5, and 6, there is a prominent transcript that
corresponds to the maximal runoff size allowed by the shortened
template. The template cleaved at 34 (lane 7)
lacks any apparent transcripts, because of promoter proximal arrest of
the transcription complex prior to the formation of transcripts 14/15
nt long (see Figs. 2 and 3). The short transcripts produced under these
conditions migrated all the way through the gel to the lower tank (data
not shown). This lane provides an internal control for the
products that resolve on lanes 4-6.
Several premature stops in elongation can be seen in lanes
4-6, indicated by shorter RNA transcripts, with the most
prominent one ~36 nt in length. For the most part, these are of the
same size in all three lanes and do not appear to display an obvious regularity of recurrence. The nature of these stops remains to be
determined, although these may indicate natural pause sites in the
template (30, 31). More importantly, an RNA band of a size that would
indicate a fixed spacing of 25-28 nt from the distal edge of the three
templates cannot be identified. These results suggest that downstream
DNA is not physically occupied after the formation of the 14th or 15th
phosphodiester bond as it is prior to that point, because full runoff
transcripts can be produced. These results are consistent with the
hypothesis that a conformation change occurs after the completion of
promoter escape at 14/15 and that the change likely involves the
release of TFIIH.
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DISCUSSION |
In the experiments described here, we utilized linear DNA
templates that differ in length by 2-nucleotide increments to obtain an
accurate measure of the downstream DNA that is required during very
early transcription. First, we determine that downstream DNA required
to carry out transcription initiation (first phosphodiester bond
formation) extends to ~34 nucleotides (Fig. 2) and that downstream DNA required for complete promoter escape extends to ~42 nucleotides from the transcription start site (Figs. 3 and 4). These numbers are
consistent with the previously estimated 35-50 range for promoter escape and 23-35 range for initiation (13). The polymerase is not
committed to elongation of transcripts until the promoter escape stage
is completed; therefore, the 42-nucleotide boundary defines the distal
end of the AdML core promoter.
The experiments presented here demonstrate that the early-elongating
polymerase requires template DNA that extends 28 nucleotides downstream
of its catalytic site, and that this requirement is most likely
continuous through the entire phase of promoter escape. Therefore, the
distal boundary of required DNA is dynamic, moving further downstream
as the polymerase synthesizes the first 14-15 phosphodiester bonds of
nascent transcripts. At the last step of promoter escape, downstream
DNA extending to positions 40/42 is required.
A particularly intriguing question is the nature of the biochemical
mechanism underlying the downstream DNA requirement. The ~28-nucleotide spacing between the position of the polymerase and the
distal end of required DNA could be used for physical contact between
RNA polymerase or other components of the transcription complex and the
DNA template. A likely hypothesis is that downstream DNA is required to
bind TFIIH, allowing its DNA helicase subunits to facilitate promoter
melting required for initiation and promoter escape. This hypothesis is
supported by evidence regarding the direct involvement of the TFIIH
helicase in initiation and promoter escape (8, 9, 12, 16, 29) and with
cross-linking experiments showing that the TFIIH helicase binds the
template downstream to RNA polymerase (27, 28). Recently it was shown
that TFIIH action in both transcription initiation and promoter escape
requires the presence of specific downstream DNA regions (29).
These findings are consistent with the idea that TFIIH binds to
downstream DNA and suggest that it slides along the template ahead of
the polymerase until promoter escape is completed. In this light, the
arrest of early elongation complexes 28 nt from the distal end of the
template can be explained in terms of the inability of the bound TFIIH
to physically translocate further downstream, thereby forming a
physical impediment to the translocation of the polymerase.
Moreover, our current and recently published results (29) are also
consistent with a model by which the conformation of the very early
transcription complex remains relatively unchanged through the
completion of promoter escape. The downstream spacing required for
initiation is very similar to that observed for the promoter escape
stage, but not identical, as some inhibition of initiation is seen when
the templates are cleaved at promoter positions 32 and 34. This
difference might be indicative of a rearrangement shortly after the
synthesis of the first phosphodiester bond and before nascent RNA
transcripts reach 8-9 nt in length. New information regarding possible
changes in composition of the early elongation complex during initial
extension of RNA transcripts would be extremely helpful in correlation
with these findings.
As it becomes apparent that downstream interactions play an important
role in early transcription, it becomes essential to find out whether
these interactions reflect a sequence-specific mechanism. To address
this question, we have taken a minimalist approach: introducing strong
perturbations in template sequence using a relatively small set of
substitutions. Because our starting point is a template where there is
strong dependence on downstream DNA in early transcription, it is
likely that such changes will have an effect if the mechanism is indeed
sequence-specific. Clearly our results shown in Fig. 5 do not indicate
that. Therefore, it is our conclusion that the downstream DNA
requirement in initiation and promoter escape reflects a
sequence-independent mechanism. This possibility is consistent with a
sliding model for TFIIH during these steps, indicating a general
mechanism that likely functions in other promoters as well.
After carefully characterizing the downstream DNA requirements during
promoter escape, an interesting follow-up question was when, during
early transcription, does the DNA ahead of the polymerase become fully
available as a template? Based on the evidence from cross-linking
studies that TFIIH directly contacts the downstream DNA region, this
information should help determine when TFIIH has left the early
elongation complex. Failure of the polymerase to use the entire
downstream template for transcription by the end of the promoter escape
stage could indicate a continued association of TFIIH with the
transcription complex. We have addressed this question in Fig. 6, and
observe no indication for a "fixed spacing" impediment as is the
case in the steps that precede the completion of promoter escape. These
results are consistent with a model in which TFIIH leaves the early
elongation complex when the promoter escape stage is complete,
i.e. the DNA-helicase activities associated with TFIIH are
no longer required.
Fig. 7 is a schematic illustration of a
model describing the continuous role of downstream DNA and TFIIH in
promoter escape. According to this model, TFIIH slides forward during
the transition from initiation through the completion of promoter
escape, while occupying a template space downstream of the polymerase
and assisting in promoter melting. Because this TFIIH action is
continuously required until the completion of promoter escape, removal
of downstream DNA, ATP, or TFIIH from the transcription complex at any
of these steps is expected to stall the transcription complex. At the
ending point of promoter escape, the nascent mRNA is ~15
nucleotides in length or larger, and ATP, downstream DNA, and TFIIH are
no longer required for its further extension. When transcription is
carried out using linear templates, the polymerase can utilize the
entire template length for transcription with no spacing required to
its distal end. Therefore, it could be expected that the early elongation complex undergo a major conformation change at the completion of promoter escape, probably involving the release of TFIIH
and TFIIE.

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Fig. 7.
A model describing proposed interactions
between TFIIH and downstream DNA during initiation and promoter
escape. During initiation and promoter escape, TFIIH slides ahead
of the polymerase, maintaining a relatively fixed spacing of 28-34
nucleotides between the RNA polymerase II catalytic site and the distal
end of the required downstream DNA. TC, transcription complex;
Cat, the catalytic site of the polymerase. The
numbers below the DNA template refer to the
nucleotide position relative to the initiation site at the promoter.
Open circles indicates suggested contacts between the
transcription complex and downstream DNA.
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