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INTRODUCTION |
The proper temporal and spatial expression of a protein encoding
gene in eukaryotes requires the formation of a large, multiprotein preinitiation complex at the gene promoter. The process begins with
sequence-specific DNA recognition of the promoter. Two primary recognition elements, TATA and initiator
(Inr),1 have been extensively
studied (reviewed in Ref. 1). Each of these elements is recognized by
specific proteins as follows: TATA by the TATA-binding protein (TBP)
and Inr by several proteins including TBP-associated factors. In
addition to recognition, these elements also play regulatory roles.
TATA has been shown to play a role in setting the rate of transcription
reinitiation (2, 3), whereas Inr can play a role in activation (4).
Although much is now known about the roles of TATA and Inr,
comparatively little is known about possible roles for other sequences within the core promoter. Ultraviolet cross-linking experiments have
shown that several transcription factors are in intimate contact with
the DNA throughout the core promoter region. TFIIA, IIB, IIE, IIF,
TBP-associated factors, and polymerase II subunits all cross-link to
regions between
50 and +15 relative to the transcription start site
(5-8), suggesting that these sequences have the potential to influence
the binding or functionality of these factors. Although no core
consensus sequence exists outside the TATA and Inr elements, there are
clear nucleotide preferences, with the majority of promoters being
GC-rich (9).
Two recent studies have investigated the roles of non-TATA, non-Inr
sequences. A GC-rich sequence upstream from TATA (the BRE) can enhance
TFIIB binding to the TBP·DNA complex and stimulate basal
transcription (10). Sequences both upstream and downstream from TATA
can affect transcription; these can alter both basal and activated
levels independently (9). The latter observations were based on
"block-swapped" promoters in which blocks of sequences were
exchanged among promoters, and transcription and factor binding were
assayed. Bandshift analyses showed that the 10-base pair blocks
flanking the TATA box play roles in both TBP and TFIIB binding. For
example, replacing either (or both) of these blocks in the more GC-rich
adenovirus ML promoter with the corresponding AT-rich sequences from
the E4 promoter lowered the level of TBP binding.
Two aspects of these results were unexpected. First, there were no
prior indications of a role for the block downstream from TATA in
transcription. Second, the TBP binding results are difficult to explain
as there is no prior evidence that TBP contacts sequences that flank
TATA; crystallographic and UV cross-linking studies have suggested that
TBP makes contacts only within the TATA box region (11-13). Of course
the effects on TBP binding and on transcription could be closely related.
One possibility for the role of these sequence blocks is that
surrounding the AT-rich TATA element with GC-rich sequences (as in the
ML case) could make the TATA box more distinctive as TBP scans the DNA
for the correct binding site. At an AT-rich promoter the TBP search
might be slowed if nearby TATA-like sequences are present, and this
would be reflected by a lower rate of association. A different
possibility is that AT-rich and GC-rich sequences take on unique
structures and that the borders between these sequences form
discontinuities. Boundary discontinuities such as heteroduplex bubbles
have been shown to stabilize TBP binding, and this effects only the
dissociation rate, making it slower (14). Below we measure the kinetics
of TBP interactions with block-swapped promoters to assess these
explicit possibilities and provide additional constraints on models for
the roles of flanking sequences. The data strongly favor the
discontinuity effect.
The prior data also left unexplained an observed variation in the
effect of TFIIB. In all cases the amount of TBP·TFIIB·TATA complex
formed correlated with the level of basal transcription. However, the
effect of TFIIB on driving TATA DNA into complexes was substantially
greater in one context: when the upstream block was GC-rich and the
downstream block was AT-rich. We searched for a source for this
phenomenon by studying the kinetics of TFIIB·TBP·DNA complexes,
which had not been done previously. The outcome provides an explanation
in terms of TFIIB having different effects on the rates of formation
and rates of decay of these complexes depending on promoter context.
Overall, the data suggest how blocks that flank TATA can influence the
kinetics of factor binding in unique ways and thus enhance the
potential for diversity in transcriptional regulation.
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MATERIALS AND METHODS |
Nucleic Acids and Proteins--
Probes for gel shifts were made
by annealing pairs of 49-nucleotide-long oligonucleotides (Operon) with
20 base pairs of complementarity at their 3' ends (see Ref. 9).
Equimolar (1 nmol) amounts of each oligonucleotide were annealed in 20 mM Tris-Cl, pH 7.5, 10 mM MgCl2,
100 mM NaCl in a 25-µl volume by rapid heating to
95 °C and slowly cooling to room temperature in a polymerase chain reaction thermocycler (MJ Research). The overhanging ends were filled
by mixing the 25-µl annealing reactions with 7.5 mM
dithiothreitol (DTT), 33 µM each dNTP, and 5 units of
Klenow enzyme (Life Technologies, Inc.) in a final volume of 50 µl
and incubating for 1 h at room temperature. DNA was precipitated
with sodium acetate and ethanol, dissolved in 8 M urea,
0.5× TBE and purified on 12% urea-polyacrylamide gel electrophoresis
(8 M urea, 29:1 ratio of acrylamide to bisacrylamide, 0.5×
TBE). The highest molecular weight bands were visualized by UV
shadowing and excised from the gel. The gel slices were crushed to
beads and soaked overnight in 1 ml of 0.3 M NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA at 37 °C. The
beads were then spun down in a centrifuge and the supernatant
recovered. DNA was again precipitated and dissolved in Tris-Cl, pH 8.0. The concentration of DNA was determined by heating the DNA to 95 °C
to denature and taking the absorbance at 260 nm. The absorbance
was multiplied by 33 µg/ml to determine the total concentration of
single strands. The probes were labeled by mixing 1.8 pmol of total
single-stranded DNA with 25 pmol of [
-32P]ATP and 3 units T4 polynucleotide kinase (Promega) in 25 µl and incubating at
37 °C for 1 h. Labeled DNA was separated from free nucleotides
though a 1-ml G-50/TE spin column. The eluates were mixed with 80 mM NaCl, 10 mM HEPES, pH 7.9, in a final volume of 45 µl, and polymerase chain reaction was annealed as above. The
final concentration of double-stranded probe was 20 nM. The annealed products were run on 5% native gels (59:1 ratio of acrylamide to bisacrylamide, 0.5× TBE) to verify the annealing and on 12% urea-polyacrylamide gel electrophoresis (see above) to verify the
Klenow extension. These probes contain the ML and/or E4 promoter sequences from
41 to +18 flanked by restriction sites.
Full-length, His-tagged human TBP (15) was provided by A. Berk (UCLA).
Full-length, C-HMK-tagged human TFIIB (16) was provided by M. Carey (UCLA).
Gel Shifts--
Electrophoretic mobility shift assays were
performed as described previously (9). Briefly, 1 fmol of labeled probe
was mixed with the indicated amounts of TBP and TFIIB in a 10-µl
reaction that contained 12 mM HEPES, pH 7.9, 12% glycerol,
60 mM KCl, 0.12 mM EDTA, 0.6 mM
DTT, 8 mM MgCl2, 50 ng of poly(dG·dC), and
500 ng of bovine serum albumin (Sigma). Proteins were bound at 30 °C
for the indicated times and loaded directly onto prechilled 5% native
gels (59:1 acrylamide:bisacrylamide, 5% glycerol, 2 mM
MgCl2, 1 mM DTT, 0.5× TBE) and run in
prechilled 0.5× TBE, 2 mM MgCl2 buffer. Gels
were run in Bio-Rad mini-Protean 3 units at 400 V for 30 min. The
temperature of the inner tank and gels never exceeded 25 °C. The
gels were dried and exposed to PhosphorImager screens (Molecular
Dynamics) for 16 h. Time course experiments were initiated by
adding protein and probe to reaction mixtures containing all the
remaining components (see above). Time points were initiated at
intervals so that they could all be loaded on the gel at the same time,
ensuring that any dissociation of complexes in the gel would be
constant for all samples.
On-rate Determination--
The indicated amounts of protein were
bound to DNA for various times before loading on gels. The appropriate
shifted complexes were quantitated using ImageQuaNT software (Molecular
Dynamics). The raw data (counts) were fitted by nonlinear least squares
regression to Equation 1,
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(Eq. 1)
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using the SigmaPlot (SPSS, Inc.) program for PC.
kobs and Ffinal are the
calculated first-order rate constant and calculated fraction bound at
the completion of the reaction, respectively. In many cases the raw
data at each time point were normalized to
Ffinal for plotting. The half-time of
association is given by Equation 2,
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(Eq. 2)
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Second-order rate constants for TBP binding were determined by
plotting the reciprocal of the kobs determined
at each concentration of TBP versus the reciprocal of the
TBP concentration. kon was then taken as the
reciprocal of the slope of this plot.
Off-rate Determination--
The indicated amounts of protein
were bound to DNA for 1 h and then challenged with 20 ng/µl
poly(dI·dC) for the indicated times. As above, each time point was
initiated at intervals and then challenged after 1 h so that all
time points could be loaded simultaneously. Addition of poly-(dI·dC)
prior to probe results in no detectable binding (data not shown). The
bands were quantitated and fit by nonlinear least squares regression to
Equation 3,
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(Eq. 3)
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where F0 is the calculated starting
fraction bound and koff the calculated rate
constant. The half-life of the complex is given by
t1/2 = ln(2)/koff, and the dissociation constant for TBP is given by KD = koff/kon.
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RESULTS |
We showed previously that replacing either or both of the 10-base
pair GC-rich blocks flanking the ML TATA box with the more mixed
sequence blocks from the adenovirus E4 promoter led to a significant
decrease in the level of TBP binding (9). TBP does not contact these
sequences directly, leaving the source of the effect unknown. To help
distinguish between various models (see Introduction), we measured
whether the altered binding was due to changes in on-rate or off-rate
or both.
TATA-flanking Blocks Alter TBP Off-rates but Not On-rates--
We
performed on-rate experiments using the promoter constructs shown in
Fig. 1. Standard bandshift experiments
were used to assess the level of TBP binding at various times after
mixing with promoter DNA. An autoradiograph from one such experiment is
shown in Fig. 2A. With TBP at
20 nM, binding becomes detectable as early as 1 min after
addition of probe and continues to rise until reaching saturation at
about 20 min. The half-time to saturation is ~5 min. The bands were
quantified by PhosphorImager analysis and fit to Equation 1, see under
"Experimental Procedures." The data and best fit curve are plotted
in Fig. 2B. This reaction appears to follow first-order
kinetics with a kobs of 2.7 × 10
3 s
1. The
concentration of TBP in this experiment (20 nM) is
comparable to the KD for TBP dimerization (10 nM, see Ref. 17), and yet we do not see the apparent
two-phase kinetics that might be expected if dissociation of TBP dimers
were rate-limiting (17). This first order behavior may be related to
the limiting amounts of DNA used in these assays.

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Fig. 1.
The ML swap series. The 78-base pair
double-stranded probes contain the sequences shown. Not shown are
sequences that are common to all four probes, including linker sequence
upstream and ML promoter sequences downstream to position +15.
M represents the wild-type ML promoter. ML sequences are
shown in regular type while E4 sequences are shown in
bold. Blocks 1 and 2 are indicated
above the sequences.
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Fig. 2.
Time course of TBP binding.
A, the ML promoter probe (0.1 nM) was mixed with
TBP (20 nM) as described. Time points were initiated at
intervals so that all samples could be loaded on the gel at the same
time. Arrows indicate the bound complex and free probe.
B, the bands in A were quantified, fit to
Equation 1, and plotted. The calculated rate constant
(kobs) for this experiment is 2.7 × 10 3 s 1. The
half-time is 4.3 min.
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On-rates for all four probes were determined at 20 nM TBP,
and the values are shown in Table I. Only
promoter M-E12 shows a difference at 20 nM TBP, and this
effect is small. The experiment was repeated at different
concentrations of TBP. The changes in the observed on-rates are
proportional to the changes in TBP concentration, but this occurs
similarly for each promoter. We determined the apparent rate constant
kon for TBP binding for each promoter, and these
values are also shown in Table I. The kon for
the parent ML promoter of 1.3 × 105
M
1 s
1
is comparable to published values (14, 18, 19). The
kon values for the swapped promoters are not
significantly different from the parent ML promoter. We conclude that
the TATA-flanking blocks have little effect on the association rate
constant for TBP binding.
Since the TATA-flanking blocks have no apparent effect on the on-rate
of TBP binding, we tested whether they affect the off-rates. We chose
to use TBP at 5 nM since that is the concentration used in
the prior study (9). TBP·DNA complexes were formed at 30 °C for 60 min to ensure that binding had reached equilibrium and then challenged
with 20 ng/µl poly(dI·dC). Addition of this competitor prior to
adding TBP prevented binding to the probe so it serves as an effective
sink for dissociated TBP (data not shown). Examples of off-rate
experiments are shown in Fig.
3A. The data from several such
experiments were fit to Equation 3 and averaged. The average koff for the parent ML probe is 1.3 × 10
4 s
1. This rate
and the dissociation constant (KD) of 1 × 10
9 M (Table I) are both
consistent with published data (14, 18, 19).

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Fig. 3.
Decay rate of TBP·DNA complexes.
A, the ML and M-E12 probes were bound by TBP for 1 h.
Poly(dI·dC) (20 ng/µl) was added for the indicated times.
B, the bands in A were quantified and fit to
Equation 3. The calculated off-rate (koff) and
half-life for ML are 1.7 × 10 4
s 1 and 70 min, respectively. For M-E12 they
are 2.7 × 10 4
s 1 and 42 min. Closed circles,
MLP; open circles, M-E12.
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The experiment was repeated using promoters in which the flanking
sequence blocks had been swapped. A sample autoradiograph comparing two
promoters is shown in Fig. 3A with the data analysis in Fig.
3B. The difference between the two curves (closed
circles, ML; open circles, M-E12) is approximately a
factor of 2. A similar 2-fold difference was seen with the other
block-swapped promoters (compare off-rates in Table I). The half-life
of TBP on the ML promoter is nearly 90 min while the half-lives for the
other promoters range from 40 to 50 min. This indicates that TBP is
less stably bound to the block-swapped promoters than to the ML parent
and demonstrates a kinetic role for both flanking sequences. We
conclude that the sequences that flank TATA affect the stability of TBP binding without altering the rate at which TBP locates the TATA box.
TFIIB Affects Both the On-rate and the Off-rate with the On-rate
Being Sequence-dependent--
The sequence of the block
upstream from TATA (block 1) is known to influence TFIIB binding (10).
In general, TFIIB binding enhances the binding of TBP to the TATA box.
It is not known whether these stabilizing effects are due to faster
on-rate, slower off-rate, or both. In the series of block-swapped
promoters studied here, the sequence of the ML block 1 is expected to
bind TFIIB better than block 1 from E4 as it provides a much better
match to the GC-rich consensus (10). The effect of TFIIB on TBP binding
is known not to be uniform among this promoter set (9). The greatest contribution of TFIIB to TBP binding is made with promoter M-E2, which
has the ML sequence upstream from TATA and the E4 sequence downstream.
To learn why this sequence-specific effect occurs and to locate the
general kinetic sources of the TFIIB effect on TBP binding to TATA, we
established rate assays for formation of the ternary complex containing
TBP, TFIIB, and TATA DNA.
We looked first at the effect of TFIIB on off-rates. The kinetic
analysis is potentially complicated because either TBP or TFIIB could
be released first. However, if TBP were released first then TFIIB would
effectively be released concurrently because it cannot bind these DNA
templates on its own (9). As shown below the dissociation also does not
include a pathway in which TFIIB only is released. Thus we can monitor
dissociation by following the joint loss of TFIIB and TBP. The
experiment is similar to that described above, adding poly(dI·dC) to
preformed complexes as a sink for released TBP. Although this has not
been done previously for the TFIIB·TBP·DNA complex, a similar
experiment was used to measure the bulk off-rate of TFIIA and TBP from
a promoter containing the ML TATA box (20).
TBP, TFIIB, and labeled DNA were preincubated for 60 min to ensure that
binding had reached equilibrium. The complexes were then challenged
with poly(dI·dC). At no time during the decay phase of these
reactions is a TBP:DNA intermediate observed (Fig. 4A), and TFIIB does not bind
to these promoters in the absence of TBP (9). This verifies that the
poly(dI·dC) challenge measures the bulk off-rate of TBP + TFIIB and
demonstrates that these two proteins come off the DNA as a unit.

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Fig. 4.
Decay time course of the ternary
complex. A, sample off-rate gel using TBP, TFIIB, and
the M-E2 probe. The ternary complex and free probe are indicated.
B, TFIIB; T, TBP. B, the data from
A were plotted as described in the legend to Fig. 4. Data
for the decay of TBP:M-E2 are also shown. The half-lives of these
complexes are 92 (TFIIB·TBP·M-E2) and 40 min (TBP·M-E2).
Closed circles, TBP:M-E2; open circles,
TFIIB:TBP:M-E2.
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Fig. 4B shows that TFIIB (open circles) decreases
the rate of decay compared with TBP alone (closed circles).
The TFIIB·TBP·DNA complex decays at about half the rate of the
TBP·DNA complex. The kinetic effect shows no promoter specificity;
the rates of decay (koff) for all four promoters
are decreased similarly by TFIIB with an average decrease of 2.3-fold
(compare off-rates in Table II for TBP + TFIIB to those in Table I for TBP alone). Two conclusions are drawn.
One is that the well known ability of TFIIB to enhance TBP binding has
a source in the slowing of the TBP decay rate. The second is more
surprising. Because TBP can bind TATA well in the absence of TFIIB one
would expect it to remain bound after TFIIB dissociates. However, that
was not observed. It appears that the TFIIB stabilization is
accompanied by a change in state that requires that the two proteins
dissociate jointly.
These effects appear to be sequence-nonspecific. Therefore, one aspect
of the prior data remains unexplained. The TFIIB effect on TBP promoter
binding is greater for M-E2 than at other promoters (9). As the data
show that the source of this was not the off-rate (2.4-fold stimulation
for M-E2 versus a 2.3-fold average), we investigated the
on-rate.
TFIIB·TBP·DNA complexes were assembled under the conditions used
previously (9): 5 nM TBP, 20 nM TFIIB and 0.1 nM DNA were mixed and incubated at 30 °C for various
times. An example of an autoradiograph from such a time course is shown
in Fig. 5A. Lane 1 contains
only TBP (60 min) as a marker. Arrows indicate the locations
of TBP·DNA and TFIIB·TBP·DNA complexes. No band is seen in the
position expected for the TBP·DNA binary complex, indicating that the
ternary complex forms cooperatively.

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Fig. 5.
Time course of ternary complex
formation. A, TBP (5 nM), TFIIB (20 nM), and probe M (0.1 nM) were mixed and
incubated for the indicated times. Lane T contains only TBP
and DNA (1-h time point). The binary and ternary complexes are
indicated. B, data from several on-rate time courses for all
four promoters were normalized and averaged. The half-time to
saturation is 7.7 min for M-E2 and roughly 15 min for the others.
Closed circles, ML; open circles, M-E1;
closed triangles, M-E2; open triangles,
M-E12.
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A comparison of M-E2 and the ML parent is shown in the association rate
curves of Fig. 5B, and normalized data for association with
each of the four promoters are shown in Table II. TFIIB causes a very
slight acceleration of binding on three of these promoters, but the
effect on M-E2 is much greater, nearly 3-fold (compare on-rates in
Table II to those for 5 nM TBP in Table I). Combined with
the 2-fold effect on off-rate, this accounts for roughly a 6-fold
increase in binding on M-E2. This compares with the observed 8-fold
increase in overall binding seen previously when TFIIB was added to TBP
at this promoter (9). Thus in addition to a nonspecific stabilization
against decay, TFIIB can assist TBP binding via a sequence-specific
acceleration of binding rate. This latter occurs with only one of the
four promoters studied.
Kinetic Interplay between TATA and the Flanking Blocks--
These
data indicate two roles for TFIIB as follows: a general strengthening
of bound TBP against decay, and a sequence-dependent enhancement of the rate of TBP binding to TATA. The M-E2 promoter that
has the enhanced on-rate has a unique combination of elements upstream
and downstream from TATA. Its upstream block from the ML promoter has a
resemblance to the consensus TFIIB-binding site (BRE). Its downstream
block from the E4 promoter weakens TBP binding (9). How TFIIB can
selectively enhance the binding on-rate for promoter M-E2 is not clear,
but two possibilities can be considered. One is that the unique
combination of ML block 1 with E4 block 2 creates a novel site that
binds TFIIB rapidly. Alternatively, the loosely bound TBP, destabilized
by the E4 block 2 sequence, may be configured in a manner that can bind
TFIIB rapidly if a strong BRE is present. We sought to mimic this
second possibility by directly weakening TBP binding via TATA mutation
within the parent ML promoter. The question is, will the BRE, silent
when embedded in the ML but not the M-E2 promoter, now also show
function in the ML context with a nonconsensus TATA box?
To examine this possibility a new series of promoters was constructed
(see Fig. 6A). The 2T mutation
replaces the two adenosines at positions
27 and
28 in the ML TATA
box with thymidines (underlined Ts in Fig. 6A) in
the context of the parent ML promoter (to form the ML-2T promoter). The
substitutions were chosen to reduce TBP affinity without causing the
drastic decreases in binding associated with "weak" TATA boxes.
This mutation decreases the lifetime of TBP binding (21) without
affecting the association rate (19). The reduction in affinity is
comparable to that caused by the M-E1 substitution. We measured the on-
and off-rates for TBP and TBP + TFIIB on this mutant parent and the
series of block-swapped promoters also containing the TATA mutation
(Fig. 6A).

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Fig. 6.
Kinetic interplay between TATA and the
flanking sequences. A, the 2T series of promoters
differ from ML in the substitution of two thymidines for the adenosines
at positions 27 and 28 (underlined). The consensus
sequence for the BRE is shown (10). The block swaps are also
illustrated as in Fig. 1. B, decay of binary
(upper) and ternary (lower) complexes for the
ML-2T promoter. C, association of binary (upper)
and ternary (lower) complexes for ML-2T.
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Initial control experiments confirmed that the 2T mutation led to more
rapid decay of TBP from binary complexes (see Fig. 6B, upper
row and TBP off-rates in Table III).
The rate of TBP decay from ML-2T is comparable to the rate from M-E1,
indicating that the 2T mutation does indeed destabilize TBP binding.
Further destabilization was not seen for the block-swapped 2T promoters indicating that the effects of TATA and the adjacent sequences are not
cumulative in this context. Next, TFIIB was added to the mutant
promoter, and the rate of decay of ternary complexes was determined
(Fig. 6B, lower row). The TATA mutation led to
destabilization of ternary complexes (Table III) as it had for the
binary complexes at the same promoter. Yet again this effect was not
seen when the flanking sequences were changed.
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Table III
Kinetics of factor binding to 2T series of promoters
Measured at 5 nM TBP, 20 nM TFIIB. Values shown
are averages of 2 trials each. Error fitting curves is 20% or less.
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Next the effect of TFIIB on the on-rate was measured at the ML-2T
promoter with the weakened TATA box (Fig. 6C, upper row for
binary complex and lower row for ternary complex). The data in Table III show that the ternary complex forms nearly 3-fold faster
than the binary complex. As this acceleration was not seen when the
TATA box retained the wild-type sequence, we infer that the TATA
mutation makes the adeno-ML promoter susceptible to a stimulating
effect of TFIIB on the rate of ternary complex formation. This
acceleration by TFIIB was eliminated when the BRE was replaced with an
AT-rich sequence (2T-E1 and 2T-E12 in Table III, Ratios). It was
retained when the BRE was retained (promoter 2T-E2). Overall, the data
favor the possibility that there are at least two requirements for the
TFIIB effect via on-rate. First, TBP binding must be weakened, either
by TATA mutation (as shown here) or by inserting a weakening sequence
block downstream (as shown above). Second, a strong TFIIB-binding site
must lie upstream of TATA. The implications of obtaining similar
effects on general factor assembly in the context of the natural
variations within promoter sequences will be discussed below.
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DISCUSSION |
Many promoters have been sequenced so far, and within an organism
no two have identical core DNA sequences (Eukaryotic Promoter Data base, see Ref. 9). In general, core promoter sequences are
GC-rich, but there is very considerable diversity. Prior studies have
indicated that elements both upstream and downstream from TATA can
influence the level of transcription and its regulation (9, 10). The
above results help to understand the origin of these effects. The
flanking sequences are shown to affect the manner in which
preinitiation complex assembly is originated to recruit RNA polymerase.
Although the TATA-flanking elements do not contact TBP directly, their
sequences are shown to influence the rate at which TBP is released from
TATA. In addition certain combinations of element types can influence
the rate at which TFIIB associates with the bound TBP. The nature and
implications of these results are discussed below.
TBP Binding and Flanking Sequences--
The data show that the
TATA-flanking sequences influence the decay rate of bound TBP but not
the rate at which TBP locates and binds the TATA box. The GC-rich
flanking sequences of the adenovirus ML promoter increase the lifetime
of the bound protein by roughly a factor of 2. It seems likely that
among naturally occurring promoters there will be flanking sequences
that yield both greater and lesser quantitative effects. Two possible
sources of this effect were considered (see Introduction). The lack of effect on the association rate makes it unlikely that surrounding the
TATA box with GC-rich sequences highlights it, making it easier for TBP
to locate it and bind. The measurable effect on decay rate makes it
more likely that flanking the AT-rich region with GC-rich blocks
creates unique structures at the edges of the TATA element,
i.e. kinks or bends, which stabilize TBP once it is bound.
Structural studies suggest how this might occur. When TBP binds a
consensus TATA box, i.e. the ML TATA, it bends the DNA
roughly 90° (11, 13, 21), and the stability of binding correlates with changes in the bend angle at the promoter (21, 22). The minor
grooves at the TATA box boundaries are widened (13), and when
artificial heteroduplex bubbles are placed at these boundaries TBP
binding is stabilized (14). The ML promoter sequences that flank TATA
both contain homopolymeric G runs which juxtapose to the
homotetrameric A run within the ML TATA box. Such homopolymeric runs
may form rigid helices (23, 24) forcing discontinuities at the borders
(25). Thus it seems that promoters with flanking elements that
juxtapose such sequences could create bent structures that assist TBP binding.
We assessed the tendency of promoters to include this extreme
juxtaposition using data from the Eukaryotic Promoter Data base. Approximately 5% of these promoters were found to have pairs of juxtaposed tetrameric and homopolymeric (separated by no more than one
base pair), roughly 10 times the expected frequency. At the other
extreme there are expected to be promoters whose flanking sequences
resist bending (9) at the TATA boundaries that would be expected to
bind TBP less tightly than the average. Overall, this should lead to
considerable diversity in TBP binding affinity, complementing the
diversity within the TATA box itself. Indeed, promoters with the same
affinity for TBP could have different properties as a consequence of
this effect; AT-rich flanking sequences can enhance the sensitivity to
induction (9), whereas nonconsensus TATA box sequences can decrease the
continuous re-initiation events that determine promoter strength (2, 3,
26).
TFIIB Binding--
The data show new aspects of TFIIB binding that
complement knowledge concerning the central role of TFIIB in
preinitiation complex assembly (27-32). In light of prior knowledge it
is not surprising that TFIIB stabilizes bound TBP against decay at all promoters. That is, TFIIB cross-links to both flanking blocks 1 and 2 (6) and can recognize sequence elements within block 1 (10). However,
two observations were surprising. First, the decay of the ternary
complex was cooperative in that TBP and TFIIB dissociated essentially
simultaneously. This is surprising because TFIIB has a far lower
affinity for DNA than does TBP and thus decay would be expected to
initially dissociate TFIIB leaving TBP bound. The observation implies
that TBP may be bound differently to DNA in the ternary complex than in
binary complex. A conformational change in this main DNA binding
protein may influence transcription, especially since evidence has
accumulated that the DNA within preinitiation complexes may have a
defined topological superstructure (6, 7, 33-35).
A second surprising observation was that sequences flanking the TATA
box can influence the rate at which ternary complexes assemble. This
rate is enhanced only when the stronger ML BRE sequence is present
upstream of TATA and TBP binding is weak. The data show that weakening
can occur either by making the TATA box nonconsensus or by placing the
more AT-rich E4 sequence downstream from TATA. The source of this more
rapid rate may have its origin in an unstably bound less bent form of
the TBP·DNA complex present when the TATA is nonconsensus (21, 22) or
the promoter contains AT-rich DNA downstream (see above). TFIIB may
bind the strong upstream BRE sequence and drive the TBP-binding
reaction to completion. This would be in addition to the sequence
nonspecific stabilization of TBP against decay. The sequence dependence
of the on-rate means that it will differ among promoters that could
have regulatory implications as forming a complex with bound TFIIB is
central to transcriptional regulation.
Overall then the current data indicate that the diversity of
sequences that flank TATA could have multiple influences in
contributing to the diversity of transcription through altering how
preinitiation complexes assemble at promoters. The affinity for TBP
will be set by both TATA and flanking sequences, and this will
influence many central properties of the promoter. There will be a
further influence of the flanking sequences on how TFIIB works. Their effect on TBP binding could range from weak (upstream sequence AT-rich)
to very strong (GC-rich sequence upstream but not downstream). These
effects are likely to be relevant directly to basal transcription that
relies centrally on formation of the TFIIB·TBP·DNA complex. They
should also be relevant to activated transcription (9), but the manner
in which this occurs and the effect of initiator sequences remains to
be determined. Basically, one can view the core promoter as a
contiguous sequence of nearly 100 base pairs (4-6, 8, 10, 36), all
parts of which are designed to work together to give appropriate
amounts of properly regulated transcription.