From the
The TATA binding protein (TBP) is required at RNA polymerase I,
II, and III promoters that either contain or lack a TATA box. In an
effort to understand how TBP might function at such a wide variety of
promoters, we have investigated the specific and nonspecific DNA
binding properties of human TBP. We show that TBP has less than a
10
The TATA binding protein (TBP)
If TBP utilizes TATA to anchor transcription complexes to DNA, then
what is the role of TBP at TATA-less promoters? Recent reviews have
suggested dual functions for TBP
(10, 11) . At TATA, TBP
is bound tightly, while at TATA-less promoters, TBP is proposed to be
either loosely bound to the DNA or bound indirectly via protein-protein
interactions. If TBP is maintained in the transcription complex of
TATA-less promoters primarily through protein-protein interactions,
then is TBP only a linchpin between proteins of the preinitiation
complex? Since many different proteins can link components within the
preinitiation complex of all three nuclear RNA polymerases and yet not
be conserved, the invariant requirement for TBP suggests that TBP does
more than simply string proteins together. The universal requirement
for TBP also argues for a common conserved function at TATA-containing
and TATA-less pol I, II, and III promoters. If this common function is
to anchor transcription complexes to DNA as it does at TATA, then how
can TBP perform this function in the absence of TATA?
TBP binds to
the minor groove of the TATA box (prototype sequence,
TATAAAAG)
(12, 13, 14, 15) . The minor
groove of DNA has a paucity of functional groups with which to provide
sequence specificity. The TBP
A
lack of precise definition of the biochemical mechanism by which TBP
binds DNA limits our understanding of the role of TBP in TATA-less (and
TATA-containing) transcription. The DNA binding surface of TBP is
sufficiently unique that paradigm DNA binding mechanisms, such as that
for Escherichia coli RNA polymerase
(20) , lac repressor
(21) , and cro repressor
(22) may
be of limited relevance. We have set out to define the mechanistic
basis of TATA and TATA-less transcription by exploring the dynamics of
TBP interactions with TATA and nonspecific DNA. Using a nitrocellulose
filter binding assay, we have measured the apparent equilibrium
constant for TATA and nonspecific DNA binding and have found a very low
degree of sequence specificity. The low specificity of TBP binding was
particularly striking when examined by DNase I footprinting. DNA
binding did not appear to be cooperative. We explored the mechanistic
basis for this low specificity by measuring the dissociation kinetics
of TBP bound to TATA and nonspecific regions of the footprinting probe.
Surprisingly, the rate of dissociation from nonspecific DNA was very
slow and not significantly different from the rate of dissociation from
TATA. When multiple TBP molecules were present on the same footprinting
probe, net dissociation from TATA did not proceed until adjacent
nonspecifically bound TBP reached a critical minimum. One plausible
interpretation of the data is that TBP stably translocates along the
DNA prior to dissociating into the bulk solution. To probe whether the
TBP
On-line formulae not verified for accuracy When measured by EMSA, S
For DNase I footprinting experiments, where titration of a
protein protects a specific region of the probe from cleavage by DNase
I, the fractional occupancy of a particular region (F) was
determined by Equation 2.
On-line formulae not verified for accuracy S
For filter binding experiments where
dissociation of a protein-DNA complex was being measured as a function
of time, the equation used to describe the fraction of the protein-DNA
complex remaining (F) is given by Equation 3.
On-line formulae not verified for accuracy Each of the terms is defined above, except that the subscripts
x, 0, and f denote units of time instead of protein
concentration. With this equation, the initial conditions are set such
that F = 1. The end point signal
(S
For DNase I footprinting experiments
where dissociation of TBP from a particular region (such as from TATA,
the medium or low affinity regions) was being measured, the equation
used to describe the fraction (F) of complex remaining is
given by Equation 4.
On-line formulae not verified for accuracy
Apparent dissociation equilibrium constants,
K
On-line formulae not verified for accuracy where P represents the uncomplexed protein concentration
(in this case, TBP or Sp1). P is related to total active
protein concentration (P
The apparent dissociation rate constant
(k
On-line formulae not verified for accuracy
To determine
the apparent K
If TBP behaves like a
classical site-specific DNA binding protein, then we expect to observe
a log-linear dissociation of TBP from TATA, the medium affinity region
and the low affinity region as soon as excess TATA competitor is added.
Moreover, since the sequence of the three regions differ, we expect
k
In
Fig. 5A, dissociation of TBP from the low, medium, and
TATA sites, once initiated, appeared to proceed at approximately the
same rate (t =
A dissociation time course equivalent to the one shown in
Fig. 4
was performed at 55 and at 70 °C (Fig. 6). These
temperatures inactivate unbound TBP in a matter of seconds.
We have examined the equilibrium binding properties and
dissociation rates of the human TBP with a TATA box and nonspecific
DNA. We report the following observations. 1) TBP has a 740-fold higher
affinity for the 8-bp TATAAAAG sequence (K
The low specificity of TBP leads us to propose that the
TBP
The
rapid preequilibrium and isomerization segments of the binding reaction
have been proposed previously by Hawley and co-workers
(17) for
TATA binding. The two-step model was based on the finding that
k
The evidence that TBP rapidly diffuses along the DNA relative to its
dissociation into the bulk solution is derived from the observed
biphasic kinetics of dissociation. When multiple TBP molecules are
present on the footprinting probe, net dissociation of TBP from high
affinity sites such as TATA proceed only when TBP bound to lower
affinity sites reaches a critically low level. When flanking DNA
sequences are removed, or if the initial TBP concentration is reduced,
the lag to dissociation also diminishes. If TBP does not translocate
along the DNA, then a log-linear decay of TBP bound to various sites
would be expected immediately upon addition of excess competitor to
initiate net dissociation from the probe. Since we do not observe
cooperative binding between TBP molecules, we conclude that TBP
dissociates into the bulk solution independent of other TBP molecules
bound to the probe. The simplest interpretation of the lags prior to
log-linear dissociation is that with every dissociation event, TBP
constantly redistributes on the DNA according to its range of
affinities for different sequences. For example, if TBP dissociates
from TATA or any other site, then that site is rapidly refilled by TBP,
which has translocated from a nearby lower affinity site. TBP is
presumably dissociating from TATA or any other site at the same rate
whether the probe contains a singly-bound TBP or is saturated with TBP,
but net dissociation from TATA does not occur until the
density of TBP on the DNA lattice has reached a sufficiently low level.
The binding mechanism outlined in Fig. 9has a number of
implications. First, sequence-specific DNA binding would be achieved,
in part, when k
If TBP binds
quite stably to nonspecific DNA, then how is TBP prevented from
becoming sequestered by the vast excess of nonspecific DNA present in
the nucleus? Part of the explanation might lie in the sequence
dependence of k
We thank T. Kodadek, B. T. Nixon, K. A. Johnson, and
members of the Pugh lab for many helpful suggestions. We also thank H.
M. Zuang for his efforts on the project.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-fold preference for binding a TATA box (TATAAAAG) than
for an average nonspecific site. In contrast to TBP, which binds to the
minor groove of DNA, major groove binding proteins typically display
binding specificities in the range of 10
. Once TBP is bound
to DNA, whether it be a TATA box or nonspecific DNA, binding is quite
stable with a t of dissociation in the range of 20-60
min for a 300-base pair DNA fragment. In this binding state, TBP
appears to be capable of stable one-dimensional sliding along the DNA.
Sequence-specific binding can be accounted for, in part, by different
rates of sliding. Additional findings demonstrate that specific and
nonspecific DNA impart upon TBP an enormous and equivalent degree of
thermal stability, suggesting that the TBP
DNA interface on
nonspecific DNA is not radically different from that on TATA.
Consistent with this notion, we find that nonspecifically bound TBP is
competent in establishing pol II transcription complexes on DNA.
Together, these finding provide a plausible mechanistic explanation for
the ability of TBP to function at TATA-containing and TATA-less
promoters.
(
)
binds to
the TATA box of eukaryotic promoters, assembles a transcription
complex, and anchors it to DNA
(1, 2, 3) . While
promoters that contain TATA require it for proper function, many
promoters lack TATA altogether and yet still require
TBP
(4, 5, 6) . For these TATA-less promoters,
there appears to be little specific sequence requirement around the
-30 region where TATA normally resides
(7, 8) .
TATA-less promoters, however, do rely on other cis elements such as
Sp1-binding sites and initiator elements
(5, 9) .
Presumably, TBP is recruited through direct and indirect
protein-protein interactions with other sequence-specific factors.
TATA crystal structure indicates
that monomeric TBP binds TATA primarily through hydrophobic and van der
Waals' interactions with the bases and
sugars
(14, 15, 16) . These types of interactions
could provide the bulk of the affinity of TBP for TATA, but in the
absence of extensive hydrogen bonding, it is not evident how they might
provide a high degree of specificity for TATA over nonspecific DNA. The
equilibrium dissociation constant (K
) of
the yeast TBP
TATA complex has been reported to be 3
10
M (17) and 2
10
M(18, 19) , while the
K
for nonspecific (yeast genomic) DNA was
5
10
M(19) , indicating a
few thousand-fold lower affinity of yeast TBP for nonspecific DNA.
DNA interface is similar to the TBP
TATA interface, we
performed thermal stability tests on specifically and nonspecifically
bound TBP. We find that while TBP is extremely unstable when not bound
to DNA, it appears to have a very high and equivalent degree of thermal
stability on nonspecific DNA as it does on TATA. TBP bound
nonspecifically to DNA also appears functional in assembling pol II
basal transcription complexes. The data presented here lead us to
conclude that TBP has the intrinsic capacity to bind quite stably to
DNA and to establish a functional transcription complex, whether the
underlying DNA is TATA or TATA-less.
Proteins, DNA, and Buffers
TBP was purified to
homogeneity from recombinant E. coli cells using a three-step
procedure involving polyethylenimine precipitation, phosphocellulose
chromatography, and ammonium sulfate precipitation as described in the
accompanying paper
(33) and in Ref. 23. The final purified
fraction is shown (see Fig. 1A). Different TBP
preparations ranged from 50 to 70% active. TBP concentrations were
determined by total amino acid analysis, and had an A of 0.057 µg
ml, which can be referenced
against bovine serum albumin (Sigma, fraction V, dry weight) having an
A
of 0.086 µg
ml or
-globulin (Pharmacia Biotech Inc., provided as dry weight
standard) having an A
of 0.043
µg
ml in side-by-side reactions, all in 0.9
Bradford reagent (Bio-Rad). The concentration of active TBP was
determined by titrations of TBP against known concentrations of TATA,
which were well above the K
for TATA
binding. Sp1 was purified from HeLa cells infected with a recombinant
vaccinia virus containing the Sp1 gene as described
previously
(6) . The 28-bp TATA oligonucleotide used in this
study was chemically synthesized. The 300-bp G
TI
footprinting probe was generated by restricting the plasmid
pS-G
TI-141 with XbaI, end labeling with kinase,
and cutting with PvuII. The probe was isolated using a
polyacrylamide gel and quantitated spectroscopically using 1
A
= 50 µg/ml. The
S
TI probes, containing either wild-type or
mutant TATA, were generated by polymerase chain reaction. For
transcription assays, pS-G
TI-141 was linearized with
PvuII.
Figure 1:
K for the TBPTATA
complex. Panel A, silver-stained protein gel of purified human
TBP. Panel B, determination of the fraction of TBP that is
active for TATA binding. Increasing concentrations of TBP (total
protein), as indicated, were incubated with 53 nM
P-labeled 28-bp TATA oligonucleotide shown inside the
figure. Bound complexes were separated from free by EMSA, as shown.
This and additional data were quantitated and the fraction (F)
of TATA bound determined as described under ``Experimental
Procedures.'' F is plotted as a function of the molar
ratio of total TBP to TATA. At F = 1.0, the reciprocal
of x axis intercept represents the fraction of TBP that is
active for TATA binding, assuming a binding stoichiometry of 1. This
value multiplied by the total TBP concentration represents the
concentration of active TBP. Panel C, binding reactions were
set up as described under ``Experimental Procedures,'' using
0.05 nM 28-bp TATA oligonucleotide. Samples were analyzed
either by EMSA (
) or by filter binding (
). F and
K were determined as described under ``Experimental
Procedures.''
Binding Conditions
In addition to TBP and probe,
binding reactions contained 20 mM Tris acetate (pH 7.5, 80%
cation), 5% glycerol, 4 mM spermidine, 1 mM
dithiothreitol, 75 mM potassium glutamate, 0.1 mM
EDTA, 4 mM MgCl, 13.6 µM
(nucleotides) poly(dG-dC), 0.01% Nonidet P-40, and 5 µg/ml bovine
serum albumin in a volume of 50 µl.
Nitrocellulose Filter Binding Assay
This assay is
used extensively for measuring protein-DNA complex formation and is
described in more detail in Ref. 24. Briefly, reactions containing
radiolabeled DNA were rapidly (5 s) filtered over nitrocellulose
contained within a 96-well dot-blot apparatus (Bio-Rad) using a vacuum
applied via a house water aspirator. The filters were washed and dried,
and the radioactivity was quantitated using a Betascope (Betagen).
EMSA
Samples (10 µl) were loaded onto a native
4% polyacrylamide gel (prerun at 25 mA for 1-2 h at 4 °C) and
electrophoresed under the ``prerunning'' conditions for 15
min. Gels were dried, and the radioactivity present was quantitated
through a Betascope imaging system.
DNase I Footprinting
Reactions were diluted with
50 µl of DNase I solution (containing 0.012 units or 5 ng of DNase
I (Worthington), 10 mM MgCl, and 5 mM
CaCl
), and the incubations continued for 30 s at 30 °C.
Reactions were terminated with 3 M ammonium acetate and 125
µg/ml carrier tRNA. The DNA was precipitated with ethanol,
subjected to electrophoresis on 8% polyacrylamide, 7 M urea
gels, and the radioactivity was visualized by autoradiography.
Immunodepletions and Transcription
HeLa nuclear
extracts were immunodepleted of TBP and TAFs with polyclonal anti-TBP
antibodies bound to protein A-Sepharose as described
previously
(6, 25) . Transcription reactions were
performed essentially as described previously
(6, 25) and involve the incorporation of [-
P]ATP into the body of the nascent RNA. RNAs were
purified by phenol/chloroform extraction and electrophoresed on a 6%
polyacrylamide, 7 M urea gel. The gel was dried, and the
radioactivity was visualized by PhosphorImaging (Molecular Dynamics).
Calculations
For the filter binding and EMSA
experiments where formation of a complex between the protein (in this
case, TBP or Sp1) and radiolabeled DNA was being measured as a function
of protein concentration, the fraction (F) of the DNA probe
bound by the protein is described by Equation 1.
is the
radioactivity present in the region corresponding to the shifted
protein-DNA complex at protein concentration x. If measured by
filter binding, S
is the amount of
radioactivity retained on the filter paper at protein concentration
x. S
is the corresponding average
radioactivity present when x = 0 (i.e. the
absence of protein), and S
is the average
amount of radioactivity present when F becomes independent of
x (i.e. when the reaction reaches the titration end
point).
is the amount of radioactivity
present in the protected region of the probe at protein concentration
x. S
and S
are defined above.
) was generally found to be less than
5% of S
.
, of the protein-DNA complexes were
calculated from Equation 5 using Kaleidagraph software
) by P = P
- PD,
where PD is the concentration of the protein-DNA complex. The
apparent association equilibrium constant,
K
, is the reciprocal of
K
. Error determinations represent
standard errors.
) was determined from Equation 6 using
Kaleidagraph software.
K
Recombinant
human TBP was purified to homogeneity from E. coli cells
(Fig. 1A). The fraction of TBP that was active for TATA
binding was determined through EMSA by titrating known concentrations
of TBP (determined by total amino acid analysis) against a known
concentration of a TATA oligonucleotide (determined by UV
spectrometry), which was well above the K for TATA
for the TBP
TATA complex (Fig. 1B). Assuming
a stoichiometry of 1:1 for the TBP
TATA complex, the TBP
preparation shown in Fig. 1B appeared to be 55% active.
Other preparations ranged between 50 and 70% active.
for the TBP
TATA
complex, TBP was titrated into reactions containing a radiolabeled
28-bp TATA oligonucleotide (5
10
M). DNA binding was measured by two different assays,
EMSA and nitrocellulose filter binding (Fig. 1C). Both
assays yielded identical estimations of the apparent
K
, which was (4.6 ± 0.3)
10
M. We consider this value provisional,
since TBP dimerization (K
=
4
10
M) represents a competing
reaction (see accompanying article
(33) ). A more accurate
determination of the K
for dimerization
will provide a better estimate of the true K
for TATA binding. Since both EMSA and the nitrocellulose filter
binding assays yielded identical results, which were no different than
that reported for yeast TBP
(17) , we assume that the filter
binding assay provides a valid reflection of the binding properties of
TBP.
K
A
plasmid representing nonspecific DNA was cut into numerous different
sized fragments and then radioactively end-labeled. The DNA fragments
(each fragment present at 5 for Nonspecific DNA
10
M)
were titrated with either TBP or Sp1 (for comparison) and allowed to
reach equilibrium. After passage of the reactions over nitrocellulose,
the bound DNA fragments were eluted and size separated by gel
electrophoresis. The apparent association equilibrium constant
(K
) for either TBP or Sp1 binding to each
fragment was determined and plotted as a function of the number of
nonspecific DNA binding sites on that fragment. As shown in
Fig. 2
, a linear relationship between the apparent
K
and the number of nonspecific sites on
each fragment was obtained. The slope of each line represents the
affinity for each nonspecific site, which in terms of apparent
K
, was determined for TBP to be 3.4
10
M and for Sp1 to be 3.0
10
M. The scatter in the TBP data set
showed precise reproduction upon multiple repeats and thus is most
likely due to sequence-specific differences in intrinsic affinity for
each fragment. As with our estimations for the
K
of the TBP
TATA complex
(Fig. 1C), the apparent K
values for the nonspecific DNA fragments are considered estimates
since TBP dimerization is a competing reaction. Since smaller
nonspecific DNA fragments require higher amounts of TBP to achieve
binding, the competitive effect of dimerization becomes more pronounced
on these shorter fragments. As such, the true K
for the smaller DNA fragments is probably higher than the
apparent value. This deviation might be responsible for the negative
y-intercept for the TBP plot. With this caveat, TBP appears to
have <10
more affinity for the TATAAAAG sequence than
for an average nonspecific site. For typical major groove binding
proteins, the K
for specific binding is
in the range of 10
M, and for nonspecific
binding it is in the range of
10
-10
M, which
represents a specificity factor of 10
-10
.
Figure 2:
Determination of the K for
nonspecific DNA binding. The plasmid pU-STI, which is
essentially pUC118 with a few promoter elements, was restricted with
HpaII,
P-end labeled, and purified. The resulting
population of 15 fragments ranged in size from 501 to 26 bp. Binding
reactions were performed as described under ``Experimental
Procedures'' with 5
10
M of
each fragment and over a range of TBP or Sp1 concentrations. After
reactions were allowed to reach equilibrium (1 h, 30 °C), samples
were filtered through nitrocellulose. The radioactive DNA fragments
retained on the nitrocellulose were eluted with 0.1% SDS. The fragments
were then size fractionated by electrophoresis on a native 8%
polyacrylamide gel. The gels were dried, and the radioactivity in the
following sized (bp) fragments was quantitated: 501/489 (combined),
431, 404, 369, 242, 190, 147, 131, 110, 67. A TATA box on this plasmid
was present on a smaller fragment that was not considered in this
analysis. The K values for each fragment were determined as
described under ``Experimental Procedures.'' The reciprocal
of the K (K) for the TBP-fragment complex (
)
and the Sp1/fragment complex (
) were plotted as a function of the
number of nonspecific binding sites/DNA fragment. Since a protein can
nonspecifically bind double-stranded DNA in two directions, the number
of nonspecific sites was taken as twice the number of base pairs. In
addition, the number of sites that did not consist of at least 8 bp
(due to their presence near the end of the DNA) was subtracted from
total number of sites. The average K for a nonspecific site
was taken as the slope of the line.
DNase I Footprint Analysis of Specific and Nonspecific
Binding
DNase I footprinting allows the binding of TBP to a
large number of sites to be compared with TATA within the confines of a
single reaction. The 300-bp probe contains a TATA box and six 70%
GC-rich Sp1 binding sites (Fig. 3A). Apart from the TATA
box, none of the sequences were intentionally designed to contain TBP
binding sites. As expected, 16 bp of DNA centered around TATA became
protected at low TBP concentrations (Fig. 3B). As more
TBP was added, a nonuniform protection pattern developed throughout the
probe. A secondary medium affinity site bound TBP with only slightly
lower affinity. At the highest level of TBP tested (34 nM),
the entire probe became protected. Interestingly, based on calculations
from the apparent K for nonspecific
binding (Fig. 2), TBP should have a K
for a 16-bp stretch of DNA (the length of a footprint), in the
range of 10 nM. From visual inspection of
Fig. 3B, this is the approximate TBP concentration where
substantial nonspecific binding is observed. The correspondence of the
filter binding and footprinting data suggest that the two assays are
detecting the same phenomenon.
Figure 3:
DNase I protection of specific and
nonspecific DNA by TBP. Panel A, sequence of the upper strand
of the footprinting probe used in this study (except panel D).
The 300-bp probe was 5`-P-end labeled (indicated by the
asterisk). Arrowheads correspond to DNase I cleavage
sites that generated bands used to quantitate TBP binding to TATA and
arbitrarily selected low and medium affinity sites. The underlined sequences correspond to the protected region encompassing the TATA
box and the medium affinity site. The 8-bp TATA box is
italicized. The 70-bp stretch preceding TATA contains the SV40
early promoter 21-bp repeats. These Sp1 sites are 70% GC-rich and would
be expected to have very low affinity for TBP. Panel B,
titration of TBP. Increasing nanomolar concentrations of active TBP, as
indicated above each lane, were incubated with 0.5
nM footprinting probe at 30 °C. Probe indicates no TBP or
DNase I added. The radioactivity in the regions corresponding to the
groups of bars in panelB was quantitated
using a Betascope imaging system. The same regions were also used in
Figs. 4-7. The increased nonspecific protection observed at high
TBP concentration is not due to artifactual DNA-independent inhibition
of DNase I by the TBP preparation, inasmuch as addition of an excess
quantity of competitor TATA oligonucleotide sequesters TBP and renders
the footprinting probe completely susceptible to DNase I attack (see
for example, Fig. 4). Panel C, sequence of the
S
TI footprint probe. Panel D,
titration of TBP on the S
TI probe containing a
wild-type (TATAAAA) or mutated (TATGAAA) TATA, as indicated. The two
probes are compositionally identical to each other except for the
indicated point mutation, but compositionally different from the probe
shown in panelA. Both probes were generated by
polymerase chain reaction using the same radioactive primer, thereby
ensuring that the probe concentrations were equivalent, inasmuch as
equal amounts of radioactivity were present in both reactions. The
region of the probe containing the wild-type or mutant TATA box is
indicated by the graybox. DNA recovery markers are
indicated by the arrows.
The binding of TBP throughout the
probe at relatively low TBP concentrations might be a consequence of
cooperative interactions with TATA-bound TBP, or the fortuitous
presence of many strong TBP binding sites scattered throughout a sea of
very weak sites. Indeed, yeast TBP has been shown to bind numerous
different sites
(26) . To address these issues, we constructed a
completely different footprinting probe in which we inserted either a
wild-type (TATAAAA) or a mutated (TATGAAA) TATA box
(Fig. 3C). This AG transition had been previously
reported to be one of the most deleterious single-point mutations of
TATA
(27) . When the two probes were titrated with TBP, the
wild-type TATA box became protected at a lower TBP concentration than
the mutant, as expected (Fig. 3D). As more TBP was
added, the nonspecific regions uniformly became protected. The
protection of the nonspecific DNA occurred at the same TBP
concentration that protected the compositionally different probe,
G
TI, shown in panelsA and B.
Therefore, TBP is unlikely to be binding to only a few high affinity
sites fortuitously scattered throughout the probe. Most importantly,
the mutated and wild-type TATA probes achieved equal levels of
nonspecific protection at the same TBP concentration. Thus, the binding
of TBP to nonspecific DNA did not appear to involve cooperativity with
TATA-bound TBP. Indeed, as discussed above, the high degree of
nonspecific protection at relatively low TBP concentration can be
accounted for by the independently measured K
for nonspecific DNA (see Fig. 2).
Mechanism of DNA Binding
The mechanistic basis for
the apparently low specificity of TBP for TATA was investigated by
examining the dissociation kinetics of TBP from TATA and nonspecific
DNA. TBP was loaded onto the 300-bp footprinting probe so as to give
complete DNase I protection (Fig. 4, lane0).
The reactions were then challenged with a vast excess of unlabeled TATA
oligonucleotide so as to initiate a net dissociation of TBP. Any TBP
that dissociated from the probe into the bulk solution would become
sequestered by the TATA oligonucleotide. After a period ranging from 0
to 180 min, the reactions were subjected to a 30-s DNase I treatment.
As shown in Fig. 4, the low affinity region was the first to
dissociate, followed by the medium affinity region and finally TATA.
While the order of dissociation was not surprising, the high degree of
stability on nonspecific sequences was unexpected. For instance, after
10 min, most of the low affinity region was still bound by TBP. The 70%
GC-rich Sp1 binding sites and the medium affinity region exhibited
significant levels of protection even after 30 min. Apparently, TBP has
the intrinsic capacity to bind quite stably to nonspecific DNA
sequences.
Figure 4:
TBP dissociation from TATA and nonspecific
DNA. 21 nM active TBP was preincubated with 0.5 nM
footprinting probe for 40 min at 30 °C under the conditions
described under ``Experimental Procedures.'' 1
µM unlabeled 28-bp TATA duplex oligonucleotide was then
added, and incubations were continued at 30 °C for the time
indicated above each lane (0-180 min). The reactions were then
subjected to DNase I treatment, processed, and analyzed. NoTBP indicates reactions performed in the absence of TBP.
indicates reactions in which the competitor TATA
oligonucleotide was added before TBP. This latter reaction represents
the final equilibrium level of dissociation (S). probe indicates no DNase I or TBP added.
In addition to qualitatively addressing the stability of
TBP on TATA and nonspecific DNA, the data in Fig. 4provide clues
on the kinetic mechanism governing TBP dissociation from TATA and
nonspecific DNA. Paradigms for dissociation of sequence-specific major
groove binding proteins from DNA have been well established. Proteins
such as the lac and cro repressors dissociate from
their cognate site into the bulk solution via two
steps
(21, 22) . Step one is a rate-limiting dissociation
of the specifically bound protein to a nonspecific site located on the
same DNA molecule. The second step involves the rapid dissociation of
the nonspecifically bound protein into the bulk solution, where it can
interact with other DNA molecules. According to this simple first-order
dissociation scheme, the fraction (F) of initial protein-DNA
complexes remaining over time (t) decays logarithmically
according the equation ln F =
-kt , where k
is the apparent first-order dissociation rate constant for the
entire dissociation process from a particular site. Since
k
is dominated by the rate at which the protein
breaks sequence-specific contacts, a protein that binds to more than
one site manifests different k
values from each
of the various sites
(22, 28) .
to also differ. Contrary to these predictions,
when the natural log of the fractional occupancy of the low, medium,
and TATA regions were plotted as a function of time, a quite different
pattern emerged (Fig. 5A). While binding to the low
affinity site decayed in a log-linear manner, the dissociation kinetics
from the medium affinity site and TATA appeared biphasic. Dissociation
from TATA and the medium affinity site appeared to wait until most of
the TBP had dissociated from the lower affinity sites. Once net
dissociation from TATA and the medium affinity site began, it proceeded
in a log-linear manner (see also Fig. 7A), which did not
appear to be significantly different from that of the low affinity
site. Thus, dissociation of multiply-bound TBP from DNA reveals two
unique phenomena: sequence-specific lags and apparent
sequence-independent rates of dissociation.
Figure 5:
Kinetic profile of dissociation from TATA
and nonspecific DNA. Panel A, binding to regions corresponding
to TATA (), the medium (
), and the low (
) affinity
regions of the dissociation time course shown in Fig. 4 were
quantitated as described in Fig. 3 and under ``Experimental
Procedures.'' lnF is the natural log
of the fraction (F) of each region protected. F is
plotted to the right of each graph. Values for
k
are plotted in Fig. 7B. Panel B, dissociation time course was performed as in Fig. 4, except that 3
nM active TBP was used (
). Panel C, 21
nM active TBP was preincubated with 0.5 nM labeled
28-bp TATA oligonucleotide at 30 °C for 40 min under the conditions
described under ``Experimental Procedures.'' To maintain a
concentration of nonspecific DNA equivalent to that in panelA, reactions were supplemented with unlabeled pBR322. Net
dissociation was initiated by the addition of 1 µM
(50-fold molar excess over TBP) of unlabeled 28-bp TATA
oligonucleotide. The fraction of TATA bound was determined through
filter binding assays. Identical results were obtained with the gel
shift assay. The dissociation rate constant, k
,
was determined to be 1.1
10
s
(t = 100 min). This value agrees well with the
value determined by Hoopes et al. (17) for yeast TBP using
EMSA on a 110-bp TATA probe. However, by DNase I footprinting on the
300-bp probe k
for TATA was determined to be 5.2
10
s
(t =
22 min, panel B). At this point, we do not know the basis for
this apparent difference.
Figure 7:
Temperature and sequence independence of
k. Panel A, binding to regions
corresponding to TATA (
), the medium (
), and the low
(
) affinity regions were quantitated as described in Fig. 3 and
under ``Experimental Procedures.'' Panel B, the
k
values for TATA (
), the medium (
),
and the low (
) affinity regions were determined from Figs.
4-7, and 0 °C data (not shown), and plotted as a function of
temperature. The TATA box, being entirely composed of A and T might be
partially or entirely melted at 70 °C, and thus might contribute to
the few-fold enhanced dissociation at this
temperature.
If TATA-bound TBP
``waits'' to dissociate until TBP has dissociated from other
sites on the same DNA molecule, then two simple predictions can be
made. 1) When the concentration of nonspecifically bound TBP is
reduced, a corresponding reduction in the lag to TBP dissociation from
TATA should be observed. Indeed, as shown in Fig. 5B,
under lower TBP concentrations where only TATA and a portion of the
medium affinity site were protected (3 nM TBP), the lag to
dissociation was substantially reduced, while the rate of dissociation
remained essentially unchanged. 2) In the absence of extensive flanking
DNA sequences, i.e. on a shorter probe (28 bp
P-labeled TATA oligonucleotide), even at a high TBP
concentration, the lag to dissociation ought to be eliminated. As
measured by filter binding (Fig. 5C), no lag to
dissociation was observed on this 28-bp probe, and dissociation
proceeded in a log-linear manner (although with somewhat slower
kinetics). Thus, with longer DNA molecules, the presence of
nonspecifically bound TBP seemed to delay the net dissociation of TBP
from TATA. Apparently, net dissociation from TATA occurred only when
nonspecifically bound TBP fell below a critical level. The only
interpretation that we are aware of that can explain this data is that
TBP constantly redistributes along the DNA prior to dissociation.
20 min). If TBP rapidly
translocates along DNA prior to dissociation, then the binding sites
measured by DNase I footprinting are not necessarily the sites of
dissociation into the bulk solution. TBP might instead dissociate into
the bulk solution from a common point that, if rate-limiting, would
yield sequence independent kinetics.
Temperature Effects on k
Thus far,
the data suggest that TBP interacts with nonspecific DNA much in the
same way as it interacts with TATA. To provide further evidence for
this interpretation, we sought to identify a solution variable that
might probe the TBPDNA interface. One such probe is temperature.
TBP is known to be exquisitely temperature sensitive in its binding to
TATA. Simply preincubating TBP at 47 °C for 15 min in the absence
of DNA totally and irreversibly abolishes its DNA binding
activity
(29) . Since the intimate contact between a protein and
DNA can stabilize a protein against
unfolding
(30, 31, 32) , temperature acts as a
useful probe for comparing protein-DNA interfaces. If the nonspecific
TBP
DNA interface was more temperature sensitive than the
TBP
TATA interface, we would expect to observe rapid loss of TBP
bound to nonspecific sites relative to TATA at elevated temperatures.
(
)
Surprisingly, the pattern of dissociation at 55 °C was
no different than at 30 °C, and only a few-fold faster at 70
°C. The presence of DNA, whether it be TATA or nonspecific,
provided a dramatic and unprecedented increase (we estimate
>1000-fold) in the thermostability of TBP. The magnitude of
thermostability imparted by DNA provides a strong argument for the TBP
interface with nonspecific DNA being qualitatively no different from
its interaction with TATA.
Figure 6:
Thermostable TBPDNA interactions.
Dissociation reactions were set up exactly as described in the legend
to Fig. 4. Equilibrium reactions at 30 °C were shifted to either 55
or 70 °C for 5 min to allow temperature equilibration. (95%
temperature equilibration occurred within 2 min) Excess 28-bp TATA
oligonucleotide was then added, which defines the zero time point. At
the time (in min) indicated above each lane, the 55
°C reactions were footprinted with 0.0084 units of (3 ng) DNase I
for 30 s at 55 °C as described under ``Experimental
Procedures.'' At the indicated time, the 70 °C reactions were
shifted to 30 °C and footprinted as described under
``Experimental Procedures.'' Control reactions, in which TBP
was briefly incubated at the indicated temperature in the absence of
DNA, then added to DNA at 30 °C showed no binding (data not shown).
In these experiments, undigested probe ran as two bands, which can be
seen in the lane marked probe as well as many of the
other lanes. In native gels, the probe runs as a single band. We note
that only full-length probe (which is more prevalent in reactions where
TBP has not yet dissociated) behaves in this anomalous way. This
anomaly is particular to the gel composition and does not affect
interpretation of the data.
Quantitation of TBP dissociation from
TATA, the medium, and the low affinity sites at 55 and 70 °C
(Fig. 7A) revealed the same sequence-specific lag and
the same sequence-independent rates of dissociation evident at 30
°C. Thus, even at these elevated temperatures, the same reaction
mechanism seemed to hold. In Fig. 7B,
k, for TATA, the medium, and the low affinity
regions were plotted as function of temperature, ranging from 0 to 70
°C. k
appeared surprisingly
temperature-independent and sequence-independent over the extremely
broad temperature range of 0-55 °C, and increased by less
than a factor of 3 at 70 °C.
TBP-dependent Nonspecific Transcription
A
functional test for nonspecific DNA binding is whether nonspecifically
bound TBP is competent to assemble and initiate pol II transcription
complexes. To examine both specific and nonspecific transcription, a
plasmid containing 2.7 kilobase pairs of nonspecific sequences and the
synthetic GTI pol II promoter (see Fig. 2A)
was linearized 141 bp downstream of the transcriptional start site. We
employed a run-off transcription assay using a HeLa nuclear extract,
which was immunodepleted of nearly all endogenous TBP (and
TBP-associated factors or TAFs)
(6, 25) . As shown in
Fig. 8
, lane1, pol II in this TBP-depleted
nuclear extract has little intrinsic ability to transcribe DNA, either
specifically or nonspecifically. The residual transcription observed in
lane1 is not due to pol II since it is insensitive
to 2 µg/ml
-amanitin (see lane5). When pure
TBP was titrated into the reaction, a 141-nucleotide transcript
corresponding to TATA-mediated transcription of the G
TI
promoter was observed, as expected (lanes2-4).
In addition, a large amount of nonspecific transcription was observed
and increased as more TBP was added. Both specific and nonspecific
transcription were sensitive to 2 µg/ml
-amanitin, indicating
that the transcripts were pol II-derived (lane5). As
expected, both specific and nonspecific transcription were
template-dependent (lane6). Although, the TBP used
in this study was greater than 98% pure, it remains possible that the
nonspecific pol II transcription was induced by a minor contaminant in
the TBP preparation. To address this possibility, the TBP preparation
was immunodepleted using affinity purified antibodies directed against
either TBP or a control antigen (yeast BRF protein). As shown in
lane7, the TBP preparation, which had been
immunodepleted with anti-TBP antibodies, failed to support specific and
nonspecific transcription, while the TBP preparation treated with the
control antibodies fully supported both specific and nonspecific
transcription (lane8). This finding confirms that
the nonspecifically bound TBP is the species responsible for assembling
functional pol II transcription complexes and is perhaps qualitatively
acting no differently than if it were bound to TATA.
Figure 8:
TBP-dependent nonspecific pol II
transcription. The PvuII-cut plasmid pS-GTI-141
(200 ng) was incubated with active TBP as indicated above each
lane and under the binding conditions described under
``Experimental Procedures.'' Reactions were incubated at 30
°C for 15 min in a volume of 15 µl. HeLa nuclear extracts (3
µl, 100 µg) were then added, and incubations at 30 °C were
continued for 5 min. Transcription reactions were initiated with the
addition of nucleotide triphosphates (including
[
-
P]ATP). Reactions were terminated after
15 min, and the radiolabeled RNA was analyzed as described under
``Experimental Procedures.'' Since these extracts were also
depleted of TAFs, and TAFs are required for basal pol III and pol I
transcription, we do not expect and do not observe any TBP-dependent
transcription arising from pol I and III (data not shown). It is well
documented that TBP can assemble functional pol II transcription
complexes in the absence of TAFs (6, 43,
44).
= 0.5 nM) than for an average nonspecific 8-bp
sequence (K
= 370 nM). 2)
TBP binding to DNA does not display cooperativity. 3) TBP dissociates
into the bulk solution from TATA at approximately the same rate as it
does when bound to flanking nonspecific DNA sequences. 4) When multiple
TBP molecules are present on the same DNA molecule, net dissociation of
TBP proceeds first from the lowest affinity sites and last from the
highest affinity sites. 5) Both TATA and nonspecific DNA equivalently
stabilize the DNA binding activity of TBP against thermal denaturation.
6) Nonspecifically bound TBP is active in assembling functional pol II
transcription complexes. The most parsimonious interpretation, which
can accommodate all of the data is that the TBP
DNA interface is
very similar at TATA and nonspecific sites, allowing TBP to function
equivalently. The kinetics of dissociation also suggest that TBP binds
DNA in an unusual multistep pathway, which includes stable
one-dimensional diffusion along the DNA. Sequence-specific DNA binding
appears to be achieved, in part, through sequence-specific rates of
diffusion along the DNA.
Specific and Nonspecific DNA Binding
Sequence-specific
and nonspecific DNA binding have been well characterized for major
groove DNA binding proteins. Nonspecific interactions typically involve
electrostatic interactions between positively charged residues of the
protein and the negatively charged DNA phosphate backbone.
Sequence-specific DNA binding involves additional interactions, most
notably, hydrogen bonding with specific bases. The TBPTATA
crystal structure provides a sharp contrast to these paradigms. TBP
binds the TATA box though predominantly hydrophobic and van der
Waals' contacts with the minor groove. Few hydrogen bonds are
made. Moreover, the minor groove lacks the array of functional groups
necessary for a protein to discriminate among bases with high
specificity. Typical major groove binding proteins such as cro repressor have 10
-fold higher affinity for a specific
site than for an average nonspecific site
(22) . For TBP, we
obtain a specificity factor of less than 10
. Possible
mechanisms by which TBP achieves specificity are described further
below.
DNA interface is very similar regardless of whether the
sequence is TATAAAAG or nonspecific. A second observation in support of
this proposal is that the rates of dissociation of TBP from TATA and
flanking nonspecific sites into the bulk solution are nearly equivalent
(t = 20-60 min). The slow dissociation rates
reflect an intrinsically high stability on specific and nonspecific
DNA. Major groove DNA binding proteins dissociate from nonspecific DNA
6 orders of magnitude faster (t estimated to be in the range
of 1 µs)
(22) . Third, the rates of dissociation into the
bulk solution from TATA or nonspecific sites display very similar
temperature insensitivity. In the absence of DNA, the TATA binding
activity of TBP is rapidly inactivated at elevated temperatures. At 70
°C, unbound TBP inactivates within seconds.
Yet when
bound to TATA or nonspecific DNA, the DNA binding activity of TBP is
very stable at these elevated temperatures. Either TBP is bound to
nonspecific DNA in the same manner as it is bound to TATA or some other
highly stable conformation exists on nonspecific DNA. There is no
evidence to date that supports the latter possibility. Fourth, the
biphasic kinetics of dissociation suggest that TBP bound to TATA is in
dynamic equilibrium with TBP bound to nonspecific DNA, and maintains
this equilibrium without dissociating into solution. The simplest
interpretation of this kinetic data requires that TATA and nonspecific
binding be qualitatively similar. Fifth, nonspecifically bound TBP
appears to be active in assembling functional pol II transcription
complexes. Finally, in all cases, crystal structure determinations of
TBP with altered TATA boxes yields essentially the same structure as
with TATAAAAG
(45) . These combined results provide a strong
argument for the interaction of TBP with TATA to be qualitatively no
different than its interaction with nonspecific DNA.
Mechanism of DNA Binding
In Fig. 9, we
propose a general mechanism for the interaction of TBP with DNA. The
model proposes that TBP dimers (T ) must dissociate into monomers
(T) prior to DNA binding. TBP monomers then rapidly
preequilibrate with DNA (D) to form an unstable complex
(TD) at an arbitrary site n. The
unstable complex then isomerizes into a stable complex
(TD
). Once TBP is stably bound, it is free
to randomly diffuse along the DNA to an adjacent site n + 1 (TD
).
Figure 9:
A
mechanism for TBP association with DNA. T represents TBP dimers
(discussed in more detail in the accompanying article (3)). T+ D represents a TBP monomer and DNA, respectively.
TD corresponds to an unstable TBP
DNA
complex at arbitrary position n.
TD
represents a stable TBP
DNA
complex, and TD
represents the same
complex as TD
but located one bp from
n. Each of these reaction segments are described in more
detail in the text.
Evidence for TBP
dimerization as a competitive reaction with DNA binding is presented in
the accompanying article
(33) . The TBP dimer interface is
proposed to overlap the concave DNA binding surface of TBP as
envisioned in the dimeric crystal structure of the apo form of TBP (34,
35). We have determined an approximate K for dimerization to be 4 nM, which is sufficiently low
so as to potentially dictate the kinetics of DNA binding. In addition,
the kinetics of dimer to monomer conversion appear to be sufficiently
slow (see Ref. 33) so as to account, in part, for the previously
observed slow kinetics of TATA binding
(17, 36) .
for TATA binding was measured to be
approximately 1000-fold lower (
10
M
s
) than that
expected of a one-step diffusion-limited reaction (
10
M
s
). However, no
direct evidence for a two-step model was presented. In fact, at
sufficiently high TBP concentrations, the rate of TBP binding became
too fast to accurately measure (t < 1 min), thereby
precluding detection of the proposed isomerization step
(17) .
These previously observed kinetics of TATA binding could also be
explained totally or in part by a slow dimer to monomer conversion of
TBP. Despite this caveat, TBP monomers might nevertheless proceed
through a rapid preequilibrium prior to stable DNA binding. This
assumption seems reasonable given that the TBP
TATA crystal
structure reveals a severely distorted DNA axis and widened minor
groove upon TBP binding. The TATA box is unlikely to stably exist in
such an extreme deformation in the absence of TBP. The proposed
isomerization step might involve such a deformation. Indeed, prebending
of the DNA has been found to enhance the binding of TBP
(37) .
is different
from k
. In other
words, the rate at which TBP slides will depend upon the underlying
sequence. For example, TBP will slide off of the TATAAAAG sequence less
often than TBP bound to the low affinity site described in this paper.
Second, the rate-determining step in dissociation from TATA or a
nonspecific site would be the proposed reverse isomerization step
described by k
. This
mechanism infers that
k
will vary depending
upon the sequence at n. Yet, the data presented here
demonstrate that TBP dissociates from TATA and nonspecific DNA at
essentially the same rate. This paradox can be reconciled by the fact
that dissociation was examined from specific and nonspecific sites
located on the same DNA molecule. If sliding
(k
) is much faster than
reverse isomerization
(k
), then dissociation
of TBP into the bulk solution from all sites, whether specific or
nonspecific, will proceed from a common site on the DNA and thus appear
to be sequence independent. Third, if
k
and
k
are
sequence-dependent, then these steps provide additional means by which
TBP discriminates among different sites.
Physiological Implications
The ability of TBP to
bind stably and functionally to nonspecific DNA suggest that TBP, in
the context of a TATA-less promoter, may be functioning no differently
than TBP bound at a TATA-containing promoter. Perhaps the only
difference between a TATA-containing and a TATA-less promoter is the
extent to which the underlying sequence contributes to promoter
specific binding. Presumably at TATA-less promoters, TBP must rely on
other factors such as TAFs and transcriptional activators to achieve
promoter specificity. Consistent with this, dTAF150 has
been reported to bind the initiator element, which is an essential
element at TATA-less promoters
(38) , and activators such as Sp1,
VP16, and NTF1 have been shown to bind
TAFs
(39, 40, 41, 42) .
and the
potentially high value of k
(see
Fig. 9
). In effect, TBP might be kinetically blocked from
associating randomly with nonspecific DNA. At TATA-less promoters,
activators might decrease k
, which would
increase the half-life of the unstable TD
complex. Unwanted nonspecific DNA binding might also be minimized
through TBP dimerization (see accompanying article
(33) ). TBP
dimers would decrease the concentration of TBP that is available for
DNA binding (TD
formation).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.