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(Received for publication, January 16,
1996) From the
A requirement for an ATP cofactor in synthesis of the first
8-10 bonds of promoter-specific transcripts by RNA polymerase II
is well established. Whether ATP is required for synthesis of the first
phosphodiester bond or at a slightly later stage in synthesis of
nascent transcripts, however, remains controversial. Goodrich and Tjian
(Goodrich, J. A., and Tjian, R.(1994) Cell 77, 145-156)
recently proposed that synthesis of the first phosphodiester bond of
promoter-specific transcripts by RNA polymerase II is independent of
ATP and general transcription factors TFIIE and TFIIH. Here we
investigate this model. Taken together, our findings indicate that ATP,
TFIIE, and TFIIH can have a profound effect on the efficiency of
transcription initiation. First, we observe that synthesis of the first
phosphodiester bond of transcripts initiated at the adenovirus 2 major
late promoter depends strongly on ATP, TFIIE, and TFIIH in a
transcription system reconstituted with RNA polymerase II, TFIIH, and
recombinant TBP, TFIIB, TFIIE, and TFIIF. Second, we demonstrate that,
in this enzyme system, ATP-dependent activation of transcription
initiation can occur immediately prior to synthesis of the first
phosphodiester bond of nascent transcripts. Finally, we demonstrate
that the activated initiation complex is unstable and decays rapidly to
an inactive state in the presence of the inhibitor ATP
Promoter-specific transcription by RNA polymerase II on linear
or relaxed DNA templates is a complex biochemical process requiring the
general transcription factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH and
a hydrolyzable ATP cofactor. A role for ATP as a cofactor in
transcription by RNA polymerase II was first suggested in studies
carried out with crude or partially fractionated transcription systems.
Using HeLa cell extracts, Weinmann and co-workers (1) initially
observed that AMP-PNP ( Although the function of ATP has not been
unequivocally established, several lines of evidence have led to the
proposal that ATP is utilized by a DNA helicase activity associated
with TFIIH to promote unwinding of the DNA template at the
transcriptional start site prior to initiation. First, using KMnO Recently, a
requirement for ATP in transcription initiation by RNA polymerase II
has been called into question. In experiments carried out with a
transcription system reconstituted with RNA polymerase II and TFIIH
purified from HeLa cells and recombinant TBP, TFIIB, TFIIE, and TFIIF,
Goodrich and Tjian (22) observed synthesis of
dinucleotide-primed, abortive AdML-specific trinucleotide transcripts
in the absence of added ATP, TFIIE, and TFIIH. In light of this
finding, they proposed that ATP and TFIIH are not essential for
synthesis of the first phosphodiester bond of nascent transcripts but,
instead, are required at a later transcriptional stage referred to as
promoter clearance. To explain the discrepancy between their findings
and those of earlier studies, they suggested that the previously
observed requirement for ATP in transcription initiation might be an
artifact resulting from impurities present in crude transcription
systems. In this report, we have investigated the role of ATP and
TFIIH in transcription initiation using an enzyme system reconstituted
with RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE, and
TFIIF. As we describe below, our findings argue strongly that ATP and
TFIIH can have a profound effect on the efficiency of transcription
initiation by RNA polymerase II, and they shed new light on the role of
ATP in this process.
Figure 1:
Requirements for synthesis of
trinucleotide transcripts from the AdML promoter. A, AdML
promoter sequence in the vicinity of the transcriptional start site.
The arrow indicates the position of the in vivo start
site. B, specificity of dinucleotide-primed abortive
transcription. Transcription reactions were performed as described
under ``Experimental Procedures.'' Reaction mixtures
contained 5 µM dATP, 500 µM of the indicated
dinucleotide primer, and 10 µCi of
[
Figure 2:
Trinucleotide synthesis depends on ATP,
TFIIE, and TFIIH. A, transcription reactions were performed as
described under ``Experimental Procedures.'' Reaction
mixtures contained 5 µM dATP, 250 µM CpA
primer, 10 µCi of [
Figure 4:
ATP
In this report, we have used the abortive initiation assay to
investigate the requirements for synthesis of the first phosphodiester
bond of AdML promoter-specific transcripts in a transcription system
reconstituted with recombinant TBP, TFIIB, TFIIE, TFIIF, and RNA
polymerase II and TFIIH purified from rat liver. As shown previously (4, 5, 22, 29, 31) RNA
polymerase II will utilize dinucleotides to prime synthesis of
promoter-specific transcripts; depending on the dinucleotide primer
provided, initiation can occur over an approximately nine-base region
centered around the normal transcription start site(31) . If
only a dinucleotide primer and the next nucleotide encoded by the
template are provided as substrates for RNA synthesis, polymerase will
efficiently synthesize abortively initiated, trinucleotide
transcripts(4, 5, 22, 30, 32) .
The dinucleotide-primed abortive initiation assay has been widely used
in studies analyzing synthesis of the first phosphodiester bond of
nascent transcripts by prokaryotic (33, 34) and
eukaryotic (4, 5, 22, 30, 32) RNA
polymerases. To assess the specificity of trinucleotide synthesis in
our reconstituted transcription system, abortive initiation reactions
with RNA polymerase II, all five general transcription factors, and
dATP were carried out in the presence of
[ To determine the requirements for synthesis of the first
phosphodiester bond of AdML promoter-specific transcripts in our
reconstituted transcription system, the abortive initiation reaction
was carried out in the presence of various combinations of RNA
polymerase II and general transcription factors, in the presence or
absence of dATP. As expected, trinucleotide synthesis was strongly
dependent on the presence of RNA polymerase II, TBP, TFIIB, and TFIIF (Fig. 1C). Surprisingly, however, efficient
trinucleotide synthesis was observed only in the presence of added dATP (Fig. 2A, lane 1) or ATP (Fig. 2B). The apparent K Consistent with the model that the
ATP/dATP-dependent step in transcription is mediated by TFIIH,
trinucleotide synthesis was strongly dependent on addition of TFIIH.
The reaction was also strongly dependent on addition of TFIIE, which
has been shown to mediate the interaction of TFIIH with the
preinitiation complex (Fig. 2A, lanes 3 and 4)(23, 35) . Thus, ATP, TFIIE, and TFIIH can
have a profound effect on the efficiency of synthesis of the first
phosphodiester bond of nascent transcripts when transcription
initiation is carried out in a highly purified, reconstituted
transcription system. To investigate in greater detail the function
of ATP in synthesis of abortively initiated transcripts, we exploited
the nucleotide analog ATP
Figure 3:
dATP-dependent activation of the
preinitiation complex prior to trinucleotide synthesis. Preinitiation
complexes were assembled as described under ``Experimental
Procedures'' with 250 µM CpA and 10 µCi of
[
To determine whether
ATP In summary, in this report we have investigated the role of ATP and
general transcription factors TFIIE and TFIIH in the synthesis of the
first few phosphodiester bonds of promoter-specific transcripts by RNA
polymerase II in a transcription system reconstituted with purified
TFIIH and recombinant TBP, TFIIB, TFIIE, and TFIIF. Our findings
indicate that ATP, TFIIE, and TFIIH can have a dramatic effect on the
efficiency of transcription initiation. In addition, we observe that
ATP-dependent activation of transcription initiation can occur
immediately prior to synthesis of the first phosphodiester bond of
nascent transcripts and, further, that the activated initiation complex
is unstable and decays rapidly to an inactive state in the presence of
the inhibitor ATP Note Added in Proof-While this manuscript was
under review, we learned that Holstege et al. (Holstege, F. C.
P., van der Vliet, P. C., and Timmers, H. T. M.(1996) EMBO J.15, in press) had obtained similar findings.
FOOTNOTES
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7245-7248
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
S (adenosine
5`-O-(thio)triphosphate), even during reiterative synthesis of
abortive transcripts.
)could not replace ATP in synthesis
of AdML promoter-specific transcripts, even though AMP-PNP is a
substrate for elongation by RNA polymerase II; these findings were
subsequently corroborated and extended by Shatkin and
co-workers(2) . In experiments carried out with partially
fractionated HeLa cell extracts, Sawadogo and Roeder (3) obtained evidence that ATP is required at an early stage in
transcription by demonstrating that ATP is essential for synthesis of
the first 8 phosphodiester bonds of transcripts initiated at the AdML
promoter. In an elegant series of experiments, Luse and Jacob (4) presented convincing evidence that, in HeLa cell extracts,
ATP is essential for synthesis of the first phosphodiester bond of
promoter-specific transcripts by demonstrating that hydrolyzable ATP is
required for synthesis of dinucleotide-primed, abortive AdML-specific
trinucleotide transcripts. These findings were recently confirmed and
extended by Gralla and co-workers(5) , who demonstrated that
ATP is required for synthesis of dinucleotide-primed, abortive
trinucleotide transcripts initiated at the adenovirus E4 promoter in
HeLa cell extracts.
as a probe for DNA melting, Gralla and co-workers (5, 6, 7) demonstrated that, in HeLa cell
extracts, ATP is needed for unwinding of a short stretch of DNA
surrounding the transcriptional start site prior to initiation. Second,
among the general transcription factors, only TFIIH has detectable
ATP-hydrolyzing activities. TFIIH has been shown to possess closely
associated DNA-dependent ATPase/dATPase(8, 9) , DNA
helicase(10, 11) , and protein kinase (9, 12, 13) activities. The TFIIH protein
kinase activity has been shown to be dispensable for transcription
under conditions where ATP is
needed(14, 15, 16) , arguing that it does not
mediate the essential ATP-requiring step in transcription by RNA
polymerase II. Finally, recent biochemical studies indicate that the
ATP cofactor is not required for transcription under a limited set of
conditions where TFIIH is dispensable for initiation; these conditions
include transcription from promoters on negatively supercoiled DNA
templates (17, 18, 19) or promoters
containing a short stretch of mismatched base pairs surrounding the
transcriptional start site(20, 21) .
Materials
Unlabeled ultrapure ribonucleoside
5`-triphosphates and dATP were purchased from Pharmacia Biotech Inc.
Dinucleotides CpA, CpU, ApC, ApG, GpA, UpU, and UpG, 
-amanitin,
and polyvinyl alcohol (type II) were obtained from Sigma. ATPS was
from Boehringer Mannheim. [
-P]CTP (>400
Ci/mmol) was purchased from Amersham Corp. Bovine serum albumin (Pentex
fraction V) was obtained from ICN Immunobiologicals. Human placental
ribonuclease inhibitor (RNasin) was from Promega.
Preparation of RNA Polymerase II and Transcription
Factors
RNA polymerase II (12) and TFIIH (23) were purified as described from rat liver nuclear
extracts. Recombinant yeast TBP (24, 25) and
recombinant rat TFIIB (26) were expressed in Escherichia
coli and purified as described. Recombinant TFIIE was prepared as
described, except that the 56-kDa subunit was expressed in
BL21(DE3)pLysS(27) . Recombinant TFIIF was expressed in
JM109(DE3) using the M13 mpET expression system and purified as
described previously(28) .Assay of Transcription Initiation
Preinitiation
complexes were assembled at the AdML promoter at 28 °C by either a
20-min (Fig. 1B, 2B, and 4) or a 45-min (Fig. 1C, 2A, and 3) preincubation of
60-µl reaction mixtures containing 20 mM Hepes-NaOH (pH
7.9), 20 mM Tris-HCl (pH 7.9), 60 mM KCl, 7 mM MgCl
, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 2% (w/v) polyvinyl
alcohol, 7% (v/v) glycerol, 6 units of RNasin, 100 ng of the EcoRI-NdeI fragment from pDN-AdML(29) ,
10 ng of recombinant TFIIB,
20 ng of recombinant TFIIF,
20 ng of recombinant TFIIE and TFIIH (
150 ng of rat
TSK
DEAE 5-PW fraction in Fig. 1B, 1C,
2A, 3, and 4B or
40 ng of rat
TSK SP 5-PW
fraction in Fig. 2B and Fig. 4A),
50 ng of yeast TBP (AcA 44 fraction), and
0.01 unit of RNA
polymerase II. As indicated in the figure legends, transcription was
initiated by addition of dinucleotide primer, ribonucleoside
triphosphates, and
-P-labeled ribonucleoside
triphosphates. Transcription was carried out at 28 °C for the times
indicated in the figure legends. Transcription was stopped by addition
of 8 µl of reaction mix to 2 µl of a stop solution containing
100 mM EDTA and 0.5 mg/ml proteinase K. Following an
incubation of at least 15 min at room temperature, 10 µl of 9 M urea containing 0.025% bromphenol blue and 0.025% xylene cyanol FF
was added, and trinucleotide transcripts were analyzed by
electrophoresis through 25% (w/v) acrylamide, 3% (w/v) bisacrylamide, 7 M urea gels as described(30) . Trinucleotide synthesis
was quantitated by PhosphorImager analysis and is expressed in
arbitrary units that represent the phosphorescence intensity measured
in a given exposure time.
-P]CTP and were incubated at 28 °C for
30 min. C, transcription factors required for synthesis of
trinucleotide transcripts. Transcription reactions were performed as
described under ``Experimental Procedures.'' Reaction
mixtures, which contained 5 µM dATP, 250 µM CpA primer, 10 µCi of [
-P]CTP, and
the indicated combinations of RNA polymerase II (Pol II) and
general transcription factors, were incubated at 28 °C for 30 min.
The reaction shown in lane 1 contained 1 µg/ml
-amanitin (-am).
-P]CTP, and
transcription factors as indicated and were incubated at 28 °C for
30 min. B, reactions were carried for 60 min as described
under ``Experimental Procedures'' in the presence of
0.05-5 µM ATP, 250 µM CpA primer, and
10 µCi of
[
-P]CTP.
S inhibits trinucleotide synthesis
when added following initiation of abortive transcription.
Transcription reactions were performed as described under
``Experimental Procedures'' in the presence of 250 µM CpA and 10 µCi of [
-P]CTP and for
the times indicated in the figure. A, reactions contained 5
µM dATP and and 100 µM ATP
S as indicated
in the figure. B, transcription reactions were performed in
the presence (
, ,
) or absence (
) of 5
µM dATP. ,
, 100 µM ATPS
was added at 20 min.
, an additional 150 µM dATP was
added at 50 min.
-P]CTP and various dinucleotide primers.
As expected from the sequence of the AdML non-template strand in the
region of the transcription start site (Fig. 1A), we
observe efficient trinucleotide synthesis when CpA or CpU but not ApC,
ApG, UpU, and UpG are used as the dinucleotide primers (Fig. 1B). In addition, trinucleotide synthesis is
strongly inhibited by 1 µg/ml
-amanitin, which inhibits RNA
polymerase II but not bacterial RNA polymerases or mammalian RNA
polymerases I or III; thus the observed trinucleotide synthesis does
not result from a contaminating polymerase activity (Fig. 1C, compare lanes 1 and 2). 
for ATP
as a cofactor in the abortive initiation assay is extremely low
(250 nM, Fig. 2B). The apparent K
for dATP was somewhat higher (500
nM) (data not shown).
S. In a previous study, we observed that
ATP
S is a potent inhibitor of ATP/dATP-dependent transcription by
RNA polymerase II in a partially fractionated transcription system from
rat liver, and we obtained evidence that ATP
S inhibits a
reversible, ATP/dATP-requiring step that occurs prior to synthesis of
4-9 nucleotide, Sarkosyl-resistant transcripts in this
system(29) . Here we demonstrate that ATP
S is also a
potent inhibitor of dATP-dependent trinucleotide synthesis in the
purified, reconstituted transcription system. When added to reaction
mixtures either with dATP, CpA, and [
-P]CTP
(see Fig. 4A) or with dATP and prior to addition of CpA
and [
-P]CTP, trinucleotide synthesis is
very strongly inhibited (Fig. 3, first two lanes of inset). The ATP-dependent step can occur prior to initiation
and is reversible, since preincubation of template, RNA polymerase II,
and the general transcription factors with dATP for 1 min prior to
addition of CpA and [
-P]CTP renders
trinucleotide synthesis resistant to inhibition by ATP
S; in the
absence of initiating nucleotides, the activated preinitiation complex
decays with a half-life of 30-60 s.
-P]CTP. All subsequent steps were carried
out at room temperature according to the procedure diagrammed at the bottom of the figure. 5 µM dATP and 100
µM ATP
S were added to reaction mixtures at the times
indicated in the figure.
S inhibits transcription once trinucleotide synthesis has been
initiated, we carried out the experiment shown in Fig. 4. In
this experiment ATP
S was added to reaction mixtures 10 min after
addition of CpA and [
-P]CTP; this resulted
in the immediate cessation of trinucleotide synthesis (Fig. 4B), indicating that the activated initiation
complex is unstable and decays rapidly to an inactive state in the
presence of the inhibitor ATP
S, even during reiterative synthesis
of abortive transcripts. Inhibition of trinucleotide synthesis was
completely reversible; following addition of excess dATP to an
ATP
S-inhibited reaction, trinucleotide synthesis resumed
immediately, and the rate of trinucleotide synthesis following dATP
addition was the same as in a control reaction containing no ATP
S.
S, even during reiterative synthesis of abortive
transcripts. Our finding that the activated initiation complex is
unstable during abortive transcription, which involves multiple rounds
of initiation by the same RNA polymerase II
molecules(4, 5) , suggests that, unlike elongating
polymerase, RNA polymerase II in the initiation complex is either
unable to maintain the DNA template in an ``open''
configuration or incompetent to catalyze synthesis of phosphodiester
bonds in the absence of continuing ATP hydrolysis. At the present time,
we do not know whether a separate ATP activation event is required for
each round of initiation or whether a single ATP activation event is
sufficient to promote multiple rounds of initiation, perhaps until the
activated initiation complex decays to an inactive state. Finally, our
findings do not exclude the possibility that additional ATP-requiring
steps may be required after synthesis of the first phosphodiester bond
of nascent transcripts but prior or concomitant to entry of RNA
polymerase II into the elongation stage of transcription. Future
experiments addressing these issues will be essential for a complete
understanding of the role of ATP in transcription by RNA polymerase
II.
hspace=3 SRC="/icons/back.GIF">
)S, adenosine
5`-O-(thio)triphosphate.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
