From the Program in Molecular and Cell Biology,
Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, the § Department of Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
73190, the ¶ Edward A. Doisey Department of Biochemistry, St.
Louis University School of Medicine, St. Louis, Missouri 63104, and the
** Howard Hughes Medical Institute, Oklahoma Medical Research
Foundation, Oklahoma City, OK 73104
Received for publication, February 14, 2001, and in revised form, March 16, 2001
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ABSTRACT |
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TFIIF, ELL, and Elongin
belong to a class of RNA polymerase II transcription factors that
function similarly to activate the rate of elongation by suppressing
transient pausing by polymerase at many sites along DNA templates. SII
is a functionally distinct RNA polymerase II elongation factor that
promotes elongation by reactivating arrested polymerase. Studies of the
mechanism of SII action have shown (i) that arrest of RNA polymerase II
results from irreversible displacement of the 3'-end of the nascent
transcript from the polymerase catalytic site and (ii) that SII
reactivates arrested polymerase by inducing endonucleolytic cleavage of
the nascent transcript by the polymerase catalytic site thereby
creating a new transcript 3'-end that is properly aligned with the
catalytic site and can be extended. SII also induces nascent transcript cleavage by paused but non-arrested RNA polymerase II elongation intermediates, leading to the proposal that pausing may result from
reversible displacement of the 3'-end of nascent transcripts from the
polymerase catalytic site. On the basis of evidence consistent with the
model that TFIIF, ELL, and Elongin suppress pausing by preventing
displacement of the 3'-end of the nascent transcript from the
polymerase catalytic site, we investigated the possibility of
cross-talk between SII and transcription factors TFIIF, ELL, and
Elongin. These studies led to the discovery that TFIIF, ELL, and
Elongin are all capable of inhibiting SII-induced nascent transcript
cleavage by non-arrested RNA polymerase II elongation intermediates.
Here we present these findings, which bring to light a novel activity
associated with TFIIF, ELL, and Elongin and suggest that these
transcription factors may expedite elongation not only by increasing
the forward rate of nucleotide addition by RNA polymerase II, but also
by inhibiting SII-induced nascent transcript cleavage by
non-arrested RNA polymerase II elongation intermediates.
Biochemical studies of transcription by RNA polymerase II have led
to the identification and purification of a collection of transcription
factors that promote elongation through direct interactions with
transcribing polymerase. These elongation factors fall into two
functional classes based on their mechanisms of action.
One class includes TFIIF (1-5), ELL (6-8), and Elongin (9-11), which
are all capable of activating the overall rate of elongation by RNA
polymerase II by suppressing transient pausing by the enzyme. Previous
studies have shown that bacterial and eukaryotic RNA polymerases are
susceptible to pausing for varying lengths of time at each step of
nucleotide addition. Because the duration of pausing is often greater
than the rate of phosphodiester bond formation
(kcat), it has been proposed that elongating RNA
polymerases cycle between active and inactive conformations at each
step of nucleotide addition (1, 12-18). TFIIF, ELL, and Elongin are all capable of increasing the rate of elongation by RNA polymerase II
at extremely low ribonucleoside triphosphate concentrations when the
time required for nucleotide addition can vary from seconds to minutes,
suggesting that these transcription factors increase the rate of
elongation not by decreasing kcat but by
decreasing the fraction of time polymerase spends in an inactive
conformation (7, 8, 11, 19).
The other class of RNA polymerase II elongation factors includes
members of the SII family of transcription factors. SII promotes elongation by reactivating RNA polymerase II that has become arrested at a variety of impediments including specific DNA sequences referred to as intrinsic arrest sites (20-24). Unlike paused RNA polymerase II,
which resumes transcription even in the absence of elongation factors,
arrested RNA polymerase II is in an inactive state and resumes
transcription only with assistance from SII.
Evidence from a variety of studies suggests that transcriptional arrest
and pausing are mechanistically related and result from aberrant
backward movement of RNA polymerase II on the DNA with concomitant
displacement of the 3'-end of the nascent transcript from the
polymerase catalytic site, an event that is either spontaneously reversible in the case of pausing or not in the case of arrest (24).
Current evidence is consistent with the model that SII reactivates
arrested RNA polymerase II by inducing a polymerase-associated endoribonuclease activity, which cleaves the nascent transcript 7-10
nucleotides upstream of the displaced 3'-end (21, 25), thus creating a
new 3'-end that is properly aligned with the enzyme's catalytic site
and can be reextended. Notably, SII also promotes nascent transcript
cleavage by paused but non-arrested RNA polymerase II elongation
complexes; in this case, transcript cleavage typically occurs in two
nucleotide increments (25, 26).
The mechanism(s) by which TFIIF, ELL, and Elongin suppress transient
pausing by transcribing RNA polymerase II are not completely understood. In a previous study, Reines and co-worker (17) observed that TFIIF can decrease the rate at which RNA polymerase II falls into
arrest, raising the possibility that TFIIF can inhibit backsliding of
polymerase and consequent displacement of the 3'-end of the nascent
transcript from the polymerase catalytic site. In addition, we
previously observed (i) that TFIIF, ELL, and Elongin can all dramatically increase the ability of RNA polymerase II to bind to and
extend DNA primers in a DNA template-directed reaction and (ii) that
TFIIF mutations that reduce TFIIF elongation activity also reduce this
TFIIF activity, consistent with the idea that the mechanisms by
which TFIIF, ELL, and Elongin promote extension of DNA 3'-ends and
suppress transient pausing by RNA polymerase II may be related
(27).1 As suggested by
Salzman and co-workers, the DNA template-directed addition of
ribonucleotides to the 3'-ends of DNA by RNA polymerase II may occur in
a reaction that mimics formation of the RNA polymerase II ternary
elongation complex (28). In this case, RNA polymerase II would bind to
the 3'-hydroxyl terminus of DNA, just as the enzyme binds to the 3'-end
of an elongating RNA molecule; the polymerase catalytic site would then
add ribonucleotides to the DNA 3'-hydroxyl terminus as if it were the
3'-end of a nascent transcript. Accordingly, our observation that
TFIIF, ELL, and Elongin can promote extension by RNA polymerase II of
DNA primers is consistent with the model that these transcription
factors may facilitate proper positioning of the 3'-ends of these DNA primers in the polymerase catalytic site and, by extension, that these
transcription factors may suppress transient pausing by polymerase by
facilitating proper positioning of the 3'-end of nascent transcripts in
the enzyme's catalytic site.
In light of this model and because SII-induced cleavage of nascent
transcripts by paused or arrested RNA polymerase II elongation intermediates is believed to occur when the 3'-end of nascent transcripts becomes misaligned with the polymerase catalytic site, we
investigated the possibility of cross-talk between SII and transcription factors TFIIF, ELL, and Elongin. These studies led to the
discovery that TFIIF, ELL, and Elongin are all capable of inhibiting
SII-induced cleavage of transcripts by non-arrested RNA polymerase
II elongation intermediates. Here we present these findings, which
bring to light a novel activity associated with TFIIF, ELL, and Elongin
and suggest that these transcription factors may expedite elongation by
RNA polymerase II not only by increasing the forward rate of nucleotide
addition by polymerase but also by inhibiting SII-induced nascent
transcript cleavage by non-arrested RNA polymerase II elongation intermediates.
Materials--
Unlabeled ultrapure ribonucleoside
5'-triphosphates,
3'-O-MeGTP,2 and
[ Preparation of DNA Templates for Transcription--
The
~310-base pair DNA fragment containing the AdML promoter was obtained
by digestion of the plasmid pDN-AdML (29) with EcoRI
and NdeI and by purification of the
EcoRI-NdeI fragment by agarose gel
electrophoresis. The oligo(dC)-tailed pCpGR220 DNA template was
prepared as described (30).
Preparation of RNA Polymerase II and Transcription
Factors--
RNA polymerase II (31) and TFIIH (rat
Recombinant human SII was expressed in E. coli and purified
as follows. The pT7-7Met expression vector encoding the full-length human SII open reading frame was obtained from Dr. Caroline
Kane, (Berkeley, CA) and expressed in E. coli BL21 (pLysS)
cells as described (37). SII was purified by chromatography on
consecutive phosphocellulose and TSK phenyl 5-PW columns.
Briefly, cell lysates were centrifuged 30 min at 31,000 × g, and the supernatant was applied to a phosphocellulose
column (Whatman P-11) preequilibrated in 40 mM Hepes-NaOH
(pH 7.9), 1 mM EDTA, 1 mM DTT, 10% (v/v)
glycerol, and 0.1 M KCl. SII was eluted with the same
buffer containing 0.33 M KCl. Active fractions were pooled
and precipitated with 60%
(NH4)2SO4. The pellet was dissolved
in 100 mM Hepes-NaOH (pH 7.9), 0.1 mM DTT, and
5% (v/v) glycerol, dialyzed against the same buffer containing 1.5 M (NH4)2SO4 until the
conductivity was equivalent to that of 1.5 M
(NH4)2SO4, and applied to a
TSK phenyl 5-PW high pressure liquid chromatography column
(75 × 7.5 mm; Bio-Rad) preequilibrated in 100 mM
Hepes-NaOH, pH 7.9, 0.1 mM DTT, 5% (v/v) glycerol, and 1.5 M (NH4)2SO4. SII was
eluted with a 36-ml linear gradient from 1.5 M to 0 M (NH4)2SO4 in the same buffer.
Preparation of Paused RNA Polymerase II Elongation
Complexes--
For a single assay of promoter-specific transcription,
preinitiation complexes were assembled at the AdML promoter at 28 °C by a 30-min preincubation of 30 µl of reaction mixtures containing 20 mM Hepes-NaOH (pH 7.9), 20 mM Tris-HCl (pH
7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM
DTT, 5 mM MgCl2, 0.5 mg/ml bovine serum
albumin, 2% (w/v) polyvinyl alcohol, 3% (v/v) glycerol, six units of
recombinant placental ribonuclease inhibitor, ~10 ng of the
EcoRI to NdeI fragment from pDN-AdML, ~5
ng of recombinant yeast TBP, ~10 ng of recombinant TFIIB, ~10 ng of
recombinant TFIIF, ~20 ng of recombinant TFIIE, ~10 ng of TFIIH,
and 0.01 unit of RNA polymerase II. Except where indicated otherwise,
transcription reactions were performed at 28 °C for 30 min in the
presence of 200 µM CpA, 0.5 µM
[
Transcription reactions were scaled up as necessary for each
experiment. Paused RNA polymerase II elongation complexes were purified
by applying ~200-250 µl of each transcription reaction mixture to
two consecutive 3-ml Sephadex G-50 spin columns prepacked in 4-ml empty
spin columns and preequilibrated in 20 mM Hepes-NaOH (pH
7.9), 20 mM Tris-HCl (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mg/ml bovine serum
albumin, 2% (w/v) polyvinyl alcohol, and 3% (v/v) glycerol, in the
presence or absence of 5 mM MgCl2. The columns
were spun for 5 min at 2000 × g in a swinging bucket rotor, and 30 µl of eluant/reaction mixture was used in further experiments. Reactions were stopped by the addition of an equal volume
of 9.0 M urea containing 0.025% (w/v) bromphenol blue and 0.025% (w/v) xylene cyanol FF. Transcripts were analyzed by
electrophoresis through polyacrylamide gels containing 25% acrylamide,
3% bis-acrylamide, 5 M urea, 89 mM Tris base,
89 mM boric acid, and 2 mM EDTA. Transcripts were quantitated using a Molecular Dynamics PhosphorImager.
To begin to explore the possibility of cross-talk between
elongation factor SII and transcription factors that suppress transient pausing by RNA polymerase II, we investigated the effect of TFIIF and
ELL on SII-induced nascent transcript cleavage by RNA polymerase II
elongation intermediates that had synthesized 16-nucleotide-long transcripts in a basal transcription system reconstituted with recombinant general initiation factors TBP, TFIIB, TFIIE, and TFIIF and
purified RNA polymerase II and TFIIH from rat liver. In these
experiments, RNA polymerase II preinitiation complexes were assembled
at the AdML promoter by preincubation of polymerase and the general
initiation factors with a DNA fragment containing the AdML promoter.
Radioactively labeled transcripts were synthesized in the presence of
200 µM of the initiating dinucleotide CpA, which directs
most transcription initiation from position
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]CTP (>3000 Ci/mmol) were purchased from
Amersham Pharmacia Biotech. Dinucleotide CpA, araCTP, polyvinyl
alcohol (type II), and Sephadex G-50 (catalog number G-50-150) were
obtained from Sigma. Acetylated bovine serum albumin and recombinant
placental ribonuclease inhibitor were from Promega. Empty 4-ml spin
columns were purchased from 5 Prime
3 Prime, Inc., Boulder, CO.
, SP 5-PW
fraction; (32)) were purified from rat liver nuclear extracts as
described previously. Recombinant yeast TBP (AcA 44 fraction; (33)),
recombinant TFIIB (34), and recombinant TFIIF (35) were expressed in
Escherichia coli and purified as described. Recombinant
human TFIIE was prepared as described (36), except that the 56-kDa
subunit was expressed in E. coli strain BL21(DE3)-pLysS.
Recombinant human ELL was expressed in E. coli and purified
from preparative SDS-polyacrylamide gels as described (8). The
recombinant 3-subunit Elongin ABC complex was expressed in E. coli and purified as described (19).
-32P]CTP, 15 µM ATP, 15 µM UTP, and 70 µM
3'-O-MeGTP.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 relative to the normal
AdML transcriptional start site (Fig.
1A), 15 µM ATP,
15 µM UTP, 0.5 µM
[
-32P]CTP, and 70 µM
3'-O-MeGTP, which prevents most transcription beyond the
first G residue of the AdML transcript at position +15. RNA polymerase
II elongation complexes were purified free of ribonucleoside
triphosphates by two consecutive Sephadex G-50 spin columns and treated
with elongation factor SII. As shown in Fig. 1B and
consistent with previous results (21-23), SII induced cleavage of
nascent transcripts by paused RNA polymerase II elongation intermediates in a dose-dependent manner (lanes
2-10). Similarly, SII induced cleavage of RNA polymerase II
elongation complexes that had synthesized 15-nucleotide-long
transcripts and had paused after incorporating the first A residue
immediately preceding the first G residue of the transcript (lane
11). SII-induced cleavage of these transcripts by RNA polymerase
II elongation complexes was dependent on Mg+2 (lanes
12 and 14); in addition, these RNA polymerase II elongation complexes were transcriptionally active because they could incorporate the RNA chain-terminating nucleotide 3'-O-MeG when
Mg+2 was added to reaction mixtures (compare lanes
11, 13, and 15).
View larger version (49K):
[in a new window]
Fig. 1.
TFIIF and ELL antagonize SII-induced nascent
transcript cleavage by non-arrested RNA polymerase II elongation
complexes. A, AdML promoter sequence in the vicinity of
the transcriptional start site. +1 indicates the position of
the in vivo start site. The site of initiation of
transcripts initiated with the dinucleotide CpA is indicated
above the template sequence. B,
Mg+2-dependent nascent transcript cleavage
activity of the SII used in these studies. Paused elongation complexes
were prepared as described under "Experimental Procedures."
Lanes 1 and 11, purified ternary complexes.
Complexes were incubated with ~2.5 ng (lane 2), ~5 ng
(lanes 3 and 4), ~10 ng (lanes 5 and
6), ~20 ng (lanes 7 and 8), or ~40
ng (lanes 9 and 10) of SII at 28 °C for 10 min. In the reaction shown in lanes 12 and 14,
complexes were incubated with ~500 ng of SII with (lane
12) or without (lane 14) 6 mM
MgCl2 for 20 min at 28 °C. In the reactions shown in
Lanes 13 and 15, complexes were incubated with 70 µM 3'-O-MeGTP for 20 min at 28 °C with
(lane 13) or without (lane 15) 6 mM
MgCl2. C, TFIIF and ELL antagonize
SII-dependent transcript cleavage in a
dose-dependent manner. Purified ternary transcription
complexes (lanes 1 and 11) were incubated for 10 min at 28 °C with 25 ng of SII without (lanes 2,
3, 12, and 13) or with increasing
amounts of TFIIF (~100 ng, lanes 4 and 5; ~1 µ g, lanes 6 and 7; ~2 µ g, lanes
8 and 9) or ELL (~1 ng, lanes 14 and
15; ~5 ng, lanes 16 and 17; or ~10
ng, lane 18).
To test the effects of TFIIF and ELL on SII-induced nascent transcript
cleavage, purified RNA polymerase II elongation complexes containing
16-nucleotide 3'-O-MeG-terminated transcripts were incubated
with a fixed level of SII with or without increasing concentrations of
TFIIF and ELL. As shown in Fig. 1C, SII-induced nascent
transcript cleavage by non-arrested RNA polymerase II elongation
intermediates was effectively inhibited by levels of TFIIF and ELL that
potently activated the rate of elongation by polymerase under our
reaction conditions (data not shown). In addition, the inhibitory
effects of ELL (Fig. 2) and TFIIF (data not shown) in these reactions could be overcome by increasing the
levels of SII in the presence of fixed ELL and TFIIF concentrations, suggesting that SII, TFIIF, and ELL function competitively in regulation of nascent transcript cleavage.
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ELL and TFIIF are both capable of stimulating the rate of elongation by
RNA polymerase II in the presence of extremely low levels of
ribonucleoside triphosphates (Refs. 7, 8, 11 and data not shown). To
address the possibility that the observed ELL- and
TFIIF-dependent inhibition of SII-induced nascent
transcript cleavage by non-arrested RNA polymerase II elongation
intermediates was due to ELL- or TFIIF-stimulated reextension of
cleaved nascent transcripts in the presence of low levels of residual
ribonucleoside triphosphates contaminating gel-filtered preparations of
paused elongation complexes, ELL and TFIIF were added back to
SII-treated elongation complexes. In these experiments, paused RNA
polymerase II elongation complexes containing 16-nucleotide,
3'-O-MeG-terminated transcripts were treated with SII for 5 min and then incubated with ELL, TFIIF, or ribonucleoside
triphosphates. As shown in Fig. 3,
addition of ELL (lanes 1, 3-5) or TFIIF
(lanes 6-8) did not promote reextension of cleaved
transcripts. In contrast, cleaved transcripts could be reextended by
addition of ribonucleoside triphosphates to reaction mixtures,
confirming that RNA polymerase II elongation complexes incubated with
SII were transcription-competent at the time ELL and TFIIF were added
to reactions (compare lanes 1 and 2).
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The results shown in Fig. 3 are consistent with the model that TFIIF
and ELL function by blocking SII-induced nascent transcript cleavage
and not by promoting reextension of cleaved transcripts. As a further
test of this model, we performed the experiment shown in Fig.
4. In this experiment, we took advantage
of the observation that, because of its ability to promote nascent
transcript cleavage, SII can reactivate elongation complexes paused
following incorporation of a chain-terminating nucleotide by promoting
removal of the chain-terminating nucleotide from the 3'-ends of nascent
transcripts. Purified RNA polymerase II elongation complexes that had
synthesized 16-nucleotide, 3'-O-MeG-terminated transcripts
were incubated with ATP, UTP, GTP, and the RNA chain-terminating
nucleotide araCTP with or without SII. If SII promotes endonucleolytic
cleavage and removal of 3'-O-MeG-residue from the 3'-end of
the transcript, the transcript will be extended to the next C residue,
resulting in the synthesis of a 20-nucleotide, araC-terminated RNA
(Fig. 4, top panel). As expected,
3'-O-MeG-terminated transcripts could not be extended in
reactions performed in the absence of SII (the small amount of
20-nucleotide transcripts synthesized in these reactions appears to
result from elongation of transcripts that had paused before
incorporation of 3'-O-MeG prior to purification of
elongation complexes (compare lanes 1 and 3 and
lanes 6 and 8)) but could be extended in
reactions performed in the presence of SII (lanes 4 and
8). Notably, the fraction of 3'-O-MeG-terminated transcripts that could be extended in the presence of SII was substantially decreased when reactions also contained either TFIIF (lane 5) or ELL (lane 9), consistent with the
model that TFIIF and ELL are capable of inhibiting SII-induced nascent
transcript cleavage by non-arrested RNA polymerase II elongation
complexes.
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On the basis of evidence that SII induces nascent transcript cleavage
by polymerases that have backtracked along the DNA template and lost
proper contact with the 3'-end of transcripts, our observation that
TFIIF and ELL block SII-induced nascent transcript cleavage raised the
possibility that TFIIF and ELL might function by helping to maintain
the 3'-end of nascent transcripts in the RNA polymerase II catalytic
site. If this model is correct, TFIIF and ELL might be expected to
stimulate not only the rate of nucleotide addition by RNA polymerase II
but also the reverse reaction, pyrophosphorolysis. Indeed, Hawley and
co-worker (22) previously demonstrated that TFIIF can stimulate the
rate of pyrophosphorolysis by RNA polymerase II elongation complexes
that had synthesized 185-350-nucleotide-long transcripts. Consistent
with their findings, we observe that both TFIIF and ELL can stimulate
the rate of pyrophosphorolysis by RNA polymerase II elongation
complexes containing 16-nucleotide, 3'-O-MeG-terminated
transcripts (Fig. 5).
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Because the experiments presented above were all performed with RNA
polymerase II elongation complexes that had initiated transcription
from the AdML promoter in the presence of the general initiation
factors, we wanted to confirm that TFIIF and ELL inhibit SII-induced
nascent transcript cleavage through direct interactions with these
elongation complexes and not indirectly through the action of one or
more of the general initiation factors present in transcription
reactions. To accomplish this, we took advantage of the
oligo(dC)-tailed pCpGR220 DNA template (30). Because the first
non-template strand (dT) residue is 136 base pairs downstream of the
oligo(dC) tail, RNA polymerase II will initiate transcription from the
oligo(dC) tail and synthesize transcripts of ~136 nucleotides in the
presence of ATP, CTP, and GTP. In these experiments, paused RNA
polymerase II elongation complexes that had synthesized ~136 nucleotide transcripts were purified by two consecutive Sephadex G-50
spin columns and treated with SII or 3 mM PPi
in the presence or absence of TFIIF, ELL, and Elongin. As shown in Fig.
6, TFIIF, ELL, and Elongin are all
capable not only of inhibiting SII-dependent nascent
transcript cleavage but also of stimulating the rate of pyrophosphorolysis by paused RNA polymerase II elongation intermediates in the absence of the general initiation factors, suggesting that these
activities are intrinsic to elongation factors that suppress transient
pausing by RNA polymerase II. In the experiment of Fig. 6A,
amounts of ELL and Elongin proteins corresponding to a molar ratio
relative to SII of ~0.1:1 were sufficient to inhibit SII-induced transcript cleavage, whereas an amount of TFIIF corresponding to a
molar ratio relative to SII of ~1.8:15 was needed. As shown in Fig.
6B, the same relative levels of ELL, Elongin, and TFIIF were
needed to stimulate the rate of pyrophosphorolysis. At the present time, we do not know whether the apparent difference in the
specific activities of ELL, Elongin, and TFIIF reflects a difference in
their intrinsic abilities to modulate the activities of RNA polymerase
II or whether it reflects a difference in the fraction of active
molecules present in our preparations of elongation factors.
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In conclusion, in this report we present evidence that transcription
factors TFIIF, ELL, and Elongin, which have all been shown previously
to activate the rate of elongation by RNA polymerase II in
vitro, are also capable of potently inhibiting SII-induced cleavage of nascent transcripts by non-arrested RNA polymerase II
elongation intermediates. In addition, we present evidence that, like
TFIIF, transcription factors ELL and Elongin are capable of strongly
stimulating the rate of pyrophosphorolysis by non-arrested RNA
polymerase II elongation intermediates. Because SII-induced cleavage of
nascent transcripts by RNA polymerase II elongation intermediates is
believed to occur when the 3'-end of transcripts becomes misaligned
with the polymerase catalytic site, our findings raise the possibility
that TFIIF, ELL, and Elongin inhibit SII-induced transcript cleavage at
least in part by helping to maintain the 3'-end of transcripts in the
enzyme's catalytic site. If this model is correct, then inhibition of
SII-induced nascent transcript cleavage by TFIIF, ELL, and Elongin
could be a manifestation of their intrinsic elongation factor activity.
Notably, in the experiments presented here we observe a correlation
between the TFIIF, ELL, and Elongin levels required to inhibit
SII-induced nascent transcript cleavage and to stimulate
pyrophosphorolysis, which is the reverse reaction of nucleotide
addition and RNA transcript elongation. Whether TFIIF, ELL, and Elongin
inhibit SII-induced transcript cleavage by controlling the orientation
of the 3'-ends of nascent transcripts or by simply interacting with the
RNA polymerase II elongation complex and sterically blocking its
interaction with SII, however, remains to be determined. Nevertheless,
our findings bring to light a novel activity associated with TFIIF,
ELL, and Elongin and suggest that these transcription factors could
expedite elongation not only by increasing the forward rate of
nucleotide addition by RNA polymerase II but also by inhibiting
SII-induced nascent transcript cleavage by non-arrested RNA polymerase
II elongation intermediates.
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ACKNOWLEDGEMENTS |
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We thank C. Kane (Univ. of Calif., Berkeley, CA) for the human SII cDNA and K. Jackson of the Molecular Biology Resource Center at the Oklahoma Center for Molecular Medicine for oligonucleotide synthesis.
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FOOTNOTES |
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* This work was supported by American Cancer Society Grant RP69921801 (to A. S.) and National Institutes of Health Grant R37-GM41628 and by funds provided to the Oklahoma Medical Research Foundation by the H. A. and Mary K. Chapman Charitable Trust (to R. C. C. and J. W. C.).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.
An Edward Mallinckrodt Young Investigator.
Associate investigator of the Howard Hughes Medical Institute.
§§ To whom correspondence should be addressed: Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 N. E. 13th St., Oklahoma City, OK 73104. Tel.: 405-271-1950; Fax.: 405-271-1580; E-mail: conawayr@omrf.ouhsc.edu.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M101445200
1 Y. Takagi, S. Tan, J. W. Conaway, and R. C. Conaway, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: 3'-O-MeGTP, 3'-O-methylguanosine 5'-triphosphate; araCTP, cytosine arabinoside 5'-triphosphate; AdML, adenovirus 2 major late; TBP, TATA box-binding protein; DTT, dithiothreitol.
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REFERENCES |
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