From the Department of Chemistry, University of
Nebraska-Lincoln, Lincoln, Nebraska 68588-0304 and the
§ Department of Biochemistry, Albert Einstein College of
Medicine, Bronx, New York 10461
Received for publication, May 22, 2000, and in revised form, November 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The TATA-binding protein (TBP) initiates assembly
of transcription preinitiation complexes on eukaryotic class II
promoters, binding to and restructuring consensus and variant "TATA
box" sequences. The sequence dependence of the DNA structure in
TBP·TATA complexes has been investigated in solution using
fluorescence resonance energy transfer. The mean 5'dye-3'dye distance
varies significantly among oligomers bearing the adenovirus
major late promoter sequence (AdMLP) and five single-site variants
bound to Saccharomyces cerevisiae TBP, consistent with
solution bend angles for AdMLP of 76° and for the variants ranging
from 30° to 62°. These solution bends contrast sharply with the
corresponding co-crystal structures, which show ~80° bends for all
sequences. Transcription activities for these TATA sequences are
strongly correlated with the solution bend angles but not with
TBP·DNA binding affinities. Our results support a model in which
transcription efficiency derives primarily from the
sequence-dependent structure of the TBP·TATA binary
complex. Specifically, the distance distribution for the average
solution structure of the TBP·TATA complex may reflect the
sequence-dependent probability for the complex to assume a
conformation in which the TATA box DNA is severely bent. Upon
assumption of this geometry, the binary complex becomes a target for
binding and correctly orienting the other components of the
preinitiation complex.
The TATA-binding protein
(TBP)1 binds to eukaryotic
class II promoters at specific sequences of DNA of the consensus
sequence TATA(a/t)A(a/t)N, nucleating assembly of the proteins required for transcription. Atomic resolution co-crystal structures of complexes
of DNA bearing consensus strong promoter sequences bound to
Saccharomyces cerevisiae (1), Arabidopsis
thaliana (2), and human (3, 4) TBPs are extremely similar,
characterized by a TBP-induced ~80° bend in the DNA helix. TBP also
binds to numerous variant TATA sequences, many of which occur naturally in promoters (5, 6). For 21 such single-point mutants of the adenovirus
major late promoter (AdMLP) TATA box sequence, in vitro
transcription activity was found to range from <1% to 107% of that
of the reference AdMLP TATA sequence (6).
The wide range of observed transcription activities suggested that TBP
does not bind similarly to all TATA elements. Gel electrophoresis circular permutation analysis of TBP·DNA complexes shows that the
electrophoretic mobility of the complexes is TATA
sequence-dependent, with bend angles from <34° to 106°
inferred from the gel mobility patterns (7). In contrast, the
co-crystal structures of 11 TATA sequence variants of varying affinity
bound to A. thaliana TBP are all very similar, with the DNA
helix bent as in the strong promoters (3, 8).
The present study was undertaken to further explore the TATA box
sequence dependence of TBP binding and DNA structure using native,
full-length S. cerevisiae TBP together with the AdMLP TATA
sequence and five single-base-pair variant sequences. End-to-end distance distributions for these duplexes, free and TBP-bound, were
extracted from measurements of time-resolved fluorescence emission in
conjunction with fluorescence resonance energy transfer (FRET). Bend
angles for the DNA within each of the TBP·DNA complexes were
determined using three models. The reference AdMLP and five variant
TATA sequences bound to TBP have significantly different mean
end-to-end distances in solution. These distances are consistent with
DNA bend angles ranging from 29.9° to 61.8° for the variant sequences and 76.2° for the native AdMLP. The latter bend angle is in
excellent accord with the bends observed in the co-crystal structures.
A strong correlation is observed between the solution bend angles and
the transcription activities (6). These findings are consistent with
the structure of TBP·TATA complexes being a principal determinant of
TATA-box-dependent transcription activity. A model is
proposed that reconciles the sequence dependence of bend angles and
transcription activities measured in solution with the DNA structures
observed in the co-crystals.
Protein, DNA, and Solution Conditions--
Full-length
S. cerevisiae TBP was prepared as described previously (9,
10). The double-labeled 14 base oligonucleotides, with 5'-TAMRA and
3'-fluorescein, were as described previously (11). The specific
sequences are shown in Table I. The corresponding single-labeled
oligonucleotides (denoted 14-mer*F) were identical except that
each had 3'-fluorescein but no 5'-TAMRA. The double- and single-labeled
probes and unlabeled complementary oligomers were synthesized by
Sigma-Genosys (The Woodlands, TX). The former two classes of oligomers
were high performance liquid chromatography/polyacrylamide gel
electrophoresis and polyacrylamide gel electrophoresis-purified, respectively. Studies were conducted at 30 ± 0.05 °C in 10 mM Tris-HCl (pH 7.4), 100 mM KCl, 2.5 mM MgCl2, 1 mM CaCl2
and 1 mM dithiothreitol.
Theory, Instrumentation, Data Acquisition, and
Analysis--
Extensive discussions of Förster resonance energy
transfer and its application to these studies have been published
(Refs. 11-13 and references therein). Very briefly, FRET is the
process whereby excited-state energy is transferred nonradiatively from a donor to an acceptor fluorophore. Because both dyes are attached to
an oligomer by flexible tethers, their distance apart is variable and
the donor decay depends upon the probability distribution, P(R), of all possible such distances (13, 14) as
follows,
Fluorescence lifetime measurements were made in the time domain using a
LaserStrobe spectrofluorometer (Photon Technology International, Inc.,
Lawrenceville, NJ) with PTI dye PL481 to generate pulsed 488-nm
excitation light. A 520-nm interference filter (Oriel Corp., Stratford,
CT) between the sample compartment and the detector isolated the
fluorescein emission. In obtaining the fluorescein decay, the following
experimental procedure was used in every case: The duplex or
duplex·TBP complex was formed in the cuvette and equilibrated ~12
min. Data acquisition was initiated, with detection beginning just
prior to fluorescence emission from the sample and ending at ~10×
the longest fluorescence lifetime, determined in preliminary
measurements. Three such complete decays were collected and averaged by
the software to generate one representative decay curve. The true
fluorescence decay was extracted from the total emission, which
included the instrument response function, using an iterative
reconvolution procedure incorporated into the minimization procedure.
Measurements of the fluorescence lifetimes of the 3'-fluorescein donor
fluorophores were made for the reference AdMLP sequence and the five
variant sequences, for both the single- and double-labeled duplexes,
and for the duplexes both free and TBP-bound. The concentrations of the
double- and single-labeled duplexes were 50 and 20 nM, respectively, with a 2-fold excess of complement and a total sample volume of 750 µl. The DNA·TBP complexes for all variants except A3
were formed by adding protein to the duplexes to final concentrations ranging from 350 to 910 nM, respectively, sufficient to
ensure >96% bound DNA in accord with the previously determined
equilibrium constants. Five replicate representative curves (each an
average of three decays) were defined as one set. Four such sets were collected for each of the following cases: free MLdpx*F,
TBP-bound MLdpx*F, free T*MLdpx*F, and
TBP-bound T*MLdpx*F. Four data sets were also obtained for
each case of the C7 sequence. Three data sets were collected for each
case of the T5, G6, and T6 sequences. Collection and analysis of data
for the A3 sequence is discussed separately.
These normalized data were fit to both bi- and tri-exponential
decay models with the relative quality of the fits assessed according
to the values of
For the bound AdMLP duplex, 16 values of
P(R) were likewise obtained from the
corresponding 4 × 4 matrix. Because all solutions of bound DNA
contained a small amount (<1% for AdMLP) of free duplex, each of the
16 probability distributions for the bound DNA was subsequently fit to
the sum of two P(R) values corresponding to bound
and free duplex. P(R)bound and
P(R)free were weighted to reflect
their respective fractional populations, determined using
Ka. The values for
The titrations of the double-labeled duplexes with TBP, conducted using
a spectrofluorimeter (Photon Technology International, Inc., Model
A-1010) and steady-state fluorescence emission, and subsequent
determination of the equilibrium binding constants (Ka) were as described (11) with the exception of
T*A3dpx*F.
The value of Ka for the A3 variant was
determined indirectly using time-resolved measurements, because the
steady-state emission of the T*A3dpx*F changed very little
upon TBP binding. Three composite bi-exponential decays characterizing
free A3dpx*F were obtained as described. To 25 nM A3dpx*F, TBP was added to final
concentrations of 153 and 178 nM (lower), 306 and 333 nM (intermediate), and 457 and 660 nM (higher),
resulting in six different mixtures of free and TBP-bound
A3dpx*F. Measurements of the fluorescence lifetimes of each
of these mixtures were made as described, to obtain one composite curve
(five data sets) for each; mean values of
To determine P(R) for free A3, three composite
bi-exponential decays characterizing free T*A3dpx*F were
determined as described. These
The determination of P(R) for TBP-bound A3
differed from that of the other sequences, because the value of
Ka was too low to achieve saturation of the duplex.
TBP was added to 50 nM T*A3dpx*F to final
concentrations of 656 and 365 nM, with the latter repeated
in two independent experiments. According to the previously determined
value for Ka, the resulting mixtures were 66 and
51% bound T*A3dpx*F, respectively, with the remainder
being free T*A3dpx*F. The observed decays,
Ida(t), from these three mixtures
were used in an expansion of Eq. 1,
To confirm that the value of R0 remained
essentially constant for all cases studied, R0
was determined independently for AdMLP and the T6 variant for both the
free and TBP-bound duplexes, using the solvent refractive index of
1.332. The overlap integrals were determined independently as described
(13) using emission spectra for free and bound MLdpx*F and
T6dpx*F and absorption spectra for free and bound
T*MLdpx and T*T6dpx. Emission and absorption spectra were collected on a steady-state fluorimeter (Photon Technology International, Inc., model A-1010) and a Hewlett-Packard diode array
spectrophotometer (model HP8452A), respectively.
To establish sufficient dye mobility consistent with Thermostability of TBP--
Because a time of ~80 min was
required for one set of five replicate fluorescence lifetime
measurements, the thermostability of TBP both free and DNA-bound was
investigated. For the latter, the complex was formed using 1:1 TBP:DNA,
both at 2.5 µM, with 25 nM
T*MLdpx*F and the remainder of the top-strand being
unlabeled DNA. (The reliability of the double-labeled duplexes as trace probes has been demonstrated previously (11).) Equilibrium was established with 95% saturation, within 1 min, as reflected by the
steady-state emission spectrum (9). The spectrum was monitored, and the
fractional saturation determined, at 5- to 10-min intervals for ~60
min. Because the double-labeled duplexes are stable for several hours
at 30 °C, any change in the spectrum of the bound complex reflected
a change in the fraction of DNA bound. The time dependence of the
fraction of bound DNA was extrapolated to obtain an estimate of the
half-time for inactivation of DNA-bound TBP.
To determine the stability of free TBP, a control experiment was done
first, as follows: TBP was added to 250 µl of buffer to a
concentration of 550 nM. To this solution was immediately added T*MLdpx*F (as a trace probe), unlabeled top strand,
and complement to 30, 520, and 890 nM, respectively, to
form 550 nM duplex. The fractional saturation was
determined from the steady-state emission spectrum at 2, 5, and 10 min
following addition of the complementary strand. The duplex had formed
and bound TBP to 90% saturation within 2 min, as reflected by the
steady-state spectrum, in precise accord with Ka.
Identical measurements were subsequently made with the 550 nM protein solution incubated at 30 °C for 5, 7, 10, 15, 30, and 60 min prior to addition of the duplex. Binding of less than
90% saturation within 2 min was attributed to inactivation of the free
protein during the preincubation. An estimate of the half-time for
inactivation of free TBP was determined directly from these data.
Simulations were conducted to demonstrate the significance of thermal
inactivation of TBP on the fluorescence measurements under the
experimental conditions of these studies. The two-intermediate linear
model and the six rate constants previously determined for this model
for AdMLP binding to yeast TBP (11) were used as the basis for the
simulations, with irreversible pathways added for inactivation of free
and bound TBP. For the latter, the first-order rate constants
determined from the measurements just described were used.
TATA Sequence-dependent DNA·TBP Affinity--
That
native S. cerevisiae TBP is monomeric at the protein
concentrations and solution conditions used in these studies has been
demonstrated by analytical ultracentrifugation (19, 20) and biochemical
studies (11). Stopped-flow binding studies conducted at stoichiometric
and equimolar concentrations of TBP and DNA (1 µM), thus
sampling the entire population of TBP molecules, are well-described by
the rate constants associated with a complex model for monomeric TBP
binding (11).2 These results
confirm the absence of a slow, rate-limiting step in binding (which
might derive from a dimer
The sequences of the reference AdMLP and five variant TATA boxes are
listed in Table I. The top strand of each
of the double-labeled 14-base pair duplexes (denoted
T*14-merdpx*F) had 6-carbon linkers to 5'-TAMRA and
3'-fluorescein identical to those used previously (11, 13). These
oligonucleotides differ only by a single base pair within the core
sequence. The equilibrium association constants (Keq) of native S. cerevisiae TBP
binding these duplexes, determined by steady-state FRET, varied over a
range of ~75× (Table I). Of the variant sequences, only T5 binds TBP
more tightly than the native AdMLP.
TBP Stability--
Control experiments were conducted to
demonstrate that a constant concentration of TBP·DNA complex was
maintained throughout the course of a set of fluorescence lifetime
measurements. The half-times for inactivation of the free and DNA-bound
S. cerevisiae TBP preparation used in these studies (9, 10)
were determined to be ~1 and ~10 h, respectively, at 30 °C under
the experimental conditions of these studies. The protein remains fully
active in DNA binding even after 24 h at 0 °C. These and similar results obtained by other
assays3,4
contrast sharply with a recent report of the loss of the "vast majority" of the DNA binding activity of S. cerevisiae TBP
after 0.3 min of incubation at 30 °C and all binding activity after 45 min even at 0 °C (21).
Numerical simulations mimicking the experimental conditions and
incorporating these rates of TBP inactivation demonstrated the effects
of TBP inactivation to be entirely negligible for at least the 80-min
time period over which fluorescence lifetime data were
acquired.5 This result is
consistent with the experimental observation that the first and last
curves were nearly identical for a given set of five replicate
fluorescence lifetime decays (Fig. 1),
showing no detectable loss of the protein·DNA complex with time.
End-to-End Distance Distributions, P(R), in Solution for Free and
TBP-bound TATA Duplexes--
The mean end-to-end distances
(
In contrast to the results obtained for the free DNA, the mean
end-to-end distances for the TBP-bound duplexes varied significantly (Table II). The largest decrease in
The values of
Control experiments were conducted to ensure that the changes in the
value of
The independently collected 3'-fluorescein emission and 5'-TAMRA
absorption spectra for the free AdMLP and T6 duplexes were invariant,
yielding identical overlap integrals and R0 = 61.0 Å. The integrals were likewise invariant for TBP-bound AdMLP and T6, with R0 = 61.2 Å, an increase of 0.3% upon
binding. These values of R0,free and
R0,bound were therefore assumed for the other
four sequences. The results of these control experiments confirm that
the sequence dependence of TATA Sequence-dependent Solution Bend Angles for
TBP-bound Duplexes--
We have shown previously that the
P(R) determined for TBP-bound
T*MLdpx*F using FRET fluorometry is generally consistent
with the bent DNA observed in the co-crystals bearing strong promoter sequences (9). The relationship of
For these three models, the smooth bend and single central bend models
(Fig. 2, C and A, respectively) correspond to the
upper and lower limits for the bend angles for a given ratio of
Because of the nature of the DNA bend observed within the TBP·TATA
complex, with sharp kinks at either end of the TATA sequence (1, 2), a
two-kink model (Fig. 2B) has also been considered. The bend
angles derived from this model are intermediate between the smooth and
single central bend models, and depend on both the total
length of the oligonucleotide and the position of the kinks. The bend
angles that derive from the ratio of
The DNA bend observed in all the co-crystal structures is relatively
smooth, with most of the total bend occurring at the flanking Phe
intercalation sites and the remainder in between. For TBP-bound
T*MLdpx*F, the solution bend angle associated with the
two-kink model, 76.2 ± 0.2°, closely corresponds to that
observed in the co-crystals (Table III). The solution angle derived
from the smooth bend model, 105.0°, is significantly larger, and from the single central bend model, 60.1°, significantly smaller. As we
show in the accompanying paper (24), the close correspondence of the
AdMLP bend in solution and in the co-crystal demonstrates clearly the
appropriateness of the two-kink model to the interpretation of the FRET data.
The values of
The similarity of the AdMLP bend angles determined in the co-crystal
and in solution using the two-kink model demonstrates the adequacy of
this model in describing the overall AdMLP conformation, without
detailed consideration of DNA structure such as helical unwinding.
Comparisons of the variant sequences to the reference AdMLP thus
focused on this model, with the larger values of
Correlation between Bend Angle and the Breadth of the
Distribution--
Because the end bases and linker arms are identical
in all oligonucleotides studied, the differences in the values of Time-resolved fluorescence resonance energy transfer
provides a rigorous approach to the determination of the structure and dynamics of macromolecules in solution. The primary experimental findings from this work are 1) the existence in solution of DNA sequence-dependent differences in the trajectory of the
DNA as it passes through TBP·TATA complexes and 2) the inverse
correlation between the observed DNA bend angle and the breadth of the
corresponding distance distribution.
DNA Bend Angles in TBP·TATA Complexes and the Corresponding
Probability Distributions Are DNA
Sequence-dependent--
The FRET data clearly demonstrate
sequence-dependent differences in the trajectory of the DNA
as it passes through TBP·DNA complexes. In sharp contrast to this
result and similar conclusions drawn from circular permutation and DNA
phasing studies (7, 25), eleven variant TATA sequences bound to TBP,
including all of the sequences in this study, have essentially
identical ~ 80° DNA bends in the atomic resolution structures
determined for TBP·DNA co-crystals (3, 8). These contrasting results
are accommodated within a two-state allosteric model, based on an
equilibrium between transcriptionally active and inactive TBP·DNA
conformations (discussed below). The apparent conundrum presented by
the solution and co-crystal structures is then definitively explained
in the accompanying paper (24).
Also important for consideration of the underlying mechanism of this
observation are the differences in the breadths of the corresponding
distance distributions provided by the time-resolved FRET data. Clearly
both structure and dynamics contribute to TBP·TATA function. The
AdMLP sequence alone shows only a slight increase in the value of
The variant sequences show a general trend toward increasingly broader
distributions as the extent of bending decreases, up to A Bi-modal Distribution Model Reconciling the Solution and
Co-crystal Bend Angles--
A two-state model is hypothesized,
unifying into a coherent perspective the sequence-dependent
solution bend angles reported herein and the x-ray results in which
only an AdMLP-like structure was crystallized. Each variant duplex
bound to TBP is proposed to exist in two conformations, one
(conformerML) with the DNA bound and bent as in the
AdMLP·TBP complex and the other (conformerTI) with the
DNA significantly less bent (Fig.
4A). Only
conformerML has the correct geometry to allow binding of
subsequent transcription proteins and effect measurable mRNA
synthesis. ConformerTI is transcriptionally inactive and
has the same overall conformation for all variant DNA·TBP complexes,
although the local structural and energetic features of the protein-DNA
interface are sequence-dependent. The presence of
conformerML is assumed for all variants, because such a
conformer is crystallized (except A3 (8)), although the solution
conditions for the crystallizations differed from those employed in the
present study. The two-state model provides a unifying and simple
relationship among the variants to explain their observed differences
in bend angle and distance distribution variance, rather than
necessitating that each variant, with a unique set of conformers, be
considered separately.7
If the equilibrium for conformerTI
Bend angles corresponding to the two-state model were then calculated
for each variant, i, using Eq. 6 with appropriate weighting for
If the exchange between conformerTI and
conformerML is fast relative to subsequent binding
processes, the transcription factors "see" and appear
macroscopically to bind to an average TBP·DNA structure that is
sequence-dependent. The model predicts that the more
AdMLP-like the average binary structure, the more efficiently transcription will proceed. Implicit in this model is a correspondence between the structure of the TBP·TATA complex and transcription activity, which is explored further below.
Minimal Correspondence of TBP·DNA Complex Lifetime to Bend Angle
or Transcriptional Activity--
Hawley and coworkers (7), inferring
bend angles from gel mobility shifts for TBP-bound AdMLP and eight
variant sequences, also observed sequence-dependent differences
in bend angles. However, for the sequences common to both studies,
those angles differ from those reported herein in magnitude, by up to a
factor of two, but more significantly, in the ordering of sequences by
decreasing bend.
Although a correlation was asserted between bend angles inferred from
circular permutation analysis and TBP·TATA complex stability (7),
careful inspection of those data reveal a minimal correspondence between these two properties. A plot of the lifetime of the TBP·DNA complex versus bend angle from Table I (7) shows no general linear correlation (correlation = 0.76, coefficient of
determination = 0.59); rather, the data form two distinct sets.
The first of these sets of five sequences is composed of unstable
TBP·TATA complexes, with lifetimes
The data of Hawley and coworkers also show a minimal correspondence
between transcription activities and the lifetimes of the TBP·DNA
complex. A plot of the lifetime of the TBP·DNA complex versus transcription activity (Ref. 7, Table I) shows
roughly two data sets, with a poor linear relationship
(correlation = 0.73, coefficient of determination = 0.54).
DNA Bends in TBP·DNA Complexes Are Highly Correlated with
Relative Transcription Activity--
The correlation between the
solution bend angles determined in the present study and the
corresponding in vivo and in vitro transcription
activities reported by Wobbe and Struhl (6) are shown in Figs.
5, A and B,
respectively. The same correlation is observed upon comparison with
either the HeLa TFIID or yeast TBP in in vitro transcription
assays. Two possible explanations for this correlation present
themselves. First, the observed differences in transcription activity
are structurally based, resulting fundamentally from the
sequence-dependent differences in the DNA bend angles in
the binary complexes, or second, they derive simply from different levels of saturation of the TATA site by TBP, due to
sequence-dependent differences in binding affinity. The
concentrations of HeLa and yeast TBP used in the in vitro
assays were reported to be saturating under the experimental conditions
of those studies (6). We therefore conclude that the >100-fold
differences observed in transcription efficiency could not have arisen
from differences in TBP·DNA affinity.
Suppose, however, that only the tightly bound AdMLP sequence was
saturated and the variant sequences were fractionally saturated in
accord with their respective binding constants, so that transcription activity did reflect differences in affinity. Then, for example, were
the AdMLP sequence 95% bound (as a lower limit), the transcription activity for the T6 sequence would be 86% that of AdMLP, based on the
Ka values shown in Table I. In contrast, the experimentally observed transcription activity for T6 was only 10%
that of AdMLP (6). Thus, several independent lines of evidence support
the conclusion that differences in TBP·DNA binding affinity cannot
account for the observed differences in transcription efficiency.
In contrast, a significant correlation is observed between the solution
bend angles and transcription activity. Wobbe and Struhl (6) similarly
concluded that the in vivo activity of a TATA element is
directly affected by the binary TBP·TATA structure. This conclusion
was based on the close similarity between the in vitro
activity of yeast TBP (and human TFIID) and transcription activity in
yeast cells. The strong correspondence between the solution geometry of
the TBP·DNA complex and transcription activity is further supported
by a comparison of Figs. 4B and 5B. The
relationship between transcription efficiency and bend angle is
strikingly similar to the relationship between the fractional
population of the allosteric conformerML and bend angle.
The extent to which conformerML is populated, for a given
sequence, thus closely corresponds to the relative transcription activity.
The relatively large values of
The trajectories of the helical axes resulting from different bends
diverge rapidly (Fig. 6). For example,
for a 14-bp duplex centered on the TATA box, the difference in the
5'-3' distance between a 40° and an 80° bend is ~4 Å. Extension
of the duplex by only 6 bp up- and downstream, for example, more than
triples that difference, from ~4 to ~13 Å. TBP-bound T6 and
AdMLP have angles of ~40° and ~80°, respectively, and 6-bp
extensions correspond generally to the flanking contact regions for
TFIIA and -B. Formation of a stable higher-order structure is thus
predicted to be less probable for the TBP·T6 complex than for the
TBP·AdMLP complex, due to the spatial requirements.
In drawing a correlation between the apparent bend angle and
transcriptional activity, however, consideration must be given to the
experimental conditions of the respective studies. The in
vitro transcription assays were performed in the presence of osmolyte (6, 35). As shown in the accompanying paper (24), the
conformations of some bound variant sequences are sensitive to the
presence of osmolyte. Because a significant correlation is observed
between bend angle and transcriptional activity both in
vitro and in vivo (Fig. 5, A and
B), it is plausible that the extremely small differences in
energy between conformers for these sequences (24) are compensated in
osmolyte by protein·protein interactions among multiple transcription
factors. How the binding of even one additional transcription protein,
in osmolyte, might affect the equilibrium among
sequence-dependent TBP·DNA conformers is not known. Thus,
effects of osmolytes on the conformation of the binary complex within
multiprotein complexes require further exploration.
However, unequivocal new insight is provided by elucidation of the
solution structures of TBP·AdMLP and TBP·A3. These binary complexes
with the high and low extremes of the observed bend angles correspond
to the high and low extremes of transcriptional activity. The solution
geometries of these two complexes are insensitive to the presence of
osmolyte (24) and establish clearly the relationship between
transcription activity and the structure of the binary complex.
Conclusions--
The geometries of the TBP-bound variant TATA
sequences in solution vary significantly and differ from their
corresponding co-crystal structures. These solution conformations are
consistent with DNA bend angles ranging from ~30 to ~76° based on
a two-kink bending model. A strong correlation between the solution
bend angles and relative transcription activity, but not with TBP·DNA affinity, is observed. This correlation is particularly notable, because efficient transcription requires complex geometric
relationships among many proteins and to summarize such complexity with
a single, simple bend angle must be, to some extent, an oversimplification.
This model contrasts with models in which the TBP·DNA binary complex
structure is conserved (8) and sequence-dependent differences in transcription efficiency derive primarily from sequence-dependent differences in the stability of that
complex (7, 8). Our results support a model in which transcription efficiency derives in significant part from the
sequence-dependent structure of the TBP·TATA binary
complex. More specifically, the distance distribution for the average
solution structure of the TBP·TATA complex may reflect the
sequence-dependent probability for the complex to assume a
conformation in which the TATA box DNA is severely bent. Upon
assumption of this geometry, the binary complex becomes a target for
binding and correctly orienting the other components of the
preinitiation complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
where Id and Ida
are the fluorescence emission intensity of the donor in the absence and
presence of acceptor, respectively, and the inverse of the
ith donor lifetime, 1/
(Eq. 1)
di, equals
(kF + kI)di
and
[1/
D*(Ro/R)6] = kt. (kF and
kI are the respective rate constants for
fluorescence and nonradiative decay, and kt is
the rate constant for energy transfer.) R0 is
the Förster distance, for which the efficiency of transfer is
0.5.
D* in Eq. 1 is the donor lifetime uniquely associated with a particular value of R0, and
remains constant as long as the acceptor absorption and donor emission
spectra remain unchanged. Thus, P(R) may be
extracted from measurements of the fluorescence lifetime decay of the
donor in the presence and absence of acceptor.
2, the Durbin-Watson parameter (15,
16), and the runs test parameter (17). The five
and
values
composing a given set were then averaged; the corresponding average
decay curve, henceforth a composite curve, characterized 15 separate
decays. Four such composite curves were thus obtained for each case of
AdMLP and C7, with three composite curves for each case of the other
sequences. For the free AdMLP duplex, a 4 × 4 matrix was then
constructed from the four composite MLdpx*F decays and the
corresponding four T*MLdpx*F decays. The resulting
16 combinations of donor/donor-acceptor decays were individually
analyzed as described (13) to obtain 16 values of
P(R)free using Eq. 1. Sixteen values
were thus obtained for
free
characterizing the distribution. These values were averaged to yield
the reported values of
free and the corresponding standard deviations.
free were
fixed at the previously determined values and the optimal values for
bound
obtained. The mean values and standard deviations for
bound were
determined as for the free duplex. The C7 data were analyzed
identically to AdMLP. The same analysis obtained for T5, G6, and T6 but
with 3 × 3 matrices.
i and
i, describing the decay for each mixture, were
determined. The observed decay, F(t), from each of these mixtures derived from both free and bound A3dpx*F
and may be described as
(Eq. 2)
where X is [A3dpx*F·TBP],
D and T are the respective total
concentrations of DNA and TBP, Ka is the association constant for the A3·TBP interaction,
(Eq. 3)
i and
i characterize the free A3dpx*F decay, and
i' and
i' characterize the TBP-bound A3dpx*F decay. The mean values for
i and
i for the free A3dpx*F were fixed in Eq. 2
at the mean values for the three composite curves previously obtained.
The decay curves for the mixtures were analyzed globally in groups of
three for four parameters: Ka,
1',
1', and
2'
(
2' = 1
1'). Parameter values were obtained for all eight possible groupings of lower, intermediate, and higher TBP concentrations, e.g. 153, 306, and 457 nM TBP. Mean values and standard deviations were determined
from these eight values for the equilibrium constant and the
values
and
values characterizing the decay from TBP-bound
A3dpx*F. This procedure was followed, rather than a single
global fit for all six mixtures, to obtain error estimates.
i and
i
values were used together with those for free A3dpx*F to
construct a 3 × 3 matrix and obtain mean values and error
estimates for
free as described.
(Eq. 4)
with the values of the fractions of free
(Ffree) and bound
(Fbound) T*A3dpx*F fixed according
to Ka. The values of
i' and
i' in the second term on the right-hand side of Eq. 4
reflect bound donor-only decay and were fixed at the mean values previously determined. P(R) was then obtained
using a matrix approach, analyzing separately all nine combinations of
the three Ida values and, in the first term on
the right-hand side, the three sets of
i and
i values from the three composite curves corresponding to free T*A3dpx*F. The resulting nine values for
bound for
T*A3dpx*F were averaged, and the S.D. was determined.
2 = 2/3, time-resolved anisotropy decays were also measured for free and
TBP-bound MLdpx*F, T*MLdpx,
T6dpx*F, and T*T6dpx. Semi cone angles for the
dyes in each of these eight conditions were determined as described
(18). Anisotropy decay measurements were made using the LaserStrobe
spectrofluorometer with 488- and 500-nm excitation light for
fluorescein and TAMRA, respectively, and with the corresponding emission isolated by a 520-nm interference and a 530-nm long pass filter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
monomer process (21)) under the
experimental conditions of this study.
The sequences of the 14-mer oligonucleotides containing the reference
AdMLP and five variant TATA sequences and the association equilibrium
constant for each binding, as a duplex, to TBP
View larger version (13K):
[in a new window]
Fig. 1.
Typical time-resolved fluorescence decays,
for T*MLdpx*F free in solution (dotted
line) and TBP-bound. The decay collected initially for
bound T*MLdpx*F (solid line), following
equilibration at 30 °C of 50 nM duplex with 440 nM TBP, is very similar to the fifth decay collected in the
data set on the same material (dashed line). The latter
curve was obtained after ~60 min and shows no trend toward the time
progression of the free duplex, due both to the stability of the bound
protein and to the large excess of TBP.
for the distribution of
distances, are listed in Table II. The values of
Values for the mean end-to-end distance, for the
distance distribution corresponding to P(R) for the AdMLP duplex and
each of the five variants, both free in solution and TBP-bound
2 of 0.97 ± 0.08. These data were subsequently analyzed to obtain the
probability distributions. P(R) was modeled in
all cases as a shifted Gaussian, determined previously from Hermite
polynomial expansions to best approximate these distributions (13, 22).
P(R) values obtained for TBP-bound DNA were refit
to P(R)bound + P(R)free, weighted using
Ka, to correct for the small amount (<4%) of free
duplex. All P(R) values fit to the summed
distributions with correlation coefficients > 0.999.
, the S.D. of the distribution, increased upon binding
for all sequences, although not uniformly (Table II). A 5% increase in
the breadth of the distribution for T*MLdpx*F contrasts
with a 144% increase for T*T6dpx*F. The range of values of
for the bound duplexes, 7.5 ± 0.1 to 10.5 ± 0.3 Å,
greatly exceeds the confidence limits of the measurements.
0, were determined for both fluorescein and
TAMRA for the AdMLP and T6 duplexes, free and TBP-bound. (The
transition moment of the fluorophore wobbles within a cone with the
vertex at the center of the transition moment. The angle
0 is half of the apical angle of this cone.) For these eight conditions, the semi cone angles ranged from 56° to 70° with
an average value of 64°. Because the error for each angle was
estimated to be ±7°, none of these angles differed significantly from the mean. The fast and slow rotational correlation times,
,
correspond to the free dye and to the macromolecule to which the dye is
attached, respectively (18). For these eight conditions,
fast = 0.15 ± 0.03 ns (free and bound),
slow,free = 5 ± 2 ns, and
slow,bound = 23 ± 2 ns. These values of
0 and
fast reflect a high degree of
rotational freedom for both dyes, free and bound, for two sequences
with disparate values for
4 base pairs both 5' and 3'. The value of
2 was therefore assumed to
equal 2/3 in all R0 calculations. (The invariance of
slow,bound further confirms the absence of
condition-dependent TBP aggregation.)
View larger version (5K):
[in a new window]
Fig. 2.
Three simple models for describing the
TBP-induced DNA bend. A, a single central bend;
B, a symmetric two-kink model; and C, a
continuous smooth bend model. The two kinks in the TBP·TATA
co-crystal structure envelop the six central core base pairs.
L2 in model B = 20.4 Å,
consistent with the structure of B DNA and also closely
approximating the distance from the midpoints of the helix between
steps 1-2 and steps 7-8 in the co-crystal structures (1, 2).
L1 = L3 = ( 20.4 Å)/2. The bend angles
reported herein corresponding to all three models are those described
by "
."
Derived solution bend angles according to three bending models for the
six duplex DNAs bound to TBP
. From the 16 (AdMLP and C7) or 9 values
(T5, G6, and T6) for both
(Eq. 5)
(Eq. 6)
where
(Eq. 7)
and L2 are as described in Fig. 2
and all distances are in angstroms. For all models, the linear distance
is assumed to be the measured
carbons) differ by
<1 Å in the TBP crystal structures with and without bound DNA bent to
80° (1, 2), the conformations corresponding to the maximum structural
distortion. The distance between the kink sites,
L2, was therefore held constant in Eq. 6 for all
sequences. Differences in
are assumed to derive primarily from the duplex DNA rather than the linker arms. The changes in
,
, upon TBP binding, rather than
itself, then provide the most informative comparison among the sequences. The relationship between
and derived bend angle is
shown in Fig. 3. The native AdMLP
sequence (T*MLdpx*F), with the largest bend angle, has the
smallest increase in
of only 0.4 Å upon TBP binding.
View larger version (11K):
[in a new window]
Fig. 3.
The correspondence for the naturally
occurring sequences between bend angle (from Eq. 6), and the change
in upon TBP binding,
diff. The sequence associated with
each data point is noted along the top axis. Because
independent variances are additive,
diff was calculated
according to
diff = (
diff reflects the broadening of
due to the
binding process; i.e. the contribution from the tethers is
removed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
the S.D. of the end-to-end distance distribution, upon TBP binding. A
plausible hypothesis is that the complementarity of the protein-duplex
interface confines the helix and restricts additional motion. This
slight increase in the breadth of the distribution for the tightly
bound AdMLP may derive from the presence of multiple conformers at
equilibrium, each with bent DNA but differing, for example, in the
extent of phenylalanine intercalation (11). An integrated hydroxyl
radical footprinting and molecular dynamics study of the TBP-AdMLP
interface supports this view of its dynamic nature (26).
= 6.2 Å for the T6 variant. The inverse correlation between bending
extent and distribution broadening may derive from the increasing
misfit along the protein-DNA interface as helical bending decreases,
including retention of solvent molecules at the interface. Indeed,
complexes of TBP with the variant duplexes may be present in multiple
conformations with the DNA bent very differently among those
conformers, as discussed further in the following section. The
broadened distribution would then result from equilibrium exchange
among such conformers occurring on a time scale that is slow relative
to the nanosecond time scale of the measurements, i.e.
microseconds. In this case, the broader distribution of distances would
not derive from any high frequency torsional and bending motions of the duplex that occur on time scales faster than
nanoseconds, because such motion would be averaged out in these
measurements (27).
View larger version (26K):
[in a new window]
Fig. 4.
A, the two-state model in which each
TBP-bound duplex is distributed between two populations,
conformerML, in which the DNA is bent ~80° as for
AdMLP, and conformerTI, in which the DNA is bent only
slightly. Keq is sequence-dependent.
Transcription proceeds from conformerML, with the DNA in
the correct geometry. B, the correspondence of the mole
fraction of conformerML with the observed ( , Table III)
and calculated (
, Eq. 9) bend angles, the latter corresponding to
the two-state model. The theoretical values derive from only one fitted
parameter,
conformerML occurs on a time scale significantly slower
than that of the nanosecond measurements, the measured fluorescence
decay for a given sequence would derive from both conformers. In fact,
the probability distributions for all of the bound variant
duplexes, P(R)i,bound, are very
well fit globally by two constrained Gaussian
distributions8 corresponding
to conformerML and conformerTI,
where i specifies the variant,
(Eq. 8)
= mole
fraction, and
TI = 1
ML. In this
analysis, the values of
ML,bound for conformerML were fixed at 47.1 and 8.5 Å (Table II), respectively. The values obtained for the two
fitted parameters were
TI,bound = 9.9 Å. The relative mole fractions of conformerML and conformerTI for each variant,
i, were determined concurrently as a function of the fitted
value of
where
(Eq. 9)
is the
observed mean end-to-end distance for a given free duplex. The
relationship of the calculated (Eq. 9) and observed bend angles (Table
III) with the mole fraction of conformerML is shown in Fig.
4B.
0.08 that of the wild
type, but with bend angles ranging from <34° to 80°. The second
set of five sequences, constituting a step function relative to the
first set, includes only severe bends, from 80° to 106°, but
lifetimes that vary 23-fold, from 0.08 to 1.85 that of the wild type.
This conclusion is further supported by the recent work of
Bareket-Samish et al. (25), who report no correlation
between TBP·TATA complex stability and DNA bend angles determined
similarly using gel phasing analysis.
View larger version (21K):
[in a new window]
Fig. 5.
The correlation between the solution bend
angles of the TBP-bound duplexes and the corresponding in
vivo (A) and in vitro
(B) transcription activities reported by Wobbe
and Struhl (6), with the activity of the AdMLP sequence set at ++ and
1.00, respectively. The bend angles were determined using the
two-kink model (Fig. 2B). For B, the
correlation = 0.98 and the coefficient of determination = 0.97. These data show a minimal relationship between transcription
activity and the association equilibrium constant (correlation = 0.88 and coefficient of determination = 0.77).
determined herein for the bound
duplexes with less favorable TATA box sequences are consistent with low
frequency DNA flexibility within the binary complexes. Such duplex
motions cannot be effectively distinguished from multiple conformations
(29). The observed correlation between the extent of DNA bending and
transcription activity thus leads us to propose that the probability
for a given TBP·TATA complex to assume the conformation required for
binding of subsequent proteins determines the corresponding
transcription efficiency. For the bound variants, as the deviation
from ~ 80° increases, severe distortions of the duplex DNA to
approach 80° become increasingly less probable. In terms of such
fluctuations, a dependence of transcription efficiency on the average
conformation of the binary TBP·promoter complex seems reasonable.
Both biochemical and crystallographic results show that flanking
sequences up- and downstream of the TATA box are contacted by TFIIA
(30, 31) and TFIIB (32-34), with TFIIB contacting both. Appropriately
bent DNA in the TBP·DNA target may thus be critical for formation of
stable ternary and quaternary complexes involving these proteins.
View larger version (13K):
[in a new window]
Fig. 6.
The helical trajectories corresponding to
40° (dotted lines) and 80° (solid
lines) DNA bends. The lighter segments of
each trajectory correspond to that part of the 14-mer duplex beyond the
5' and 3' phenylalanine insertion sites, beginning with positions 31
and
24, respectively. The
20 position downstream of the TATA box
(
) and the
38 position upstream (
) delineate the TFIIB contact
regions and the
42 position (
), the TFIIA contact region. The
distance between the up- and downstream TFIIB contacts (double
arrows) differs by ~7 Å for 40° (T6) and 80° (AdMLP)
bends.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Stephen Burley for communication of results prior to publication.
![]() |
FOOTNOTES |
---|
* This work was supported by a Fellowship from the Program in Mathematics and Molecular Biology at the University of California-Berkeley, which is sponsored by National Science Foundation Grant DMS-9406348 (to R. M. P.), by Wheeler (to J. W.) and McDonald (to R. M. P.) Fellowships from the College of Graduate Studies, University of Nebraska-Lincoln, and by National Institutes of Health Grants GM59346 and CA76049 (to L. J. P.) and GM39929 (to M. B.).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, University of Nebraska-Lincoln, 525 Hamilton Hall, Lincoln, NE 68588-0304. Tel.: 402-472-3501; Fax: 402-472-2044.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M004402200
2 R. M. Powell, manuscript in preparation.
3 E. Jamison and M. Brenowitz, unpublished observations.
4 M. Daugherty and M. Fried, unpublished observations.
5 J. Wu, unpublished data.
6
Using instead the average value of
7
This two-state model is conceptually analogous
to the allosteric model, with an R-state active form, a T-state
inactive form and L, the allosteric constant, defining the
R-T equilibrium. Myriad hemoglobins are well described by these two
states but have widely variable value of L, depending on how
substitutions alter the
1
1/
2
2 interface.
8 Whereas at least two distinct distributions are required to fit various higher-order DNA structures (28), each of the structures investigated herein were very well fit by a single distribution. It is only in the model-dependent global analysis of all bound sequences that two distributions can be distinguished, in varying proportions, accounting for all observed decays.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TBP, TATA-binding protein; AdMLP, adenovirus major late promoter; FRET, fluorescence resonance energy transfer; TAMRA, carboxytetramethylrhodamine; T*14-mer*F, TATA-bearing 14-base double-labeled DNA oligomer with 5'-TAMRA and 3'-fluorescein; T*14-merdpx*F, duplex DNA corresponding to T*14-mer*F top strand; 14-mer*F, corresponding single-labeled 14-mer with 3'-fluorescein; TFIIA, -B, and -D, class II general transcription initiation factors A, -B, and -D; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kim, Y., Geiger, J. H., Hahn, S., and Sigler, P. B. (1993) Nature 365, 512-519[CrossRef][Medline] [Order article via Infotrieve] |
2. | Kim, J. L., Nikolov, D. B., and Burley, S. K. (1993) Nature 365, 520-527[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Nikolov, D. B.,
Chen, H.,
Halay, E. D.,
Hoffmann, A.,
Roeder, R. G.,
and Burley, S. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4862-4867 |
4. | Juo, Z. S., Chiu, T. K., Leiberman, P. M., Baikalov, I. B., Berk, A. J., and Dickerson, R. E. (1996) J. Mol. Biol. 261, 239-254[CrossRef][Medline] [Order article via Infotrieve] |
5. | Hahn, S., Buratowski, S., Sharp, P., and Guarente, L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5718-5722[Abstract] |
6. | Wobbe, C. R., and Struhl, K. (1990) Mol. Cell. Biol. 10, 3859-3867[Medline] [Order article via Infotrieve] |
7. | Starr, D. B., Hoopes, B. C., and Hawley, D. K. (1995) J. Mol. Biol. 250, 434-446[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Patikoglou, G. A.,
Kim, J. L.,
Sun, L.,
Yang, S. H.,
Kodadek, T.,
and Burley, S. K.
(1999)
Genes Dev.
13,
3217-3230 |
9. | Parkhurst, K. M., Brenowitz, M., and Parkhurst, L. J. (1996) Biochemistry 35, 7459-7465[CrossRef][Medline] [Order article via Infotrieve] |
10. | Petri, V., Hsieh, M., and Brenowitz, M. (1995) Biochemistry 34, 9977-9984[Medline] [Order article via Infotrieve] |
11. | Parkhurst, K. M., Richards, R. M., Brenowitz, M., and Parkhurst, L. J. (1999) J. Mol. Biol. 289, 1327-1341[CrossRef][Medline] [Order article via Infotrieve] |
12. | Parkhurst, K. M., and Parkhurst, L. J. (1995) Biochemistry 34, 285-292[Medline] [Order article via Infotrieve] |
13. | Parkhurst, K. M., and Parkhurst, L. J. (1995) Biochemistry 34, 293-300[Medline] [Order article via Infotrieve] |
14. | Cantor, C. R., and Pechukas, P. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 2099-2101[Abstract] |
15. | Durbin, J., and Watson, G. S. (1950) Biometrika 37, 409-428 |
16. | Durbin, J., and Watson, G. S. (1951) Biometrika 38, 159-178[Medline] [Order article via Infotrieve] |
17. | Hamburg, M. (1974) Basic Statistics: A Modern Approach , pp. 285-287, Harcourt Brace Jovanovich, Inc., New York |
18. | Bucci, E., and Steiner, R. F. (1988) Biophys. Chem. 30, 199-224[CrossRef][Medline] [Order article via Infotrieve] |
19. | Daugherty, M. A., Brenowitz, M., and Fried, M. G. (1999) J. Mol. Biol. 285, 1389-1399[CrossRef][Medline] [Order article via Infotrieve] |
20. | Daugherty, M. A., Brenowitz, M., and Fried, M. G. (2000) Biochemistry 39, 4869-4880[CrossRef][Medline] [Order article via Infotrieve] |
21. | Jackson-Fisher, A. J., Burma, S., Portnoy, M., Schneeweis, L. A., Coleman, R. A., Mitra, M., Chitikila, C., and Pugh, B. F. (1999) Biochemistry 38, 11340-11348[CrossRef][Medline] [Order article via Infotrieve] |
22. | Parkhurst, L. J., and Parkhurst, K. M. (1994) Proc. Soc. Photo-Opt. Instrum. Eng. 2137, 475-483 |
23. | Koo, H.-S., Wu, H.-M., and Crothers, D. M. (1986) Nature 320, 501-506[Medline] [Order article via Infotrieve] |
24. |
Wu, J.,
Parkhurst, K. M.,
Powell, R. M.,
and Parkhurst, L. J.
(2001)
J. Biol. Chem.
276,
14623-14627 |
25. | Bareket-Samish, A., Cohen, I., and Haran, T. E. (2000) J. Mol. Biol. 299, 965-977[CrossRef][Medline] [Order article via Infotrieve] |
26. | Pastor, N., Weinstein, H., Jamison, E., and Brenowitz, M. (2000) J. Mol. Biol. 304, 55-68[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Okonogi, T. M.,
Reese, A. W.,
Alley, S. C.,
Hopkins, P. B.,
and Robinson, B. H.
(1999)
Biophys. J.
77,
3256-3276 |
28. | Yang, M., and Millar, D. P. (1996) Biochemistry 35, 7959-7967[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Naimushin, A. N.,
Fujimoto, B. S.,
and Schurr, J. M.
(2000)
Biophys. J.
78,
1498-1518 |
30. |
Lagrange, T.,
Kim, T.-K.,
Orphanides, G.,
Ebright, Y. W.,
Ebright, R. H.,
and Reinberg, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10620-10625 |
31. |
Coulombe, B.,
Li, J.,
and Greenblatt, J.
(1994)
J. Biol. Chem.
269,
19962-19967 |
32. | Lee, S., and Hahn, S. (1995) Nature 376, 609-612[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Lagrange, T.,
Kapanidis, A. N.,
Tang, H.,
Reinberg, D.,
and Ebright, R. H.
(1998)
Genes Dev.
12,
34-44 |
34. |
Tsai, F. T. F.,
and Sigler, P. B.
(2000)
EMBO J.
19,
25-36 |
35. | Sawadogo, M., and Roeder, R. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4394-4398[Abstract] |