Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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We investigated transcript initiation and early
elongation by RNA polymerase II using templates mismatched between Both Escherichia coli RNA polymerase (reviewed in Refs.
1 and 2) and RNA polymerase II (3, 4) pass through a stage of abortive
initiation between the formation of the initial phosphodiester bond and
the establishment of a stable ternary transcription complex. The RNA
chains released during abortive initiation by either RNA polymerase are
generally from 2 to 10 nt1
(see, for example, Refs. 4-6), although aborted transcripts as long as
15 nt have been reported (7). The partitioning of initiations between
abortive and productive pathways varies among promoters
(e.g. see Refs. 7-10), but the mechanistic basis for this
difference is not well understood. A number of recent results have
emphasized the importance of the strength of the RNA-DNA hybrid in
maintaining stability for transcription complexes during RNA chain
elongation (Refs. 11-16; reviewed in Ref. 17). Thus, it is possible
that the transcript-template hybrid is a major factor in determining
the relative levels of abortive initiation among promoters. However,
the study of Rice et al. (18), which involved RNase
digestion of RNA polymerase II ternary complexes, indicated that the
RNA-DNA hybrid within the transcription complex is very short ( In order to further analyze the role of template sequence in abortive
initiation, we have extended the observation that templates mismatched
in the vicinity of the transcription start site ("bubble templates") support specific transcript initiation by RNA polymerase II with the addition of only TBP and TFIIB (Ref. 23; see also Refs.
24-26). We found that priming RNA synthesis with an appropriate dinucleotide allows transcript initiation on bubble templates by RNA
polymerase II alone. We have therefore been able to examine abortive
and productive transcript initiation in the complete absence of the
general transcription factors. These studies have allowed us to
conclude that the level of abortive initiation by RNA polymerase II is
at least partly determined by interactions of the polymerase with the
transcript and/or the template, independent of transcription factors.
Our results are consistent with a model in which the strength of the
RNA-DNA hybrid is an important component of transcription complex
stability during the transition from initiation to early elongation.
RNA Polymerase II Purification--
Calf thymus was obtained
frozen from ANTEC (Tyler, TX). RNA polymerase IIA was purified through
the DE-52 chromatography step essentially as described by Hodo & Blatti
(27), followed by affinity purification on an anti-RNA polymerase II
C-terminal domain antibody column as described by Thompson et
al. (28). Briefly, 1.5 kg of thymus was ground and clarified in a
4-liter volume; nucleic acid and associated proteins were precipitated by the addition of polyethyleneimine to 0.05%. RNA polymerase II was
extracted from the polyethyleneimine pellet, precipitated with ammonium
sulfate, and resuspended in sufficient buffer D (50 mM
Tris, pH 7.9, 25% glycerol, 2 mM EDTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride) to reduce the ammonium sulfate concentration to 150 mM prior to batch adsorption at 20 mg
of protein/ml on DE52 (Whatman) for 1.5 h. The DE52 was washed
with Buffer D containing 150 mM ammonium sulfate until the
absorbance declined to base line and then step-eluted with Buffer D
containing 0.5 M ammonium sulfate. Peak fractions
containing polymerase II activity were pooled, concentrated by ammonium
sulfate precipitation, and adsorbed to 8WG16 antibody immobilized to
Sepharose (28). The antibody column was eluted four times with 1 volume
of 40% glycerol, 0.5 M ammonium sulfate (28); the last
elution, done for 12 h at 4 °C, provided all of the RNA
polymerase II used in this study. Polymerase pools were concentrated in
Centriprep 30 concentrators (Amicon) and dialyzed to 50 mM
Tris-HCl, pH 7.9, 25% glycerol, 150 mM ammonium sulfate,
0.2 mM EDTA, and 1 mM dithiothreitol prior to
storage at Templates for in Vitro Transcription--
To form bubble
templates, 86-nt oligos (from Operon Technologies; see Fig. 1) were
mixed at 2 mg/ml total DNA concentration with a 10% molar excess of
the nontemplate DNA strand in 25 mM Tris, pH 7.9, 8 mM MgCl2, 50 mM KCl. Templates were
reannealed by heating to 99 °C for 3 min followed by cooling, first
to 90 °C for 10 min and then to room temperature at 0.2 °C/min.
Reannealed templates were resolved on 12% nondenaturing polyacrylamide
gels and eluted from gel slices by diffusion in 0.5 M
ammonium acetate, 0.5% SDS, and 2 mM EDTA followed by
ethanol precipitation. To remove acrylamide and other impurities, the
templates were gel-filtered through a Sephadex G-50 spin column and
ethanol-precipitated prior to use.
In Vitro Transcription with Pure RNA Polymerase
II--
Transcription conditions were similar to those of Pan & Greenblatt (23). Bubble duplex templates (0.22 pmol) were incubated with 0.18 pmol of highly purified RNA polymerase II in a 10-µl reaction volume in transcription buffer (10 mM Tris-HCl, pH
7.9, 10% glycerol, 8 mM MgCl2, 75 mM KCl, and 5 mM
Transcription reactions were terminated by the addition of 85%
phenol/CHCl3 and digested with calf intestinal alkaline
phosphatase to reduce background levels as described (4). Reactions
containing RNase A were treated with proteinase K (Life Technologies,
Inc.) at 1 mg/ml for 10 min prior to phenol/CHCl3
extraction. RNAs were resuspended in 7.5 µl of 90% formamide loading
buffer; heated to 95 °C for 5 min; and electrophoresed in 28%
polyacrylamide (25:3 acrylamide:bisacrylamide), 7 M urea
gels with TBE (0.089 M Tris, 0.089 M borate, 2 mM EDTA, pH 8.4) running buffer at 13 watts for 16-20 h
until the bromphenol blue nearly reached the bottom of 48-cm plates.
In Vitro Transcription in Extracts--
RNA polymerase II
preinitiation complexes were prepared by incubation of either bubble
templates or double-stranded DNAs in HeLa cell nuclear extract followed
by gel filtration through Bio-Gel A-1.5m (Bio-Rad) essentially as
described (4), except that transcription buffer (see above, containing
1 mM dithiothreitol rather than 5 mM
Wet gels were scanned by a PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA) and analyzed using ImageQuant software. After drawing a
line from the transcript at C15 through the short
transcripts, the peak areas were calculated. The intensity of each band
was corrected by dividing by the number of radioactive CTP and UTP
residues present in that particular transcript, which allowed us to
calculate the relative abundance of all of the transcripts in a gel
lane. Blockage at a particular position (formally analogous to
termination efficiency) was computed by taking the number of
transcripts at that position and dividing by the sum of those
transcripts and all longer transcripts.
Our laboratory has studied abortive initiation by RNA polymerase
II at a variety of promoters using HeLa nuclear extracts as the source
of the transcriptional machinery. This work revealed that the
efficiency of clearance can vary significantly among promoters (4, 10).
In order to further explore the molecular basis of this effect, we took
advantage of the fact that the general transcription factor
requirements for initiation by RNA polymerase II are considerably
simplified with templates that are mismatched (23-26) or easily
denatured (25, 30) at the initiation site. We began our experiments
with a variant of the AdMLP in which bases 9
and +3 (bubble templates). Highly purified RNA polymerase II alone was
able to initiate transcription specifically on these templates in the
presence of dinucleotide primers. The length distribution of abortively
initiated RNAs was similar for purified RNA polymerase II on bubble
templates and polymerase II on double-stranded templates in HeLa
nuclear extracts. Increasing the U content in the initial portion of
the transcript caused similar increases in abortive initiation for
transcription of bubble templates by pure polymerase and
double-stranded templates in extracts. Thus, the level of abortive
initiation by RNA polymerase II is at least partly determined by
interactions of the polymerase with the transcript and/or the template,
independent of the general transcription factors. Substitution of
5-bromo-UTP for UTP reduced abortive initiation on bubble templates, consistent with the idea that transcription complex stability during
early elongation depends on the strength of the initial RNA-DNA hybrid.
Interestingly, transcription of bubble templates in HeLa extracts gave
very high levels of abortive initiation, suggesting that inability to
reanneal the initially melted template segment inhibits transcript
elongation in the presence of the initiation factors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 base
pairs) and not a major factor in transcription complex stability. The
RNA polymerase II initiation process on linear DNA templates requires,
at minimum, TBP, TFIIB, TFIIF, TFIIE, and TFIIH in addition to RNA
polymerase II (reviewed in Ref. 19). The interaction of these general
transcription factors with the template and/or the transcript could
also strongly influence the abortive initiation process, and indeed,
direct effects of TFIIE and TFIIH on the initiation/elongation
transition have been reported (20-22).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. The RNA polymerase II was >95% pure and
essentially all in the IIA form as assayed by SDS-PAGE. The polymerase
was free of detectable RNase activity.
-mercaptoethanol) supplemented with 200 µg/ml acetylated bovine serum albumin. After 30-min preincubations at 37 °C, transcription was initiated by adding 2.5 µl of a nucleotide mix. Final substrate concentrations in
the reactions were 1 mM of dinucleotide primer and, unless otherwise indicated, 10 µM each of CTP and UTP; in some
reactions, dATP was also present at 10 µM. In most
experiments, both CTP and UTP were 32P- labeled (NEN;
specific activity in the reaction was 40 Ci/mmol). For sarkosyl and
heparin challenge experiments, 0.5 µl of water, 1.3% sarkosyl, or
2.6 mg/ml heparin was added to the 10-µl preincubation reactions
30 s prior to the addition of the initiating or chase nucleotides,
as indicated in the figure legends. Chase reactions were performed by
adding 1.38 µl of 5 mM GTP, UTP, and CTP in transcription
buffer to a 12.5-µl reaction at the indicated times. 5-Bromo-UTP and
the sodium salts of sarkosyl and heparin were obtained from Sigma.
Nuclease digestions were performed on transcription reactions after the
addition of
-amanitin to 2 µg/ml. RNase H (U.S. Biochemical Corp.;
10 units/ml) or RNase A (Sigma; 10 µg/ml) was then added and
incubated for 10 min at 37 °C.
-mercaptoethanol) was used as the column buffer and preinitiation
complexes were assembled for 30, not 20, min. Template concentration in
the assembly reactions, for both bubble duplexes and double-stranded
DNAs, was 19 µg/ml. The double-stranded DNA template, used as the
intact plasmid, was
pML20-40.2 This construct is
based on the pML20 plasmid (29) but differs from pML20 downstream of
the DraI site beginning at +18. The sequence of pML20-40
from
41 to +45 is given in Fig. 1. Note that it is identical, outside
of the nonpaired region, to that of the adenovirus major late promoter
(AdMLP) bubble template. The gel-filtered preinitiation complexes were
either used immediately or stored for 1 day at
80 °C prior to use.
Reaction conditions and sample preparation methods were identical to
those given above for the pure RNA polymerase reactions except that
dATP was generally present at 10 µM (see Fig. 2).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9 to +3 are mispaired
(Fig. 1). This bubble duplex template was
based on the work of Pan and Greenblatt (23), who showed that an AdMLP
with a
9 to +3 unpaired region supported strong in vitro
promoter activity when assayed with RNA polymerase II, TBP, and TFIIB.
We found that RNA polymerase II alone would initiate transcription
accurately on this template, provided that a dinucleotide primer is
provided (see below). Thus, we have been able to study the effects of
template sequence on RNA polymerase II promoter clearance in the
absence of general transcription factors.
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Fig. 1.
Transcription templates used in this
study. The sequence of the double-stranded AdMLP template, from
41 to +45 relative to the normal transcription start site, is shown
at the top. The AdMLP bubble template contains essentially
the same template strand sequence but is mismatched from
9 to +3. The
AdUMut bubble template differs from the AdMLP bubble template by two T
to C changes (nontemplate strand) at +4 and +6. The sequence of the
mismatched nontemplate strands in the bubble templates
(5'-AAGCAGAAACGA-3') differs from the
9 to +3 template used by Pan
and Greenblatt (23) at the fourth base from the 5'-end, which was
changed from T to C to prevent bidirectional priming by the ApC
dinucleotide.
Pure RNA Polymerase II Directs Initiation Events on Bubble Duplex
Templates in the Absence of General Transcription Factors--
As a
control for the bubble duplex reactions, we prepared preinitiation
complexes on conventional double-stranded DNAs bearing the AdMLP.
Templates were incubated in HeLa extracts and purified by gel
filtration to remove most of the contaminating NTPs. Initiation on
these templates may be primed with either the ApC or CpA dinucleotides, which pair with the +1/+2 and 1/+1 positions on the template strand
(see Fig. 1 and Refs. 3, 4, and 31). To simplify discussion, we will
identify transcripts produced with either dinucleotide primer by their
3'-ends, using +1 as the normal start site. The addition of limiting
levels of labeled UTP and CTP to ApC- or CpA-primed preinitiation
complexes on double-stranded templates allowed initiation and
transcript elongation until +15, where a G residue is required
(C15 RNAs; lanes 11-12 and
29-30 of Fig. 2). Some
complexes continued elongation to +20, where ATP is required,
presumably because of the presence of trace levels of GTP in the other
nucleotides. All transcripts longer than 3 nt were made by RNA
polymerase II, since their synthesis was abolished by 2 µg/ml
-amanitin (lanes 13 and 31). As
expected from earlier work with preinitiation complexes assembled in
crude nuclear extracts (4), some ApCpU and CpApC synthesis was
amanitin-resistant.
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Transcript initiation by RNA polymerase II on double-stranded templates normally requires either ATP or dATP as an energy source (3, 32, 33) and is sensitive to the detergent sarkosyl at 0.05% (34) or the polyanion heparin (see, for example, Ref. 35). There was no amanitin-sensitive transcription of the double-stranded AdMLP template when dATP was absent from the reaction (Fig. 2, lanes 14 and 32) or when 0.05% sarkosyl or 100 µg/ml heparin was added before the NTPs (lanes 15, 16, 33, and 34). All of the RNAs longer than about eight bases chased efficiently when GTP and excess CTP and UTP were added to the original reactions (lane 18; data not shown for CpA priming), while the shorter RNAs appeared to be abortively initiated. These results agree with our earlier studies on this system (4, 10). The polymerase II ternary elongation complex is not expected to be sensitive to sarkosyl or heparin, and we saw no effect of these reagents on the elongation competence of complexes stalled downstream of U7 (lanes 19 and 20; data not shown for CpA priming). Transcription of the double-stranded templates was essentially complete in 5 min (compare lanes 11 and 12, and compare lanes 29 and 30).
When pure RNA polymerase II alone was incubated with the AdMLP bubble template, the dinucleotide primer ApC, and limiting levels of labeled UTP and CTP, synthesis of amanitin-sensitive short RNAs was observed (Fig. 2, lanes 1-3). Similar results were obtained with pure RNA polymerase II and the CpA primer on the bubble templates (lanes 23-25). No RNA was synthesized in the pure polymerase reactions when ATP, CTP, and UTP were used as substrates without a dinucleotide primer (data not shown). Most of the RNAs made in the pure polymerase reactions resulted from pausing at or upstream of +15. Some RNAs of >15 nt were also obtained. Possible origins for these RNA will be discussed below.
Essentially no RNA was made in the pure polymerase reactions when either 0.05% sarkosyl or 100 µg/ml heparin was added before the NTPs (lanes 5, 6, 27, and 28). This is consistent with a true initiation event. As expected, transcription by pure polymerase II on the bubble templates did not require ATP or dATP (compare lanes 2 and 4 or lanes 24 and 26). Considerably more RNA was made by pure RNA polymerase II on the bubble templates if the 5-min reactions were extended to 30 min (compare lanes 1 and 2, and compare lanes 23 and 24), in contrast to the case of transcription of the double-stranded templates in extracts. Chase of dinucleotide-primed pure polymerase reactions with GTP, CTP, and UTP resulted in production of the expected U20 product (Fig. 2, lane 8; data not shown for CpA primer). Elongation resumed for complexes with RNAs as short as 9 nt, but in no case were the pure RNA polymerase II ternary complexes fully active for continued RNA synthesis. Typically, 40-75% of dinucleotide-primed C15 complexes made by pure polymerase II on the bubble templates could resume transcription upon chase. The failure of complexes in the pure polymerase system to chase quantitatively was not simply the result of the longer reaction times used on the bubble templates, since 30-min initial reactions with double-stranded templates yielded C15 complexes that were fully active in a subsequent chase (lane 22). In contrast to initiation, transcript elongation on the bubble templates by pure polymerase II was not sensitive to sarkosyl or heparin (lanes 9 and 10; data not shown for CpA primer).
To determine whether the short transcripts that could not be chased in
the pure polymerase reactions were released from ternary complex, we
performed gel filtration with Sephacryl S-200 on ApC-primed reactions.
Ternary complexes should elute in the void volume, whereas released
transcripts should appear in the included fractions in this experiment.
As seen in the left part of Fig.
3A, RNAs 7 nt and shorter
appeared exclusively in the later eluting included fractions,
indicating that these RNAs were released from the transcription complex. Active ternary complexes were almost entirely confined to the
void volume. In the experiment in Fig. 3A, 75% of the
C15 complexes that were excluded from the column could
chase (lanes 4 and 5). Presumably, the
failure of some transcripts to chase in lane 5 resulted from ongoing transcript release by complexes halted at
C15 prior to the pooling of fractions and the addition of
chase NTPs. A very small proportion of active complexes trailed into
the initial included fractions (lanes 6 and
7). The void volume fraction contained only RNAs 7 nt or
longer, and at least some of the RNAs of each length could chase. In
contrast, essentially all of the RNAs in the included fractions failed
to chase. These results are very similar to those obtained upon gel
filtration of dinucleotide-primed RNAs made on double-stranded
templates by RNA polymerase II in extracts, except that in the latter
case all of the complexes running in the void volume were active in
transcript elongation upon subsequent chase (3, 4).
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As noted above, RNAs longer than 15 nt were observed in transcription reactions with pure RNA polymerase II in the absence of GTP. Some of these >15-nt RNAs may have resulted from GTP contamination in the other nucleotides, but not all of them can be explained in this way. In particular, many of these RNAs did not comigrate with transcripts produced by read-through of the G-stop on the double-stranded templates. All of the >15-nt RNAs must be polymerase II products, since their synthesis was amanitin-sensitive. While we have not further investigated the origins of these anomalous transcripts, we suspect that they resulted from the addition of NTPs to transcripts released from the template along with the RNA polymerase in binary complexes. This supposition seems reasonable, since Johnson and Chamberlin (36) demonstrated that binary complexes of polymerase II and longer RNAs could cleave these RNAs in the presence of TFIIS and then add a limited number of bases to the newly generated 3'-ends.
Increased Abortive Initiation by RNA Polymerase II Occurs during
Transcription of a Variant AdMLP Template with a More U-rich Initially
Transcribed Region--
We showed that a mouse -globin promoter
supported much higher levels of abortive initiation and correspondingly
lower levels of productive transcription when compared with the AdMLP
(10). In a series of subsequent experiments, we
found3 that simply making the
first six transcribed bases of the AdMLP identical to the same six
bases of the globin promoter resulted in a template that supported high
levels of abortive initiation, approaching those seen with the intact
globin promoter. Only two base changes, at positions +4 and +6, were
needed to match the AdMLP and globin promoter over this initially
transcribed region. In order to explore the role of template sequence
alone in this effect, we synthesized a variant of the AdMLP bubble
template, called AdUMut, incorporating the two base changes just
discussed (see Fig. 1). The results of transcribing this template with
either pure RNA polymerase II or HeLa nuclear extract are shown in Fig. 4. The AdMLP bubble template was
transcribed as a control.
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As in the case of the AdMLP template, transcription of the AdUMut template with either dinucleotide primer, pure RNA polymerase II, and labeled UTP and CTP resulted in elongation to the G stop at C15, with some production of longer RNAs. The striking difference between the templates was the much higher level of abortive initiation obtained on AdUMut with either primer (in Fig. 4, compare lanes 1-3 with lanes 7-9, and compare lanes 13-15 with lanes 19-21). To quantify this difference, we computed the fraction of transcription complexes that could continue RNA synthesis past a certain template location, a parameter we call blockage. Blockage is defined as the ratio of the number of transcripts stopped at a particular location to the number of transcripts at that position plus all transcripts that read through to downstream locations. These values, expressed as percentages, are given in Table I for either ApC- or CpA-primed reactions with pure polymerase II. We observed from 1.4- to 7-fold increases in abortive initiation on the AdUMut template relative to the AdMLP template within the first 8 nt of transcribed sequence. Interestingly, the maximum increase in abortive initiation with either primer occurs, not at a particular template location but at a transcript length of 8 nt (Table I).
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Since the large majority of the short (<9-nt) RNAs made by pure RNA polymerase II on the AdUMut template could not be chased, it seemed likely that they had been released from ternary complex, as was the case with comparably sized RNAs made on the AdMLP bubble templates. We again used gel filtration on Sephacryl S-200 to test this. After the initial 30-min incubation of pure polymerase II and the AdUMut bubble template, none of the transcripts stalled at U3 to U8 and 47% of the transcription complexes stalled at C15 could be chased (Fig. 3A, lane 18). Only a very small proportion of active ternary complexes survived the gel filtration on the AdUMut template as compared with the AdMLP template (compare lanes 19 and 20 with lanes 4 and 5). All of the AdUMut transcripts 8 nt or shorter were found exclusively in the later eluting excluded fractions, indicating that they had been released from ternary complex.
We also transcribed the AdMLP and AdUMut bubble templates in HeLa
nuclear extracts. For these studies, we incubated the bubble duplexes
in HeLa extracts and then purified preinitiation complexes by gel
filtration. On a given template, we observed even greater levels of
abortive initiation with extracts than with pure polymerase II
reactions under identical conditions (in Fig. 4 compare
lanes 5 and 2, lanes
11 and 8, lanes 17 and
14, and lanes 23 and 20, respectively; see also Table I). The AdUMut template supported greater
levels of abortive initiation than the AdMLP template when transcribed
by polymerase II in extracts, with either primer (compare
lanes 5 and 11, and compare
lanes 17 and 23), in agreement with
the effect seen with pure RNA polymerase II. The short transcripts synthesized in extracts were not simply due to transcriptional stalling, since the addition of chase NTPs did not cause these complexes to resume elongation (compare lanes 6,
12, 18, and 24 with unchased
reactions). The amount of transcript produced from the bubble templates
in extracts did not increase between 5 and 30 min (data not shown).
Thus, the relatively slow RNA synthesis by pure polymerase II that we
observe on bubble templates in comparison with transcription of
double-stranded templates (see Fig. 2) is not an intrinsic property of
RNA polymerase II transcribing a bubble template. In the extract
reactions, small amounts of ~11-15-nt RNAs were made even in the
presence of 2 µg/ml -amanitin (lanes 4,
10, 16, and 22); we presume that these
are transcripts by RNA polymerases I and/or III from the nuclear extracts.
Short Transcripts (<9 nt Long) Were Exclusively Released from the Pure Polymerase II Ternary Complex and from the Bubble Templates-- In some studies with pure RNA polymerases and either bubble or oligo(dC)-tailed templates, it was reported that the transcripts formed extended RNA-DNA hybrids (37-40). To determine whether the short transcripts released by polymerase II as abortive initiation products were present as RNA-DNA hybrids (RNase H sensitive and RNase A resistant) or were released from ternary complex as normally displaced RNAs (RNase H-resistant and RNase A-sensitive), we performed nuclease digestions on AdUMut transcription reactions fractionated on Sephacryl S-200. The results are shown in Fig. 3B. As in the experiment in Fig. 3A, some transcripts remained in active ternary complex in the void volume (Fig. 3B, lanes 1 and 2). RNAs in this fraction were not digested by either RNase H (lane 3) or RNase A (lane 4), presumably as a result of protection of the transcript when present in active or inactive ternary complexes. Transcripts stalled at C15 were also present in included fractions (lanes 5, 8, and 11), but these RNAs were not expected to be in ternary complex from the results in the right part of Fig. 3A. All transcripts in the included fractions were found to be sensitive to RNase A but not to RNase H digestion (lanes 6, 7, 9, 10, 12, 13, 15, and 16), indicating that these RNAs are indeed released from ternary complex, rather than being retained as RNA-DNA hybrids. In particular, the decreased stability of C15 complexes is not the result of formation of continuous RNA-DNA hybrids.
The Incorporation of a Nucleotide Analog That Should Increase the
Stability of the RNA-DNA Hybrid Reduces Abortive Initiation--
We
compared initiation by pure RNA polymerase II in the presence of either
UTP or 5-bromo-UTP (Br-UTP), a UTP analog that strengthens the RNA-DNA
hybrid. For these experiments, labeled CTP was present at 0.5 µM, and no nonlabeled CTP or labeled UTP was added. The
latter condition avoided competition between labeled UTP and Br-UTP for
incorporation into RNA (see Fig. 5). When
the AdMLP bubble template was transcribed with UTP and limiting labeled CTP for 30 min, the expected transcripts stalled at C15
were produced, as well as prominent RNAs corresponding to stops before
C incorporation at U5, U8, and U14
(Fig. 5, lane 2). A fraction of transcription
complexes stalled at C15 and U14 chased after
the addition of GTP, CTP, and UTP (lane 3) or
GTP, CTP, and Br-UTP (GU'C chase, lane 4).
Incorporation of Br-UTP caused decreased electrophoretic mobility
relative to UTP-containing RNAs (compare, for example, the 20-mer in
lanes 3 and 4). Performing initial
transcriptions on the AdMLP with Br-UTP resulted in a significant
reduction in abortive initiation (compare lanes 2 and 6 of Fig. 5), most prominently at U5.
Blockage was almost eliminated at U5 for the particular
reaction shown in Fig. 5, and it was reduced an average of 3.7-fold
over eight experiments. Blockage after position U8 was
reduced by an average of 1.7 fold over eight experiments. For reactions
with Br-UTP on the AdUMut bubble template, we used the same conditions
as for the AdMLP template except that the CpA dinucleotide primer was
substituted. Transcription for 30 min with UTP produced transcripts
stalled at C15 and U14 as well as transcripts
stopped at U3 and from U5 to U8
(Fig. 5, lane 12). Incorporation of Br-UTP during
the initial transcription reaction reduced abortive initiation, as for
the AdMLP template (compare lanes 11 and
15). Blockage was eliminated after U3
(lane 15) and reduced from 1.6- to 2.2-fold (the
average of eight experiments) from U5 through
C9.
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If abortive initiation is reduced when Br-UTP is substituted for UTP,
one might expect more full-length (U14 and C15)
transcripts to accumulate in the presence of the U analog. For the
experiment shown in Fig. 5, we computed the amount of RNA at each
transcript length, beginning at C4 for the AdMLP reactions
and U3 for the AdUMut reactions and then summed these
numbers. For the AdMLP template with UTP as substrate, 10% of the
total RNA C4 and longer was U14 or
C15, but when Br-UTP was substituted for UTP 22% of the
total RNA was U14 or C15. The effect was even
stronger with the AdUMut template. Transcription with UTP gave 6% of
the total RNA as U14/C15, while 31% of the
total was U14/C15 when Br-UTP was substituted.
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DISCUSSION |
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In order to further explore the role of the sequence of the initially transcribed region in initiation efficiency by RNA polymerase II, we have taken advantage of the simplification provided by the use of bubble templates. In pilot experiments that led to the present study, we reproduced the earlier result (23) that transcription of AdMLP bubble templates by RNA polymerase II with normal NTP substrates requires, at minimum, TBP and TFIIB (data not shown). We were somewhat surprised to discover that priming RNA synthesis with a promoter-complementary dinucleotide was sufficient to remove any initiation factor requirement (Fig. 2). The start of transcription in dinucleotide-primed pure polymerase II reactions on bubble templates is sensitive to heparin and to low levels of sarkosyl, as is the case for a normal initiation event on double-stranded templates. Also, the transcript length at which ternary complexes become stable (the close of the abortive phase of transcription initiation) is the same for both templates. Thus, we believe that the start of transcription on bubble templates by pure RNA polymerase II in the presence of dinucleotide primers represents a reasonable model of normal transcript initiation.
The ability to start transcription and escape abortive initiation on
the bubble templates without any additional proteins shows that RNA
polymerase II has no absolute factor requirement for either process.
One might argue that this point is already well established, since
templates with 3' dC extensions support transcription by polymerase II
(39). However, such templates do not provide a close structural analog
to an initiation intermediate, as the bubble templates do. Furthermore,
in most cases the large majority of transcripts on these dC-tailed
templates cannot be extended beyond about 15 nt (38). The most
important consequence of having a factor-independent initiation
reaction was the ability to compare transcription efficiencies on the
same initially transcribed sequence in the presence and absence of
factors. The change of two bases, at +4 and +6, between the AdUMut and
AdMLP bubble templates resulted in a greatly increased level of
abortive initiation by pure RNA polymerase II on AdUMut. The effect is
strongest for the fifth through the eighth bases added, with an average
increase in abortive initiation of 3-4-fold during this stage of
transcription (Table I). This result is very similar to the 5-fold
increase in abortive initiation that we reported in an earlier
comparison, using double-stranded DNAs and nuclear extracts, of AdMLP
and the AdUMut-like mouse -globin promoter (10). The fact that we
can essentially duplicate the nuclear extract/double-stranded template
result with bubble templates and pure RNA polymerase II suggests that
the efficiency with which polymerase escapes abortive initiation is to
a considerable extent determined by the transcript-template sequence alone.
What is the mechanistic basis for the increase in abortive initiation seen with AdUMut versus the AdMLP template? In approaching this question it is useful to briefly review recent findings on the extent of the RNA-DNA hybrid within the transcription complex, an area that has been somewhat controversial. Rice, Kane, and Chamberlin (18) studied this issue by treating RNA polymerase II ternary complexes with ribonucleases and then testing for retention of the 3' portion of transcripts within the complexes. They concluded that RNAs as short as 3 nt could be retained in active ternary complexes and extended upon NTP addition (18). While this finding argues against any extensive RNA-DNA hybrid, several other recent studies have reached the opposite conclusion. Nudler et al. (12) incorporated a cross-linkable U analog at various positions within transcripts made by defined E. coli ternary complexes. This analog cross-linked to the template A residue with which it was presumably base-paired, and only to that residue, as long as the U analog was present between 2 and 8 nt upstream of the 3'-end of the RNA. When the analog was present further upstream, only cross-links to the RNA polymerase were observed. Incorporation of other nucleotide analogs that would strengthen the RNA-DNA hybrid reduced the tendency for transcriptional arrest when these analogs were placed within eight bases of the 3'-end, but no effect was seen upon incorporation further upstream. Nudler et al. (12) concluded that the RNA-DNA hybrid within their transcription complexes is 8 nt long. Essentially the same conclusion was reached by Kashlev and colleagues in two very recent reports. In the first of these (15), transcription complexes were assembled with 30 base bubble templates mismatched over the central nine bases. Short RNAs were hybridized to one strand of the bubble region and these hybrids were challenged with E. coli RNA polymerase. Functional transcription complexes were obtained by this approach with RNAs of 6 nt or longer. Six-nt RNAs gave rather unstable complexes; stability was increased with 7-nt RNAs and was maximal with 8-nt RNAs. Significantly, 8-nt RNAs that could hybridize only over the 3' six bases gave rise to unstable complexes. These results were also taken to indicate that the RNA-DNA hybrid in the transcription complex is 8 bp long. Komissarova and Kashlev (16) reached similar conclusions from an entirely different method, one which uses normal transcription complexes. They found (16) that RNase digestion of ternary E. coli polymerase complexes did not result in cleavages closer than 14-16 bases from the 3'-end of the nascent RNA. Transcripts could be truncated down to 8-10 nt, but no further, with the E. coli GreB factor or pyrophosphorolysis. Most significantly, Komissarova and Kashlev (16) were able to detect apparent RNase cleavage to within three bases of the 3'-end of the nascent RNA in active complexes, but this resulted from the failure of a denaturing agent to immediately inactivate the RNase at the end of the experiment. When this problem was eliminated, no RNase cleavages closer than 14-16 bases from the 3'-end were detected.
Our results are consistent with a model in which the stable transcript elongation complex contains an 8-base pair RNA-DNA hybrid, with RNA upstream of the hybrid interacting in some way with the RNA polymerase. First, we obtained much more abortive initiation with the AdUMut template relative to the AdMLP DNA. With the ApC primer, the greatest difference was obtained for 8-nt RNAs (Table I). This is the RNA length at which the AdUMut transcript (ACUUUUUU) has the longest continuous run of U residues at its 3'-end. In comparison, the corresponding 8-nt AdMLP transcript is ACUCUCUU. Since the U:dA hybrid is unusually weak (reviewed in Ref. 17), one would expect that abortive initiation would occur more frequently on AdUMut than on AdMLP and that the greatest difference would be seen at +8. The decrease in abortive initiation and increased yield of 15-nt RNA that we observed when Br-UTP was substituted for UTP is also consistent with the importance of RNA-DNA hybrid strength in determining the stability of the early transcription complex.
All of our results cannot be explained simply by invoking differences in hybrid strength. A striking example is the relative level of abortive initiation on the AdUMut and AdMLP templates when CpA was used as a primer instead of ApC. In this case (Table I), the greatest increase in abortive initiation with the AdUMut template compared with AdMLP was seen at U7, although the longest U-run occurs for the U8 transcript. This finding can be explained if RNA immediately upstream of the 8-base pair hybrid region enters an RNA binding channel on the RNA polymerase (see Refs. 16 and 41). The putative RNA-RNA polymerase interaction would stabilize the transcription complex and partially compensate for differences in hybrid stability at the 3'-end of the RNA. It is also important to note that while we favor an explanation of our data based on differential hybrid strength, other models are also possible. For example, if U-rich RNAs interact less well with the RNA polymerase than do other RNAs, then the difference in abortive initiation between the AdUMut and AdMLP templates may be explained without invoking an RNA-DNA hybrid longer than a few base pairs (18). Such an explanation would also require that the substitution of Br-U for U results in somewhat higher transcript affinity.
While the bubble template system has proven useful in investigating the role of transcript sequence in the initiation process, our results have also revealed some limitations to this approach. One of these is the slower rate of transcript accumulation on the bubble templates relative to fully double-stranded templates. We have performed template challenge experiments and also some preliminary assays with matrix-attached bubble templates. In all of these tests (data not shown), template commitment with the bubble templates and pure RNA polymerase II was found to be very rapid (about 2 min). Thus, the slow accumulation of transcripts on the bubble templates must result from either slow initiation by RNA polymerase II or a slow isomerization of the template-bound polymerase into an initiation-competent state. Bubble templates transcribed in nuclear extracts showed relatively rapid RNA synthesis (Fig. 4), which indicates that slow initiation is not an inherent property of bubble templates. This result raises the possibility that while transcription factors are not absolutely required for initiation by RNA polymerase II, such factors may serve to stimulate the process of initiation.
Another limitation of the bubble templates is the relative instability
of transcription complexes, such as those bearing C15 RNAs,
which have escaped the abortive initiation stage. On bubble templates,
we typically see one-third to one-half of the C15 complexes
fail to chase after a 30-min transcription reaction, while analogous
complexes on double-stranded templates retain full transcriptional
competence. The recent results of Holstege et al. (42)
suggest a possible reason for this instability. These workers
demonstrated that during transcript initiation by RNA polymerase II at
the AdMLP, the initial denatured region of 9 to
2 extends
continuously downstream until 10 bonds are made, at which point the
upstream portion of the bubble abruptly reanneals, leaving a denatured
region extending downstream from about +3. Such upstream reclosure
cannot occur on our bubble templates, which would suggest that
complexes stalled at locations just downstream of +10 might be
destabilized by an inappropriately melted upstream region. We do not
think that the simple presence of a long stretch of free template
strand upstream of the polymerase is the instability signal, because
the addition of DNA oligonucletides complementary to the otherwise
unpaired segment of the template strand upstream of
2 did not
increase the stability of C15 complexes (data not shown).
The strain imposed by the inappropriately long transcription bubble in
complexes with >11-nt nascent RNAs could cause the ejection of the
polymerase as a binary transcript/polymerase complex, thereby leading
to the limited, template-independent synthesis of RNAs >15 nt as
discussed above.
The inability to reclose the upstream region could also explain the increased level of abortive initiation on bubble templates seen in extract transcription, relative to the pure RNA polymerase reaction. This effect was particularly striking for the AdUMut template, on which almost no promoter clearance was achieved in extract transcription (Fig. 4). We can imagine that blocking reannealing with a permanently unpaired upstream region might lock initiation factors in place and prevent transcription from continuing. It is tempting to suggest that TFIIH is involved in this effect. Kumar et al. (43) recently showed that the lack of TFIIH in reactions performed with highly purified polymerase II transcription factors leads to a failure of transcription to progress beyond about +15 to +20. Thus, we can speculate that failure of the bubble to reclose might trap TFIIH in a configuration within the complex such that it actually blocks promoter clearance by RNA polymerase II.
In summary, the bubble template system has allowed us to demonstrate a
major role for the sequence of the initially transcribed region in the
extent of abortive initiation by RNA polymerase II. This approach
should also be useful in the future in analyzing the contributions of
subsets of the RNA polymerase II general transcription factors to the
polymerase II initiation process.
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ACKNOWLEDGEMENTS |
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We thank Danny Reinberg, Robert Landick, and Zachary Burton for generous gifts of transcription factors and factor expression constructs and Donna Driscoll, Richard Gronostajski, and Richard Padgett for advice during the course of these experiments. We also thank Natalia Komissarova and Mikhail Kashlev for communicating results prior to publication.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM29487.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 Molecular
Biology, NC20, Lerner Research Institute, Cleveland Clinic Foundation,
9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-7688; Fax:
216-444-0512; E-mail: lused{at}cesmtp.ccf.org.
2 I. Samkurashvili and D. S. Luse, unpublished observations.
3 J. Kitzmiller and D. S. Luse, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: nt, nucleotide(s); TBP, TATA box-binding protein; TFII, general factor for transcript initiation by RNA polymerase II; AdMLP, adenovirus major late promoter; AdUMut, variant of the AdMLP bubble template; Br-UTP, 5-bromo-UTP.
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REFERENCES |
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