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J. Biol. Chem., Vol. 275, Issue 45, 35006-35012, November 10, 2000
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,, and¶
From the Department of Chemistry, Bowling Green State
University, Bowling Green, Ohio 43403 and the
§ Department of Chemistry, Ohio Northern University, Ada,
Ohio 45810
Received for publication, May 31, 2000, and in revised form, July 3, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The TATA-binding protein (TBP) in the TFIID
complex binds specifically to the TATA-box to initiate the stepwise
assembly of the preinitiation complex (PIC) for RNA polymerase II
transcription. Transcriptional activators and repressors compete with
general transcription factors at each step to influence the course of the assembly. To investigate this process, the TBP·TATA complex was
titrated with HMG-1 and the interaction monitored by electrophoretic mobility shift assays. The titration produced a ternary
HMG-1·TBP·TATA complex, which exhibits increased mobility relative
to the TBP·TATA complex. The addition of increasing levels of TFIIB
to this complex results in the formation of the TFIIB·TBP·TATA
complex. However, in the reverse titration, with very high mole
ratios of HMG-1 present, TFIIB is not dissociated off and a
complex is formed that contains all factors. The simultaneous addition
of E1A to a mixture of TBP and TATA; or HMG-1, TBP, and TATA; or TFIIB, TBP, and TATA inhibits complex formation. On the other hand, E1A added
to the pre-established complexes shows a significantly reduced capability to disrupt the complex. In add-back experiments with all
complexes, increased levels of TBP re-established the complexes, indicating that the primary target for E1A in all complexes is TBP.
The assembly of the transcriptional preinitiation complex
(PIC)1 on a promoter is the
pivotal event in the regulation of gene expression (reviewed in Refs.
1-4, 61). The successful completion of the stepwise formation of PIC
is prerequisite to the initiation of RNA polymerase II transcription.
The initial steps in assembly involve the binding of TFIID to the TATA
element in the promoter, in which recognition and binding occurs
through the sequence-specific subunit, the TATA-binding protein (TBP).
This is followed by the binding of TFIIB to form the TFIIB·TBP·TATA
complex, which represents the molecular platform for the subsequent
complexation with RNA polymerase II/TFIIF and other general
transcription factors essential for basal level transcription.
However, there are a multitude of regulatory proteins, activator and
repressor proteins and cofactors, that may actively impinge on the
assembly process, leading to either an enhancement or inhibition of the
level of transcription (reviewed in Refs. 1, 3, 5-7). Two major
targets for many regulatory proteins appear to be TBP (8, 9) and TFIIB
(10, 11). Cellular and viral proteins, which interact directly with TBP
and TFIIB include the TAFs (12-14), TFIIA (15-17), c-Myc (18),
HMG-1 (19), p53 (20, 21), NC1 (22, 23), human Dr1·DRAP complex
(identical to NC2) (24-26), c-Rel (27), adenovirus E1A (17,
28-32), and VP16 (33, 34).
Repressors exhibit a number of mechanisms to effect their action (6,
35). HMG-1 and NC2 represent general repressors, in that they both
interact with TBP to block PIC formation (19, 25, 26). The ubiquitous,
abundant, and highly conserved HMG-1 protein has been further
implicated in the regulation of transcription, exhibiting both positive
and negative effects on transcription (9, 19, 36-39). HMG-1 has been
reported to bind to the TBP·TATA complex, which inhibits subsequent
TFIIB binding, resulting in incomplete PIC assembly and thereby
inhibiting transcription. Interestingly, the addition of increasing
levels of TFIIB in in vitro transcription assays was unable
to restore activity (19). The multifunctional adenovirus E1A
oncoprotein, which like HMG-1, exhibits no sequence-specific DNA
binding activity (40-42), has been shown to serve as an activator of
viral gene expression (5), while exhibiting inhibitor or activator
activities with specific cellular promoters (43). The E1A product has
been reported to effect its action in some cases by binding to TBP (28,
29, 31), whereas the 12S E1A product is reported to bind Dr1, thereby facilitating its dissociation from the TBP·TATA complex (26).
In this work, we used a sensitive gel shift assay and provide direct
evidence that HMG-1 forms a stable EMSA complex with TBP·TATA. The
complex exhibits an increased mobility relative to the TBP·TATA
complex, contrary to EMSA findings with other reported DNA·protein
complexes. The TFIIB·TBP·TATA complex is stable in the presence of
low levels of HMG-1, but conditions in which there are high excesses of
HMG-1 produce a complex that contains both HMG-1 and TFIIB.
Furthermore, adenovirus E1A inhibits the formation of the TBP·TATA,
HMG-1·TBP·TATA, and TFIIB·TBP·TATA complexes, while the
pre-established complexes resist dissociation by E1A. The addition of
excess TBP re-establishes the complexes, indicating that TBP is the
primary target for E1A action in all complexes.
Isolation, Purification, and Characterization of
Proteins--
Calf thymus HMG-1 was purified in non-denaturing
conditions using salt extraction, differential ammonium sulfate
precipitation, and fractionation by HPLC using a MonoQ column as
outlined previously (44). The expression vector, pET-His6-hTBP,
provided by F. Pugh, was transfected into Escherichia coli
BL21(DE3) cells and the protein purified by polyethyleneimine
fractionation, phosphocellulose chromatography, and ammonium sulfate
precipitation as described by Pugh (45). The expression vector, phIIB,
obtained from D. Reinberg, was similarly used to obtain TFIIB, which
was purified using phosphocellulose chromatography (46). The GST-E1A
(13 S; 289 amino acid residues) fusion protein was expressed in BL21 cells containing the pGEX-E1A (obtained from T. Shenk) and was purified
by binding to glutathione agarose, elution by glutathione, followed by
dialysis into reaction buffer (47). All proteins were greater than 80%
pure as evidenced by Coomassie staining of gels run on
SDS-polyacrylamide gel electrophoresis.
EMSA Studies--
Oligonucleotides that make up the adenovirus
major late promoter (Ad MLP;
For supershift experiments, the reaction mixtures were incubated for 30 min at 30 °C, and then the antibody (polyclonal HMG-1 was reported to bind to TBP both as a GST-TBP fusion protein
and when TBP was complexed with the TATA element in the adenovirus
major late promoter (Ad MLP) (19). To further characterize this
interaction, the titration of the TBP·TATA complex with HMG-1 was
followed by EMSA. Preliminary experiments showed that the glutamate
buffer system consistently produced an EMSA-stable TBP·TATA complex
and provided an essential "window" to determine if competing factors affected the TBP·TATA interaction. Fig.
1A shows that the addition of
HMG-1 produced a concentration-dependent decrease in the
original band for the TBP·TATA complex, with the concomitant increase
in a single band for an HMG-1·TBP·TATA complex. Interestingly, the
new band exhibited an increased mobility relative to that for the
TBP·TATA complex. Fig. 1B shows that this complex required the presence of both TBP and HMG-1 and could be titrated away with cold
oligonucleotide containing the TATA sequence, whereas it was unaffected
by the addition of an oligonucleotide containing the GCTA sequence in
lieu of TATA. Fig. 1C confirms the presence of HMG-1 in the
new complex, because the band was supershifted by anti-HMG-1, whereas
the anti-TFIIB control produced no effect. These data indicate that the
HMG-1 binding was dependent on both the TATA sequence and TBP and the
HMG-1·TBP·TATA complex exhibited an anomalous increased mobility.
The unusual mobility is not unique to this electrophoretic buffer
system, because the increased mobility of the complex was also observed
using other buffers (data not shown). Therefore, this electrophoretic
behavior is a characteristic of this complex under a variety of
conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 to 1) were purchased from National
Biosystems and 32P-end-labeled. The DNA (approximately 0.4 ng) was reacted with human TBP (approximately 1 nM unless
indicated otherwise), with and without other transcription factors, in
binding buffer (24 mM Tris acetate, pH 8.0, 10% glycerol,
4 mM magnesium acetate, 50 mM potassium
glutamate, 0.1 mM EDTA, 1 mM dithiothreitol,
0.01% Nonidet P-40, 4 mM spermidine, 5 µg/ml
poly(dG-dC), and 50 µg/ml BSA) for 30 min at 30 °C or 4 °C, as
indicated. The reaction mixture was loaded on a 4% polyacrylamide gel
in 0.35× TBE buffer containing 0.05% Nonidet P-40, and the
electrophoresis was carried out at 4 °C. The gels were then dried
and exposed to x-ray film at 80 °C. Oligonucleotides (Santa Cruz
Biotechnologies) used in competition studies for TBP binding with the
Ad MLP DNA contained either the wild-type or mutated TATA motif.
Binding studies were carried out by either simultaneous addition of all
components, or alternatively, the final transcription factor was added
30 min after the initial complex was established and reaction was
continued for an additional 30 min.
-HMG-1 from
R. Roeder or affinity-purified -TFIIB from Santa Cruz
Biotechnologies) was added at 4 °C for 10 min before loading the gel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
[in a new window]
Fig. 1.
HMG-1 interaction with the TBP·TATA
complex. A, HMG-1 forms a stable ternary complex with
TBP bound to the Ad MLP. Increasing amounts of HMG-1 were added to the
TBP·TATA complex. HMG-1 concentrations in lanes 1-6 are
0, 0.6, 1.7, 5.0, 15, and 45 nM. B, Sequence
specificity of TBP·TATA and HMG-1·TBP·TATA complexes. Unlabeled
oligonucleotides were used to compete in complex formation with
32P-labeled Ad MLP. All lanes contain mixtures of TBP and
32P-labeled Ad MLP, with lanes 4-6 also
containing approximately 40 nM HMG-1. No competitor
oligonucleotide was added in lanes 1 and 4. A
25-fold molar excess of unlabeled oligonucleotide with a TATA sequence
(lanes 2 and 5) or with a GCTA sequence in lieu
of TATA (lanes 3 and 6) was added in competition.
The TATA-containing oligonucleotide is
GCAGAGCATATAAAATGAGGTAGGA. C, HMG-1·TBP·TATA
complex is supershifted by -HMG-1. The HMG-1·TBP·TATA complex
(lane 1) was treated with two levels of -HMG-1
(lanes 2 and 3) or with -TFIIB control
(lane 4). The asterisk indicates the band
position for the TBP·TATA complex; the arrow points to the
supershifted band.
Fig. 2 compares the relative stability of
the TBP·TATA and HMG-1·TBP·TATA complexes at a number of TBP
levels. At equal levels of TBP, the band intensity for the
HMG-1·TBP·TATA complex was significantly greater than that for the
corresponding TBP·TATA (and also in relation to the free DNA band
intensity) at all levels of TBP examined. This indicates that HMG-1
binding enhanced complex formation, by at least 10-fold, leading to an
increased population of the HMG-1·TBP·TATA complex. In similar
experiments in which individual structural domains of HMG-1 (the A-box
and B-box domains; residues 1-89 and 86-165, respectively) were
reacted with the TBP·TATA complex, the mobility and the band
intensity of the TBP·TATA complex was unchanged, even at [HMG-1
box/TBP] mole ratios as high as 2000 (data not shown). The addition of
the individual A- and B-boxes together also exhibited no changes (data
not shown).
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The addition of TFIIB to the TBP·TATA complex forms the TFIIB·TBP·TATA complex, which, like the HMG-1·TBP·TATA complex, also exhibits a greater stability in gel shift experiments than does the TBP·TATA complex (48).2 However, unlike the HMG-1·TBP·TATA complex, this ternary complex exhibits a reduced mobility relative to the TBP·TATA complex.
It was of interest to determine if TFIIB and HMG-1 compete for
overlapping sites on the TBP·TATA complex or whether these two
factors bind simultaneously to TBP·TATA and form a stable complex.
This electrophoretic buffer system provides a convenient means to
resolve this question, because the two complexes exhibit opposite
mobilities in the EMSA system. Fig.
3A shows that the addition of
increasing amounts of TFIIB to the TBP, TATA, and limiting HMG-1
(sufficient for complex formation) mixture resulted in the formation of
the TFIIB· TBP·TATA complex. At a [TFIIB/HMG-1] mole ratio of
0.2 (lane 3), there was a 50/50 mixture of the two complexes, whereas only the TFIIB·TBP·TATA complex was detectable at or above a mole ratio 0.6 (lane 5). This indicates that
TFIIB bound much more effectively than HMG-1 to the TBP·TATA
complex.
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In the reverse titration (lanes 8-14), limiting amounts of TFIIB were used to permit TFIIB·TBP·TATA complex formation, with the addition of increasing levels of HMG-1. The addition of HMG-1, up to mole ratios of less than 0.4 (lane 10) had no effect on the complex, consistent with the data in lanes 1-7. However, as the levels of HMG-1 were increased enormously, with the [TFIIB/HMG-1] mole ratio in the range of 0.3-0.03 (lanes 12-14), a band of intermediate mobility was produced, indicating that a simple HMG-1·TBP·TATA complex was not formed.
Fig. 3B shows that the band in lane 7 in 3A was not supershifted by anti-HMG-1 (lane 2) but was supershifted by the addition of anti-TFIIB (lane 3), indicating that the complex contained TFIIB·TBP·TATA. Fig. 3C shows similar results that examined whether TFIIB remained in the complex after TFIIB·TBP·TATA was reacted with large excesses of HMG-1. Lane 2 shows that anti-TFIIB supershifted this complex (lane 1 is the same complex from lane 14 in Fig. 3A). Under these conditions of limiting TFIIB and huge excesses of HMG-1, TFIIB remained in this complex, with the resultant formation of a TFIIB·HMG-1·TBP·TATA complex.
The adenovirus E1A protein can either activate or repress transcription
presumably by interacting with coactivators, the PIC, or components at
different stages in the assembling preinitiation complex. Fig.
4 shows the effect of the GST-E1A fusion
protein on disrupting the TBP·TATA (Fig. 4A),
TFIIB·TBP·TATA (Fig. 4B), and the HMG-1·TBP·TATA
(Fig. 4C) complexes. This experiment was carried out by
either (a) the addition of GST-E1A after the complexes were
established (lanes 1-6) or (b) by simultaneous
addition of all proteins (lanes 7-12). Fig. 4 reveals that
similar results were observed for all complexes examined. The presence
of E1A was able to completely inhibit complex formation if
E1A was added simultaneously with all components. On the other hand,
E1A was unable to effect complete complex dissociation of
the established complexes, but did partially disrupt them at the
highest GST-E1A levels (approximately 80 ng). The control reaction in
which GST was added alone exhibited no effect.
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To begin to understand the mechanism of E1A action and investigate the
primary target of E1A, add-back experiments were carried out. In these
experiments, sufficient E1A was added to just obviate the formation of
the original three complexes, followed by addition of increasing
amounts of TBP, HMG-1, or TFIIB to the respective complexes, in an
attempt to compete with E1A and, as a result, re-establish the
complexes. Fig. 5 (A,
B, and D) shows that the addition of increasing
levels of TBP to the mixture of E1A, DNA, and TBP (A); TFIIB
and TBP (B); or HMG-1 and TBP (C) re-established each of the three complexes at about the same level. In each case, it
requires an 8-fold increase of TBP. Of importance is that the [TBP/E1A] mole ratio at the point of the re-established complexes was
about 0.3 in all cases. In the case in which increasing levels of TFIIB
were added back to the TBP, TFIIB, E1A, oligonucleotide mixture (Fig.
5C), a [TFIIB/E1A] mole ratio of greater than 60 (last lane) had very little effect. In the case of adding
increasing levels of HMG-1 back to the corresponding HMG-1, TBP, E1A,
oligonucleotide mixture (Fig. 5E), a [HMG-1/E1A] mole
ratio of greater than 50 (last lane) was found to have no
significant effect. These results indicate that the primary interaction
of E1A was with TBP in all the complexes examined.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The action of regulatory proteins on the formation of the transcriptional preinitiation complex determines the rate at which transcriptional initiation will ensue, and indeed, the fate of the committed complex. The regulation of PIC assembly itself, by a dynamic balance in the binding of general factors and activator and repressor proteins, is an important element in transcriptional control. We have examined the early stages of PIC assembly, with the focus on the ternary complexes containing TBP·TATA and either HMG-1 or TFIIB and the effect of E1A on the course of the assembly process.
Reaction of TBP·TATA with increasing concentrations of HMG-1 produced
a distinct EMSA complex, which is dependent on the presence of both TBP
and a TATA-containing oligonucleotide, with the band being supershifted
by anti-HMG-1. Identification of this complex as HMG-1·TBP·TATA is
in accord with a previous report (19). HMG-1 occurs as a monomer in
solution and, although the stoichiometry of the complex is not known,
HMG-1 is presumed to bind as a monomer. Interestingly, the mobility of
the HMG-1·TBP·TATA complex is increased relative to that for
TBP·TATA complex and differs from the mobility of the complex in the
previous study (19). As far as we know, the increased mobility
observed, as a result of another protein complexing with TBP·TATA,
has not been observed previously for any other EMSA complexes. This
unexpected mobility may be influenced by the electrophoretic buffer but
is fundamentally a result of either the large net negative charge on
HMG-1 (9), the ability of HMG-1 to alter the bend angle of DNA in the
complex and/or shape of the complex (49), or contributions from both.
Previous studies have shown that the sequence-specific binding affinity of a number of regulatory factors, including steroid receptor proteins (50, 51), HOXD9 protein (37), p53 (53), and the Oct-POU domains of Oct-1, -2, and -6 (54), was stimulated in the presence of HMG-1. In these cases, although the original EMSA band for the complex did increase in intensity, its position did not shift in the presence of HMG-1. The behavior of HMG-1 in these systems contrasts with our findings that indicate the formation of an EMSA-stable HMG-1·TBP·TATA complex.
The stability of the HMG-1·TBP·TATA complex is greater than that for the TBP·TATA complex as evidenced that, at equivalent amounts of TBP, there is significantly more HMG-1·TBP·TATA complex than TBP·TATA complex. This enhancement of complexation is similar to that observed for both TFIIB and/or TFIIA bound to the TBP·TATA complex. In addition, we find no evidence that the A- and B-boxes of HMG-1 bind with the TBP·TATA complex (data not shown), suggesting that the stable HMG-1 binding may involve multiple-site interactions between HMG-1 and the TBP·TATA complex. The requirement for multiple-site interactions is quite common in stable protein-protein interactions and has been proposed, for example, in the binding of E7 protein to TBP and for E1A protein binding to the retinoblastoma protein (55, 56). Added support for this proposal comes from competition experiments in which HMG-1 is not competed off the HMG-1·TBP·TATA complex by high levels (600-fold excess) of A- and B-boxes, either individually or together (data not shown). These findings may also suggest that the C-terminal domain of HMG-1, which contains a highly acidic tail, may take part in the stable interaction with TBP. This possibility has been suggested previously (19).
It was reported that addition of increasing levels of TFIIB could not restore basal level transcription in an HMG-1-inhibited in vitro assay (19). However, as shown in Fig. 3A (lanes 1-7), increasing levels of TFIIB, in the context of limiting HMG-1, does effectively prevent HMG-1 binding in the complex, resulting in the formation of TFIIB·TBP·TATA. Fig. 3B shows that this band is supershifted by anti-TFIIB, but not by anti-HMG-1, indicating that the band corresponds to the TFIIB·TBP·TATA complex. The reverse titration, done in conditions of limiting TFIIB, showed that low levels of HMG-1 had no effect on TFIIB·TBP·TATA complex formation (in agreement with the previous titration). The addition of very high levels of HMG-1, however, produced a band, intermediate in mobility to the TFIIB·TBP·TATA and the HMG-1·TBP·TATA complexes. The presence of TFIIB in this complex was confirmed, because the addition of anti-TFIIB produced a supershift of the band (Fig. 3C). These data indicate that the band represents a complex that contains both TFIIB and HMG-1 and, under our conditions, this complex is observed only at high HMG-1 levels that were used in an attempt to compete off TFIIB. An EMSA complex assumed to be HMG-1·TFIIB·TBP·TATA was reported previously, but no evidence for the presence of TFIIB in the complex was presented (57). This finding indicates that there are conditions in which an intermediate complex can be formed, which contains TBP·TATA, with the simultaneous and stable binding of both HMG-1 and TFIIB. It should be pointed out that the transcription factor, TFIIB, is conformationally pliable as indicated by both structural and biochemical studies. It has been shown that transcriptional activators, such as VP16 and Pho4 (58, 59), induce a conformational change in TFIIB, and this behavior is consistent with what was observed in conditions of very different levels of HMG-1 in our experiments.
The finding that TFIIB is very effective in competing limiting amounts of the transcriptional inhibitor, HMG-1, from the HMG-1·TBP·TATA complex is consistent with both factors competing for the same or overlapping binding sites on the TBP· TATA complex. The crystal structure for the TFIIB·TBP·TATA complex shows that TFIIB binds to residue 289 at the junction of S2'-S3' in the second stirrup of TBP (26, 70), suggesting this region as a potential target. In addition, TFIIB interaction also involves binding to DNA, both upstream and downstream of the TATA element (8, 58-60), which HMG-1 binding may also include. Another aspect of this, which may not be mutually exclusive, is that HMG-1 binding to TBP may lead to an alteration of the bond angle of the DNA.
The presence of the E1A protein is devastating to the assembly of all complexes investigated. It does not just inhibit HMG-1 or TFIIB from associating with TBP on the TATA element, it completely inhibits the formation of the TBP·TATA, HMG-1·TBP·TATA, and the TFIIB·TBP·TATA complexes (when all factors are added simultaneously). Because E1A is known to bind to TBP (17, 28-32), this is consistent with E1A·TBP complexation in solution, resulting in the inhibition of TBP binding to the TATA sequence. This may also suggest that TBP is a highly specific target for E1A, with binding to the other factors being less significant in this context. Consistent with this proposal, approximately the same level of E1A was effective in inhibiting formation of all three complexes. On the other hand, if these three complexes are established before E1A addition, E1A exhibits only a weakly disruptive effect. Although there is some reduction in the level of complex, a significant amount of complex remains even at the highest levels of E1A. These data indicate that E1A is significantly less effective in dissociating or disrupting the preformed complex than it is in inhibiting the assembly of the factors before complexation.
HMG-1 shares some common features with the general transcriptional repressor, Dr1 (24-26). HMG-1 and Dr1 repressor proteins bind directly to TBP, both in solution and in the TBP·TATA complex, leading to a reduced transcriptional activity due to inhibition of PIC formation. However, although Dr1 inhibition cannot be overcome by increasing the concentrations of TFIIA, RNA polymerase II, or the other general transcription factors (25), inhibition of transcription by HMG-1 can be reversed by increased levels of TFIIA, but not TFIIB (19). Future studies must be done to reveal further the extent of similarity of these two repressors.
To gain some initial insight into the mechanism of action and the
primary targets for E1A in these complexes, add-back experiments were
performed. The addition of increased levels of TBP to a solution with
sufficient E1A to inhibit TBP·TATA formation (Fig. 5A) led to the re-establishment of the TBP·TATA complex. The complex was completely re-established at about an 8-fold increase in TBP. This was
consistent with previous findings that TBP is a target for E1A (24, 25)
and that the addition of excess TBP in these experiments overcomes
TBP·E1A complexation and permits productive TBP·TATA complex
formation. This effect of TBP is a strong one in that it occurs at a
low mole ratio (0.3) of [TBP/E1A]. TBP is likewise able to
re-establish the TFIIB·TBP·TATA and the HMG-1·TBP·TATA complexes (Fig. 5, B and D) at approximately this
same mole ratio of [TBP/E1A]. On the other hand, the addition of
comparable mole ratios of TFIIB/E1A was ineffective in re-establishing
the TFIIB·TBP·TATA complex (Fig. 5C). In fact, a
[TFIIB/E1A] mole ratio of greater than 60 produced no significant
TFIIB·TBP·TATA complex formation. The same was true for attempts to
re-establish the HMG-1·TBP·TATA complex (Fig. 5E), in
which a [HMG-1/E1A] mole ratio of greater than 50 had only a small
effect. These collective findings indicate that E1A is specific for
targeting TBP, with neither TFIIB nor HMG-1 being targets for E1A
binding. In comparison with similar competition studies in which the
12S E1A product was used with Dr1, TBP, and a TATA-containing
DNA, enormous excesses (microgram quantities) of E1A were
required to inhibit assembly of the Dr1·TBP·TATA complex (26).
Nonetheless, our results indicate that E1A has the capability of
preventing the initial stages of PIC formation and, therefore, may lead
to inhibition of transcription.
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ACKNOWLEDGEMENTS |
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We thank F. Pugh, D. Reinberg, and T. Shenk for the expression vectors for TBP, TFIIB, and GST-E1A, respectively, and to R. Roeder for polyclonal antibody to calf thymus HMG-1.
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FOOTNOTES |
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* This work was supported by Grant R15 from the National Institutes of Health (to W. M. S.) and by grants from the Ohio Cancer Research Associates and the American Cancer Society, Ohio Division.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 Chemistry, Bowling Green University, Overman Hall, Bowling Green, OH 43403. Tel.: 419-372-8293; Fax: 419-372-9809; E-mail: wscovel@bgnet.bgsu.edu.
Published, JBC Papers in Press, July 5, 2000, DOI 10.1074/jbc.M004735200
2 W. Lu, R. Peterson, A. Dasgupta, and W. M. Scovell, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
PIC, preinitiation
complex;
TBP, TATA-binding protein;
TAFs, TATA-binding
protein-associated factors;
HMG-1, high mobility protein-1;
EMSA, electrophoretic mobility shift assay;
his-tagged hTBP, N-terminally
hexahistidine-tagged human TBP;
GST-E1A, glutathione
S-transferase-E1A fusion protein;
Ad MLP, adenovirus major
late promoter;
S2'-S3', segment containing 
-sheets 2'-3'.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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