(Received for publication, July 8, 1994; and in revised form, October 12, 1994)
From the
The role of nucleotides in activated RNA polymerase II
transcription was studied. Permanganate footprinting confirmed that
there is a strict nucleotide requirement for forming open promoter
complexes that cannot be overcome by the addition of a dinucleotide
primer corresponding to the start site sequence. However, higher
concentrations of other nucleoside triphosphates can substitute for ATP
in catalyzing open complex formation. Opening catalyzed by these
nucleotides is inhibited by the ATP analogue adenosine
5`-O-(thiotriphosphate), suggesting that they may function
through cross-binding to the ATP site. The K for ATP for opening and the involvement of other nucleotides
in opening differs from the characteristics reported for TFIIH helicase
and C-terminal domain kinase activities. This raises the possibility
that opening does not involve these activities. The results alleviate
very significantly the considerable current uncertainty concerning the
role of ATP in the mammalian mRNA transcription initiation pathway.
Transcription initiation by RNA polymerase II proceeds through a
multistep pathway. First, protein factors interact with DNA elements to
form a closed promoter complex. Then, the closed complex isomerizes
into an open complex in which the DNA template strand is exposed for
transcription. This is followed by the initiation of RNA synthesis.
Sometime after RNA chain initiation has begun, RNA polymerase leaves
the promoter and progresses into the transcription elongation mode
(sometimes termed promoter clearance). This pathway involves a common
set of general transcription factors (activator, TFIID, A, B, pol II, ()F, E, H and J), but the individual roles of most of these
factors remain unsettled. The factors can be assembled onto the DNA in
an ordered or stepwise pathway (for review, see Conaway and
Conaway(1993) and Zawel and Reinberg(1993)), or alternatively,
pre-assembled multifactor complexes can be recruited onto the promoter
(Koleske and Young, 1994; Kim et al., 1994). In the presence
of nucleoside triphosphates these assemblies catalyze the transcription
initiation cycle.
Many studies have concluded that hydrolysis of the
-
bond of ATP is required for pol II transcription initiation
(Bunick et al., 1982; Sawadogo and Roeder, 1984; Conaway and
Conaway, 1988; Jiang et al., 1993). However, there is
considerable uncertainty about what steps involve ATP hydrolysis. A
number of steps have been proposed as requiring ATP, including open
complex formation (Wang et al., 1992a, 1992b; Jiang et
al., 1993) and promoter clearance (Goodrich and Tjian 1994). ATP
is also required for helicase and kinase activities possessed by
factors associated with transcription initiation (Feaver et
al., 1991; Lu et al., 1992; Serizawa et al.,
1992, 1993; Schaeffer et al., 1993). The relationships between
these various ATP-requiring processes and those ATP-dependent enzymatic
activities are not known but have been the subject of much discussion.
The open complex step has been studied quite intensively and has been found to require ATP at a variety of promoters that depend on transcription activators (Wang et al., 1992a, 1992b; Jiang et al., 1993). Recent transcription studies have raised the possibility that in certain systems ATP may not be required to open the transcription start site (Goodrich and Tjian 1994; Timmers 1994). In an attempt to reconcile these various experiments, we have adopted selected conditions used in the minimal systems and directly measured pol II open complex formation. The data show that promoter opening has a strict nucleotide requirement but that high concentrations of other nucleotides can substitute for ATP, possibly by cross-binding. Other results argue against, but do not exclude, that the use of ATP for opening is via the activity of the TFIIH helicase or of the C-terminal domain (CTD) kinase.
High pressure liquid chromatography-purified ribonucleoside
triphosphates and deoxyribonucleoside triphosphates were purchased from
Pharmacia Biotech Inc. Elution profiles, provided by the manufacturer,
demonstrate that the nucleotides are 99% pure and show no
cross-contamination of other nucleotides with ATP. Three different
batches of nucleotides gave similar results. -Amanitin was from
Sigma and was present at a concentration of 1 µg/ml. Hela cell
nuclear extract was prepared as described (Dignam et al.,
1983). Several batches of nuclear extract were used in these
experiments. All showed an absolute requirement for nucleotide in the
opening reaction, with ATP acting at the lowest concentration. The
absolute values of the K
values, however,
varied somewhat with the extract preparation.
The DNA template is as described (Jiang and Gralla, 1993; Wang et al., 1992a; Carey et al., 1990a, 1990b) and is a supercoiled plasmid containing nine GAL4 binding sites upstream of truncated Adenovirus E4 promoter, which retains its core promoter element and transcription start site region. In some experiments, plasmid template was linearized by BglI digestion, as indicated.
Potassium permanganate footprinting was as described (Wang et al., 1992a, 1990b; Jiang et al., 1993, 1994; Jiang and Gralla, 1993). First, the transcription mixture was set up which contains 25 µl of Hela nuclear extract (6 mg/ml protein concentration in D buffer: 20 mM Hepes, pH 7.9, 100 mM KCI, 0.1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), 8.25 mM magnesium chloride, 200 ng of carrier plasmid DNA, 10 ng of supercoiled DNA template or 12 ng of linearized DNA template. GAL4-VP16 or GAL4-AH was added as transcription activator. This mixture was incubated for 30 min to allow the assembly of preinitiation complexes. Then dATP or ATP was added for 2 min to drive promoter opening. Potassium permanganate was added at 6 mM for 3 min to probe the single strand region of DNA. 2-Mercaptoethanol was used to quench the potassium permanganate. Proteinase K was added to digest proteins, which was followed by phenol, phenol/chloroform, chloroform extraction. DNA was precipitated and redissolved in water and was passed through a Sephadex G50-80 spin column to desalt. Taq polymerase was used to extend a radiolabeled primer repetitively, and the products were resolved on a 6% sequencing gel. Potassium permanganate hypersensitive sites, indicative of DNA melting, were revealed on the autoradiograph.
The
investigation of Mg and nucleotide requirements for
open promoter complex formation basically was modeled on the above
procedure. Mg
was omitted from the transcription
mixture to study the Mg
requirement. Other
nucleotides or analogs were added at concentrations as indicated.
Lanes1 and 2 of Fig. 1are repeats of prior experiments and confirm that opening
does not occur in the absence of ATP or in the absence of activator
(compare the absence of signals to the positive control in lane6). Lane3 shows that opening also does
not occur in the absence of divalent magnesium ion. The figure also
shows that opening occurs in the presence of ATP (lane6) but does not occur using the nonhydrolyzable ATP
analogs AMP-PNP (lane4) or ATPS (lane5). Therefore, in agreement with prior studies, pol II
open complex formation requires ATP
-
phosphoanhydride bond
hydrolysis. The experiment also shows that opening requires magnesium
ion, which is an established co-factor in ATP-dependent reactions.
Figure 1:
Pol II open promoter complex formation
requires hydrolyzable ATP and Mg. 10 ng of
supercoiled G9E4T DNA template were incubated with HeLa nuclear
extract, GAL4-AH, and 8.25 mM Mg
for 30 min
to allow the assembly of preinitiation complexes. Then, dATP was added
at 500 µM for 2 min to drive promoter opening (lane6), followed by potassium permanganate probing.
Comparisons include: lane1, without any nucleotide; lane2, without activator; lane3,
without Mg
; lane4, 1 mM AMP-PNP was added instead of dATP; lane5; 500
µM ATP
S was added instead of dATP. The start site
region is bracketed and the -10 position permanganate
sensitivity is indicated. Lanes1 and 2, 3 and 4, and 5 and 6 were from
three separate experiments.
Next we determined whether opening could also occur at very low
concentrations of ATP. Fig. 2shows a titration using various
concentrations of ATP or dATP and either of 2 activators: GAL4-AH and
ATP (Fig. 2A), GAL4-VP16 and dATP (Fig. 2B). In both cases, the opening reaction is not
detectable at 2.5 µM ATP or dATP (Fig. 2, A and B, lanes3) and is quite strong at
10 µM ATP or dATP (Fig. 2, A and B, lanes1). Other experiments (not shown,
but see below) indicate that the reaction is not quite complete at 10
µM and suggest a K of 5-10
µM for ATP or dATP. Because the opening reaction is shown
to have an absolute requirement for added low concentrations of ATP,
the endogenous amounts of ATP must be extremely low or absent.
Figure 2: Titration of ATP in pol II open complex formation. Preinitiation complexes were assembled for 30 min and then various concentrations of ATP or dATP were added for 2 min before potassium permanganate probing. A, GAL4-AH was added as transcription activator. Lane1, 10 µM ATP; lane2, and 5 µM ATP; lane3, 2.5 µM ATP; lane4, 1.25 µM ATP; lane5, without ATP. B, GAL4-VP16 was the transcription activator, and dATP was used instead of ATP, otherwise lanes are as in A. The start site region is bracketed, and the -10 position permanganate sensitivity is indicated.
One
possibility is that opening can occur due to the presence of a
dinucleotide primer; this could conceivably trigger opening by binding
to the polymerase-promoter complex and forcing the strands apart as it
hybridized to the template. To test this possibility we used UpU, which
is complementary to the transcription start site of the adeno E4
promoter used here. Lanes2 and 3 of Fig. 3show that even 2 mM UpU dinucleotide fails to
trigger opening (compare with lane1 in which opening
is driven by dATP). Thus a complementary dinucleotide primer cannot
substitute for ATP in driving promoter melting even at a concentration
that is approximately 200 times greater than the K for ATP.
Figure 3:
UTP but not UMP or dinucleotide UpU can
substitute for ATP to drive promoter opening. Closed preinitiation
complexes were assembled for 30 min with or without -amanitin, and
then different nucleotides were added before potassium permanganate
probing. Lane1, 500 µM dATP was added
for 2 min; lane2, 2 mM UpU was added for 2
min; lane3, 2 mM UpU was added for 8 min; lane4, 500 µM ATP was added for 2 min; lane5, 2 mM UpU and 125 µM UTP
were added for 2 min; lane6, 125 µM UTP
was added for 2 min; lane7,
-amanitin was added
before 2 min of 125 µM UTP addition; lane8, 1 mM UMP was added for 2 min. The start site
region is bracketed, and the -10 position permanganate
sensitivity is indicated. In the absence of
-amanitin, when UpU
together with UTP or UTP alone used to drive promoter opening, the
potassium permanganate hypersites reach +18 position as indicated. Lanes1-3, 4 and 5, 6 and 7, and lane8 were from separate
experiments.
Next we added 125 µM UTP to the reaction containing UpU primer to simulate conditions that would allow RNA chain initiation to begin. Transcripts made in the presence of UpU and UTP can begin with the sequence UpUpU. Permanganate probing shows that a series of changes is induced by the inclusion of UTP in a UpU-primed reaction (lane3 with UpU compared with lane5 with UpU + UTP). Very strong opening is indicated by the strong permanganate signal in lane5, which includes the usual start site region (indicated by brackets). The signal includes additional bands downstream, within the initial transcribed region, up to +18. However, comparison experiments show that this opening reaction does not depend on the presence of dinucleotide UpU; lane6 of Fig. 3shows that both start site and downstream opening can be triggered by 125 µM UTP in the absence of UpU.
This result suggests that
UTP can trigger opening by virtue of it being a hydrolyzable substrate
as was the case with ATP. This is supported by other experiments.
Neither UpU or UMP trigger the opening reaction (UpU, lanes2 and 3; UMP, lane8). In
addition, -amanitin does not inhibit the UTP-dependent opening
over the start site (lane7), showing that initial
RNA synthesis is not required under these conditions.
-Amanitin
does change the pattern, however, in restricting the signal to the
start site as opposed to the initial transcribed region (compare lane7 with lane6). This suggests
that the part of the signal that lies within the initial transcribed
region corresponds to transcription bubbles associated with the
beginning of RNA synthesis (further discussed below). However, the part
of the signal that corresponds to the typical open complex over the
start site does not disappear, confirming that UTP triggers opening
independent of RNA synthesis.
Because the opening triggered by UTP
is not dependent on the formation of a transcript, it is possible that
other nucleotides that do not correspond to the initial transcript
could also trigger opening. The data in Fig. 4show that as ATP,
125 µM of either GTP, CTP, or UTP, can also drive promoter
opening (lanes 2-5versuslane1 without nucleotide added). Because -amanitin was added to
these reactions before nucleotides were added, the downstream
hyper-sites triggered by UTP, which reflect RNA chain initiation, are
not observed (lane3). The data show that there is a
strict nucleotide requirement for opening but that this can be
satisfied by high amounts of other nucleoside triphosphates.
Figure 4:
All four NTPs can be used for promoter
opening and ATPS inhibits open complex formation driven by UTP,
CTP, and GTP. Closed preinitiation complexes were assembled for 30 min
and then
-amanitin was added before a 2-min nucleotide addition
and probing. Lane1, without any nucleotide; lane2, 125 µM ATP; lanes3 and 7, 125 µM UTP; lanes4 and 9, 125 µM CTP; lanes5 and 11, 125 µM GTP; lane6, 125
µM ATP
S was added before 125 µM UTP
addition; lane8, 125 µM ATP
S was
added before 125 µM CTP addition; lane10, 125 µM ATP
S was added before 125
µM GTP addition. The start site region is bracketed, and
the -10 position permanganate sensitivity is
indicated.
These same results were obtained with several different batches of nucleotides purified by fast protein liquid chromatography. Elution profiles showed no cross-contamination with ATP and a purity greater than 99%. Since no signal is seen even when 2.5 µM ATP is added (above) neither cross-contamination nor endogenous ATP can account for the opening seen when other nucleotides are added.
In an
attempt to determine whether these nucleotides use the same enzyme site
as does ATP, we added ATPS to each reaction before UTP, CTP or GTP
was added (lanes6, 8, and 10).
Recall that ATP
S does not have a
-
phosphoanhydride bond
that is easily hydrolyzed and thus does not trigger opening (Fig. 1, lane5). This property has made
ATP
S a useful competitive inhibitor of many enzyme reactions that
rely on the binding and hydrolysis of ATP. The results show that the
125 µM ATP
S inhibits opening driven by equal amounts
of UTP, GTP, or CTP (Fig. 4, lanes6, 8, and 10versuslane 7, 9, and 11). This suggests that all nucleotides use a
similar mechanism to open the start site and that in fact they may all
cross-bind to the same enzyme site, where ATP
S also binds.
Figure 5:
ATP is the preferred substrate for pol II
open complex formation. A, DNA template (10 ng) was added as
supercoiled plasmid. In the presence of -amanitin, open complexes
were assayed in a standard protocol using 10 µM nucleotide; ATP (lane1), GTP (lane2), CTP (lane3), and UTP (lane4). B, the DNA template (12 ng) was linearized
prior to addition to the HeLa extract; 10 µM ATP (lane1), GTP (lane2), CTP (lane3), and UTP (lane4). The start site
region is bracketed, and the -10 position permanganate
sensitivity is indicated.
Recent experiments have
shown that the requirements for transcription can differ depending on
whether linear or supercoiled DNA is used (Parvin and Sharp, 1993;
Timmers, 1994; Goodrich and Tjian, 1994). In addition, it is well
established that the open complex formation by prokaryotic polymerases
can be facilitated by DNA supercoiling (Meiklejohn and Gralla(1989) and
for review, see Pruss and Drlica(1989)). In these and in all of our
previous experiments, the template was added to reactions in the
supercoiled form. The state of the template during the subsequent
incubation has been followed and it has been found that it is gradually
converted to forms with lower superhelical density and to concatamers. ()To investigate whether these results are influenced by
changes in DNA topology, the experiments were repeated using DNA that
had been linearized prior to incubation. The result shows that the
preference of ATP is retained using a linear DNA template (Fig. 5B).
These open complex patterns of melting can be compared with those
seen when initial transcription is allowed. When UTP is used in the
absence of -amanitin, short transcripts ranging from 2 to 6
nucleotides can theoretically be produced, corresponding to initiation
at any of 6 adjacent thymines of the E4 promoter transcription start
site. The data shown that the use of UTP leads to changes in the
pattern of the permanganate sensitivity; the strongest region of
sensitivity now extends from -3 and +18 (Fig. 3, lanes5 and 6). This 21-base pair region is
expected to include complexes associated with transcription of the 6
thymines in the start site region. Thus it likely represents a mixture
of up to 6 overlapping transcription bubbles. These bubbles would each
need to be 15 base pairs long to occupy cumulatively a 21-base pair
region. This suggests that the bubble maintains a constant length of
14-15 base pairs during the transition from open complex to
initial transcribing complex. The results suggest similarities with
prokaryotic transcription, which has a similar bubble size (Siebenlist etal., 1980; Kirkegaard etal.,
1983; Carpousis and Gralla, 1985; Sasse-Dwight and Gralla, 1989) which
remains constant during transcription (Gamper and Hearst, 1982).
It has been known for many years that the elongation
substrate AMP-PNP, an analog of ATP that contains a nonhydrolyzable
-
bond, cannot substitute for ATP in pol II transcription
(Bunick et al., 1982). Inclusion of dATP in a AMP-PNP reaction
restores accurate pol II transcription (Sawadogo and Roeder, 1984;
Conaway and Conaway, 1988; Jiang et al., 1993). These
observations demonstrate the involvement of ATP and the hydrolysis of
its
-
bond in transcription initiation by RNA polymerase II.
However, recent studies involving minimal transcription systems have
found conditions where ATP does not appear to be required for
initiation (Goodrich and Tjian 1994; Timmers 1994).
It was shown previously that formation of a pol II open promoter complex has the same ATP requirements as does transcription (Wang et al., 1992a; Jiang et al., 1993). In this paper we confirm that there is a strict nucleotide requirement for open complex formation. However, we also find that other nucleotides can substitute for ATP when they are present at higher concentrations. Because ATP is the preferred substrate and because the intracellular concentration of ATP is by far the highest of the nucleotides, we infer that promoter opening in vivo is driven primarily by ATP. However, in vitro, whether the promoter opens will be determined by the aggregate effects of all nucleotides that are present.
These observations may be used to rationalize some apparently conflicting observations in the literature. Recent studies have shown that transcription initiation can be detected in the absence of ATP. One case used an abortive initiation system (Goodrich and Tjian, 1994). Such systems typically involve reiterative synthesis of short RNAs, which can be 100 times more abundant than long transcript. Thus even a small fraction of DNA in a functional state could yield a detectable signal in this assay under conditions where opening and long transcripts would be much more difficult to detect. All systems (Timmers, 1994; Parvin and Sharp, 1993; Goodrich and Tjian, 1994) use fully supercoiled DNA, which allows the apparent bypass of several factor requirements, including that for hydrolyzable ATP and activator. All systems contain other hydrolyzable nucleotides, which the current results imply may partially substitute for ATP in triggering opening. This suggests that the lack of requirement for ATP in abortive initiation was related to an ability to detect minor fractions of transcription complexes formed in the presence of suboptimal amounts of substitute nucleotides.
ATP has also been suggested to be required for other steps in the transcription pathway, including promoter clearance (Goodrich and Tjian, 1994). Our results do not address this issue directly, but they support this possibility. The proposed ATP requirement for promoter clearance has two properties that appear to differ significantly from the ATP requirement for opening. The clearance step apparently cannot be facilitated by other nucleotides, whereas other nucleotides can facilitate the opening step. In addition, the ATP requirement for clearance can be bypassed by DNA supercoiling, whereas in the Hela extract the ATP requirement for opening cannot be bypassed by DNA supercoiling; however, this would need to be verified in a more purified system. Nonetheless, it seems likely that polymerase II transcription initiation involves two successive ATP-using steps that are catalyzed by different enzyme activities.
There has been
much discussion of the role of the ATP-dependent DNA helicase and CTD
kinase activities in transcription (Corden, 1990; Young, 1991; Guzder et al., 1994; Jiang and Gralla, 1994). Our results raise the
possibility that these activities might not be involved in promoter
opening. Recall that the data show that the K for
ATP is 5-10 µM for opening and that other NTPs can
partially substitute for ATP. By contrast, the helicase activity has a K
for ATP of nearly 200 µM and can
use only ATP. The CTD kinase activity has a different nucleotide
requirement than that for opening. It works with ATP and GTP, with ATP
being preferred; which differs from the promoter opening activity.
These considerations suggest that promoter opening occurs independent
of the DNA helicase and CTD kinase activities. However, this suggestion
must be tempered by the possibility that these requirements may be
properties of isolated enzymes and may not fully reflect the priorities
of the enzymes within functional transcription complexes.
Thus,
these experiments raise the interesting possibility that there may be
three independent activities associated with polymerase II
transcription that hydrolyze the -
bond of ATP:promoter
melting, CTD kinase, and DNA helicase. We have characterized that the
first ATP dependent activity is involved in the critical promoter
opening step. Future experiments will be required to learn the
relationship of the other two activities to the pathway of mammalian
mRNA transcription.