| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 34, 31685-31690, August 22, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||
From the
Department of Chemistry, University of
Nebraska, Lincoln, Nebraska 68588-0304 and the
¶Department of Biochemistry, the University of
Vermont College of Medicine, Burlington, Vermont 05405-0068
Received for publication, May 17, 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
5 nM), simultaneous binding
and DNA bending, and rapid formation of a stable human TBP-DNA complex having
DNA bent 
100°. These data allow, for the first time, a direct
comparison of the reactions of the full-length, native human and yeast TBPs
with a consensus promoter, studied under identical conditions. The general
reaction characteristics are similar for the human and yeast proteins,
although the details differ and the hTBPwt-induced bend is more
severe. This directly measured hTBPwt-DNAAdMLP
interaction differs fundamentally from a recently published
hTBPwt-DNAAdMLP model characterized by low affinity
(µM) binding and an unstable complex requiring either a 30-min
isomerization or TFIIB to achieve DNA bending. Possible sources of these
significant differences are discussed. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The 180-residue C-terminal DNA binding domains (CTD) of the TBPs from yeast (yTBP), human (hTBP), and Arabidopsis thaliana (aTBP) are highly conserved, with greater than 80% homology and with generally conservative substitutions in the remainder. The pseudo-symmetric TBPCTD contacts the duplex along the distorted minor groove via interactions that are largely non-polar and hydrophobic. The amino acid residues involved in these DNA contacts are nearly 100% conserved, with only a single variation in which Arg-204 in hTBP changes to Lys-110 in yTBP and Lys-68 in aTBP (2). The N-terminal domain is, in contrast, heterogenous between the yeast (60 amino acids (14)) and human (159 amino acids (15)) proteins and severely truncated in the A. thaliana protein (18 amino acids (16)).
How these similarities and differences among TBP molecules from different species affect the TBP-DNATATA interaction and structure remains an open question. A limited answer has emerged from crystallographic studies on the C-terminal domains of yTBP and hTBP and the (analogous) full-length aTBP, bound to DNATATA: the structures of these three TBP-DNA complexes are essentially identical in the crystalline form (2, 17, 18) and are TATA sequence-independent at least for yTBP (6). Beyond these results, however, comparisons of the behavior of yTBP, hTBP, and aTBP with DNATATA and the structures of the resulting binary complexes must be inferred from unrelated experiments conducted in different laboratories using any one of these three proteins, either as the full-length or truncated C-terminal form. In addition, although a clear understanding of the behavior of the human protein is of obvious interest, most of the work done to date has utilized Saccharomyces cerevisiae, or yeast, TBP. The extent to which these latter results also hold true for hTBP is currently largely a matter of speculation.
The recognition mechanisms of TBP for promoter DNA and the solution structures of TBP-DNA complexes have been primary research interests in our laboratory for several years (4, 7–11). Our focus has been 2-fold: 1) identifying and comparing detailed kinetic models for TBP binding to consensus (TATAa/tAa/tN) and variant promoter TATA sequences, and 2) evaluating the solution structure of the TBP-DNATATA complex. These studies utilize DNA duplexes that are labeled with fluorescent dyes that serve as a donor-acceptor pair for Förster resonance energy transfer (FRET). The methodologies employed in these biophysical studies allow the collection of kinetic data in real time as well as precise measurements of inter-dye distances that correspond to the extent of helical bending in the TBP-DNATATA complexes.
Our prior work has been conducted using full-length S. cerevisiae
TBP. Function studies conducted using three TATA sequences have shown
multistep interaction mechanisms overall with concurrent binding and DNA
bending (7,
10,
11). The associated structure
studies have revealed TATA sequence-dependent DNA bend angles for yTBP-DNA
complexes in solution, with bends varying from 30 to 80° for the
seven sequences examined. These bend angles are strongly correlated with
relative transcription activity
(8). The sequence dependence of
the yTBP-induced DNATATA bend angles in solution complexes is in
direct contrast to the invariance of 11 crystalline
yTBPCTD-DNATATA complexes: all of these complexes, in
which the TATA sequence substitutions were identical or similar to those in
the solution studies, had essentially identical crystal structures with
80° DNA bend angles
(6). In solution, only
consensus TATA sequences are bent by yTBP as severely as in the crystal
structures in the absence of high osmolyte concentrations
(9). These functional and
structural differences highlight the fact that generalizations cannot
safely be made about the behavior and architecture of TBP-promoter complexes
between full-length and truncated proteins, between complexes in solution and
in crystals, or among TBPs from diverse species and emphasize the need for
clear comparative studies.
To directly address this issue, we have expanded our biophysical studies to include the interaction of full-length wild-type human TBP with promoter DNA. The work reported herein includes the binding affinity and kinetics of hTBP with DNA bearing the adenovirus major late promoter (AdMLP) and is, to our knowledge, the first published kinetic data obtained in real time for this interaction. In addition, we estimate the bend angle induced in this consensus sequence by hTBP in solution. Our results show that hTBP rapidly and simultaneously binds and bends promoter DNA and induces a bend angle even larger than that induced by yTBP. This reaction is broadly comparable with those with the yeast protein, although the mechanistic details clearly differ, and the distortion in the helical trajectory appears to be significantly larger in the human protein-DNA complex.
Our results differ sharply with the inferences drawn from the
electrophoretic mobility shift assay studies of Zhao and Herr
(19). They recently proposed a
model in which full-length wild-type hTBP binds initially to consensus
DNAAdMLP to yield an unstable complex having unbent DNA, with
introduction of the helical bend occurring only after 30 min. The
significant differences between their results and ours are discussed.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Recombinant human TBP, containing only the native amino acid sequence, was
expressed in the Escherichia coli strain BL21(DE3) and purified, with
slight modifications, as described
(20). In the purification,
non-ionic detergents were omitted, thus avoiding any potential complications
from abnormal association/aggregation, such as those seen previously with
yeast TBP (21). Protein purity
was analyzed using SDS-PAGE with Coomassie stain, and the mass of the protein
was determined using matrix-assisted laser desorption ionization/time of
flight mass spectrometry. The protein activity was determined as described
(8) using a fluorescence assay
with stoichiometric concentrations of protein and DNA. Briefly, equimolar
concentrations of duplex DNA and protein were mixed at concentrations
>>Kd and the fractional saturation determined
from the steady-state spectra. The ratio of the observed to predicted binding
gave the fraction of active protein. The concentrations reported are for
active protein. All experiments were conducted using 800 nM
protein, concentrations at which protein dimerization is
negligible.2 All
reactions were conducted at 20 ± 0.05 °C in 10 mM Tris,
pH 7.4, 100 mM KCl, 1 mM CaCl2, 2.5
mM MgCl2, 5 mM dithiothreitol, 5
µM leupeptin, and 10% glycerol.
Theory of FRET—Förster developed the basic theory for the phenomenon of the resonant transfer of energy between a suitable donor fluorophore and acceptor chromophore (22–24). Stryer and Haugland (25) elegantly confirmed these principles experimentally and Webber advanced the ideas, techniques, and instrumentation for applications to biomolecular systems (reviewed in Ref. 26). An extensive body of literature established since that time has further addressed FRET theory and its application in myriad biomolecular systems. Very briefly, quantum mechanical coupling of the electronic transitions in two fluorophores (reflected in the overlap of the donor emission and acceptor absorption spectra) permits excited state energy in the donor molecule to be transferred directly to the excited state of the acceptor molecule without emission and re-absorption of a photon. The probability of such transfer depends on the extent to which the transition moments of these two molecules assume the appropriate relative orientations. The rate of the energy transfer process depends on the inverse sixth power of the distance separating the two dyes, conferring an exquisite dependence of the transfer rate on the inter-dye distance.
The extent to which energy is transferred is readily observable in the fluorescence emission spectra of donor and acceptor dyes attached to biomolecules; efficient energy transfer decreases the emission intensity of the donor and increases that of the acceptor. We have published detailed discussions of FRET as it is used in the present work, utilizing DNA labeled with 3'-fluorescein (donor) and 5'-TAMRA (acceptor), in a number of related studies (4, 7–11, 27–29).
Instrumentation, Data Acquisition, and Analysis—Steady-state spectra were obtained using a Photon Technologies, Inc. (Lawrenceville, NJ), model A-1010 steady-state fluorimeter together with a Coherent (Santa Clara, CA) model INNOVA 70–4 argon ion laser tuned to 488 nm with the power set at 15–20 milliwatts. DNA duplex was formed using 8 nM T*ML*F with 5x complement, shown in separate experiments to yield full hybridization of the labeled strand. The titration was conducted using seven concentrations of hTBP ranging from 0 to 175 nM. Each successive titration point was obtained using a new duplex preparation in order to minimize stirring with protein present. Spectra were collected from 500 to 600 nm and base line corrected at each hTBP concentration. Determinations of the corresponding fractional DNA saturation, the equilibrium association constant (Ka), and the titration end point have been described (7).
Fluorescence stopped-flow data were collected and analyzed exactly as
described (7,
10,
11) using 20 nM
T*MLdpx*F and 140 nM hTBP (concentrations after mixing).
Briefly, a customized stopped-flow device having a 1.8-ms dead time was used
to collect a total of 2000 points/curve over 400 s with the time constant
= 12.24 ms and the fluorescein emission isolated using a 520-nm interference
filter. Replicate curves were corrected for background and very well fit to a
tri-exponential decay model. The slowest phase, which has been shown to
correspond to fluorescein photobleaching
(4,
7), was extracted, and the
resulting decay parameters were averaged.
Previous mechanistic studies using yTBP were conducted in buffer identical to that in the present study but without 10% glycerol. To ensure an unambiguous comparison between the hTBP results and those obtained previously using yTBP, the binding isotherm and stopped-flow kinetics were re-collected for the yTBP-T*MLdpx*F interaction in the presence and absence of 10% glycerol using 200 nM yTBP.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
38 kDa
(Fig. 1), consistent with that
of the native protein. The mass obtained from matrix-assisted laser desorption
ionization/time of flight spectral analysis confirmed that the purified
protein was full length. The fluorescence assay showed the DNA binding
activity to be 
70%, slightly lower than the activity of the yTBP
determined previously using the same assay and independently confirmed by
DNase footprinting. A full characterization of the protein will be reported
elsewhere.2
|
The 14-bp duplex DNA ("T*MLdpx*F," top strand TAMRA-5'-CGCTATAAAAGGGC-3'-fluorescein) and similar probes have been used extensively in our studies with yTBP (4, 7–11, 27, 29). The fluorescence emission from the two dyes directly signals changes in the distance separating the TAMRA and fluorescein dyes, due to the corresponding changes in FRET (Fig. 2), and has been shown in our previously published studies to be highly sensitive to changes in the 5'–3' distance as the duplex is bound and bent by TBP (details in the legend for Fig. 2).
|
Steady-state spectra are shown in Fig. 2 for T*MLdpx*F alone in solution (A) and hTBP-bound (B). The latter was obtained within 1 min following addition of the hTBP to the DNA. The dramatic difference in the signal between the unbound and bound DNA derives from a large decrease in the distance between the 5'-TAMRA and the 3'-fluorescein and has been shown to directly reflect the extent of helical bending induced by the protein (8, 9). Both spectra were monitored for an additional 60 min and yielded superimposable spectra over this period (Fig. 2, A and B). This observation is particularly important for the TBP-DNA complex (Fig. 2B) because it demonstrates the invariance of the conformation of the hTBP-bound duplex throughout a 1-h period. These spectra are sufficiently sensitive to changes in the DNA structure that changes of even a few degrees in the helical bend of the protein-bound DNA can be precisely distinguished (28, 29). In addition, such superimposable spectra reflect the stability of the DNA and protein preparations.
Titrations of T*MLdpx*F with hTBP were conducted using
steady-state emission spectra. The fractional DNA saturation determined from
the spectral changes observed at each point was used to construct the
equilibrium binding isotherm (Fig.
3). These data reveal the high affinity of the human protein for
this duplex, with a dissociation equilibrium constant = 5 nM at 20
°C. By comparison, yeast TBP binds the AdMLP sequence with
Kd = 21 nM
(10), a value confirmed in the
present study to be independent of the presence or absence of 10% glycerol.
The percent decrease in the ratio of the peak emission intensities at full DNA
saturation was calculated to be 60%. This spectral change upon hTBP
binding is significantly larger than the 44 ± 1.5% decrease obtained
with yTBP binding to the same labeled DNA duplex. The corresponding control
experiments show that the emission of the donor-only labeled duplex,
MLdpx*F, is invariant for the unbound and TBP-bound duplex for both
the human and yeast proteins. The implications of these observations for
helical bending are discussed below.
|
The kinetic trace of hTBP binding to the AdMLP duplex was monitored using
fluorescence stopped-flow (Fig.
4, closed cir-cles). In these measurements, the change in
the emission intensity of the fluorescein peak is monitored as a function of
time following rapid addition and mixing of hTBP and T*MLdpx*F
(dead time <2 ms). This association curve shows the real time course of the
process in which the DNA population shifts from being completely unbound and
free in solution to being nearly fully engaged in the hTBP-DNA complex.
Several features of this trace are notable. First, at the completion of the
reaction, the decrease in the fluorescein intensity is in excellent agreement
with that predicted from the steady-state spectra (Figs.
2 and
3), showing consistency among
the data in Figs. 2,
3,
4 and confirming that
essentially the full DNA conformational change has been captured in this
reaction curve. Second, the "overall" half-time for this
association reaction is 5 s. Third, this curve displays the
T*MLdpx*F being bound and bent by native hTBP in real time, with no
observable lag phase. Taken together, these observations provide direct
evidence that full bending is achieved concurrently with binding and is
complete within 150 s. Finally, the association curve is multiphasic. A
comparison of the theoretical curves obtained using
mono-(Fig. 4, dotted
line) and bi-exponential (Fig.
4, solid line) decay models emphasize this point. Such
multiphasic decay is similar to the pattern seen for the binding of yTBP to
AdMLP (Fig. 4, inset,
dashed line) and to numerous other promoters.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
With yTBP, even single base pair substitutions in the TATA sequence result in significant differences in the binding isotherms, kinetics (7, 10, 11), and the degree of the TBP-induced DNA bend angle in solution (9, 11). These differences reflect unique recognition pathways. The three yTBP-DNATATA interactions examined in most detail differ in the kinetics and thermodynamics of the partial reaction steps and in the concomitant uptake or release of salt and water. We have proposed that these variations may derive in part from differences in the inherent flexibility of the sequences and the corresponding compatibility along the protein-DNA interface. However, the common theme remains that yTBP-promoter recognition proceeds with simultaneous TBP-DNA binding and helical bending to rapidly form very stable complexes.
The central finding of this work is that full-length wild-type human TBP rapidly binds to and concurrently bends the AdMLP consensus promoter, forming a stable hTBP-DNA complex. This result is consistent with the results that we have obtained for native yeast TBP binding consensus and variant TATA sequences, which show multiphasic association occurring with overall rates generally comparable with those obtained herein and which show binding and bending to be inseparable processes (4, 7, 10, 11). The multiphasic kinetics have been shown for the yTBP-DNATATA interactions to arise from the presence of at least two intermediate species, both of which have fully bent DNATATA. Notably, the two phases observed during the course of TBP-DNA association do not reflect partial bending of the DNA followed by completion of bending; rather, the ordinate in Fig. 4 reflects a progressive increase in the "average" bend angle for the entire DNA population as unbound duplex binds to and is bent by the TBP.
We have previously determined bend angles for seven yTBP-bound TATA
sequences from the fluorescence emission decays of the dye-labeled DNA
duplexes, resolved in the nanosecond time regime
(8,
10). Time-resolved decays for
the fluorescein donor in the absence and presence of TAMRA acceptor are used
to obtain the mean 5'–3' distance, for both the unbound and
TBP-bound duplex, from which model-dependent bend angles may then be
calculated. The present study employs one of these same probes, and it is
possible to combine the time-resolved yTBP-AdMLP data with the steady-state
hTBP-AdMLP data to estimate a bend angle for the latter; we know the mean
end-to-end distance of unbound T*MLdpx*F and also that the width of
this inter-dye distance distribution, 
, is nearly invariant for unbound
and TBP-bound duplex (8).
Furthermore, both the time-resolved and steady-state emission of the duplex
labeled only with the donor fluorescein, MLdpx*F, remain unchanged
upon addition of saturating amounts of hTBP. Consequently, the difference in
the steady-state emission of T*MLdpx*F unbound and hTBP-bound may
be attributed to a change in the inter-dye distance due to increased FRET with
bending. Finally, we know the steady-state intensity changes for this duplex
upon yTBP and hTBP binding are 44
(7) and 60%, respectively.
Taken together, this information is sufficient to reliably estimate a
12.2-Å decrease in the mean inter-dye distance on binding to hTBP. This
value leads to a 102° bend, based on a bending model having two kinks
(detailed in Ref. 8)
corresponding to the up- and downstream phenylalanine insertion sites. This
bend is significantly greater than the 80° bend induced in the same
duplex by the yeast protein (7,
10), and is to our knowledge
the most severe that has been determined for TBP-induced bending. Confirmation
of this bend estimate will come from time-resolved measurements, from which
highly precise inter-dye distances can be obtained using extensive ensemble
averaging (8,
9,
28).
Aspects of the hTBP-DNAAdMLP interaction reported herein differ from the corresponding interaction with yTBP; hTBP binds the AdMLP promoter sequence more tightly and more rapidly, induces a more severe helical bend, and the details of the kinetic traces differ. Overall, however, the human system appears to be a variation on the yeast theme; binding and bending occur simultaneously and rapidly, with no lag phase, to yield a stable binary complex having severely bent DNA. This finding is not surprising, since the C-terminal DNA binding domains of the human and yeast proteins are 81% homogeneous, with all of the DNA-contacting side chains conserved absolutely except for one conservative substitution of Lys-110 in yTBP to Arg-204 in hTBP. The observed differences must therefore derive from non-contact amino acids or, a more probable hypothesis, from differences in the non-conserved N-terminal domains.
In contrast to the results reported herein, Zhao and Herr
(19) have recently published
data and proposed a model for the hTBP-promoter interaction that suggests
remarkably different mechanisms for promoter recognition by the human and
yeast TBPs. The disparity between their conclusions and ours is sufficiently
striking to warrant careful consideration. Zhao and Herr
(19) have reported that
full-length, wild-type human TBP initially binds to the AdMLP promoter to
yield an unstable complex having unbent DNA, followed by a very slow
process of DNA bending occurring over 30 min to render a stable complex.
A model is proposed, based on those data, in which native hTBP first binds to
but does not bend promoter DNA. The reaction then proceeds along one of two
pathways: either this hTBP-DNA complex independently undergoes an 30-min
isomerization process during which the bound DNA undergoes bending, or the
general transcription factor TFIIB binds to this hTBP-DNA complex "to
induce DNA bending." One additional pathway in their model shows TFIIB
binding directly to the hTBP-DNA complex after the isomerization to the bent
DNA conformation is complete.
The implications of such a mechanism for cellular functioning in humans are
significant. In this model, formation of the hTBP-DNAunbent complex
is, along one reaction pathway, followed by the very slow introduction of a
helical bend. Because the latter is a first-order process, the rate of this
bending would be independent of the relatively high reactant concentrations in
the cell and would presumably proceed at the rate reported by Zhao and Herr
(19), over the course of
30 min. The TBP-DNA complex having bent DNA is almost certainly the
transcriptionally active form, yielding the architectural scaffolding
necessary for the assembly of subsequent transcription factors. The proposed
isomerization process would therefore be, overwhelmingly, the rate-limiting
step in formation of hTBP-DNAbent along this pathway.
Realistically, any hTBP thus bound would be effectively trapped and
unavailable for nucleating assembly of the transcription machinery. The
pathway in which TFIIB binds to hTBP-DNAbent can likewise
reasonably be neglected since this complex would be available for assembly of
the pre-initiation complex only after 30 min following the initial
binding of hTBP. This kinetic trapping would eliminate such complexes from
participating in the cellular response to the need for synthesizing
protein.
Along the other proposed reaction pathway, TFIIB would bind to hTBP-DNAunbent and induce a helical bend. The hTBP-DNATATA co-crystal structure shows that the bend in the TATA sequence results from the partial intercalation of pairs of phenylalanine residues of the hTBP into the DNA helix, with the minor groove of the DNA fully interfaced with the protein-binding site. In addition, we know from crystallography that the conformation of the TBPCTD is very similar before and after DNA binding. Furthermore, the TFIIB contacts the DNA helix both upstream and downstream of TBP, contacts facilitated by the TBP-induced bending of DNA. These facts taken together make it difficult to envision a mechanism whereby TFIIB could induce the DNA bend in a pre-formed TBP-DNAunbent complex. In addition, we observe stable complexes with fully bent DNA-TATA within 150 s of the addition of relatively low concentrations of hTBP in the absence of TFIIB. A model in which hTBP-DNAunbent complexes are kinetically trapped in a transcriptionally inactive form unless and until TFIIB binds and induces a bend in the DNA is thus called into question.
Zhao and Herr (19) cite
precedent for this slow-bending model with a previously proposed two-step
mechanism for S. cerevisiae TBP-DNA binding
(30). This comparison is
inaccurate. First, Hoopes et al.
(30) reported formation of a
stable yTBP-DNATATA complex with an association half-time of 1
min. This result is in good agreement with our data but inconsistent with that
reported by Zhao and Herr (19)
for hTBP. Second, Hoopes et al.
(30) stated specifically that
their data were not consistent with a process that included a rate-limiting
isomerization step. Finally, the data that supported this yTBP model have been
shown to be more appropriately interpreted as simply formation of a transient
encounter complex leading to successful binding rather than as a true two-step
process (7). In fact, the
hTBP-DNA data reported by Zhao and Herr
(19) differ strikingly from
the data obtained for yTBP-DNA by both Hoopes et al.
(30) and our laboratory, and
their proposed model differs fundamentally from the models for yTBP
recognition of three TATA sequences proposed previously by our laboratory
(4,
7,
10,
11).
Several observations raise concerns about the results upon which the Zhao
and Herr model is predicated. An inspection of their published gel results
shows that only a very small fraction (1–5%) of the native hTBP
used in these experiments was active in DNA binding (see Figs. 1–7 in
Ref. 19). A dissociation
equilibrium binding constant (Kd) can be
calculated from this fraction of binding and the reported concentrations,
yielding a Kd value between 0.5 and 2
µM. This value reflects binding affinity that is about 3 orders
of magnitude lower than ours and is in the range usually associated with
nonspecific binding. Such low affinity raises the possibility that the
DNA-binding properties of the small fraction of active hTBP present in those
experiments may have been compromised. Such altered functionality might also
be expected to alter the ability of the protein to induce helical bending,
since we know of no example of such low affinity binding that results in bent
DNA.
One potential area of agreement between our results and those of Zhao and Herr (19) involves their mutant hTBPs. They identified a group of hTBP molecules having single residue substitutions that rapidly bound and bent promoter DNA, behavior that is in good agreement with our data for wild-type hTBP. One possible explanation for this result is that the non-native residues remaining at the thrombin cleavage site of their GST fusion interacted adversely with the wild-type but not mutant TBP. If their mutations were in fact functionally innocuous, these modified proteins would then display normal DNA binding/bending activity. Whatever the explanation, it is clear from their results that their mutant hTBPs show much higher DNA binding activity and form far more stable complexes with DNA than does their native hTBP.
The data presented herein show unambiguously that native hTBP binds to and concurrently introduces a severe bend in T*MLdpx*F in a reaction that is in general accord with that of yTBP. The agreement between the stopped-flow and steadystate amplitudes shows that the processes of binding and bending are coincident, a particularly clear result since MLdpx*F shows no change. Thus, the concomitant binding and bending of DNA by TBP appear as a common theme in promoter recognition.
However, the differences in affinity and in the stopped-flow curves for hTBP and yTBP binding to AdMLP, together with the increased severity of the hTBP-induced bend, suggest that the recognition mechanism of these two proteins differ. Because the multiphasic association kinetics for the hTBP-AdMLP reaction are reminiscent of those for the yeast protein, it seems reasonable to hypothesize that the hTBP-DNA recognition pathway will include formation of at least one intermediate species. A detailed description of each partial reaction along this pathway requires collection and analysis of extensive thermodynamic and kinetic data sets, work that is now beginning. Because our investigations with human and yeast TBPs use the identical labeled promoter DNA and the wild-type, full-length proteins, these solution studies yield a detailed and dependable comparison of the way in which these two proteins recognize a consensus TATA sequence.
|
FOOTNOTES |
|---|
To whom correspondence may be addressed. Tel.: 402-472-5765; Fax:
402-472-9402; E-mail:
kparkhurst1{at}unl.edu.
|| To whom correspondence may be addressed. Tel.: 402-472-3501; Fax: 402-472-9402; E-mail: lparkhurst1{at}unl.edu.
1 The abbreviations used are: DNATATA, DNA bearing a TATA box
sequence; TBP, TATA-binding protein; CTD, C-terminal domain; y, yeast
(Saccharomyces cerevisiae); h, human; a, A. thaliana; FRET,
Förster/fluorescence resonance energy transfer; AdMLP, adenovirus major
late promoter; TFIIB, transcription factor IIB; T*ML*F, 14-base DNA oligomer
bearing the AdMLP TATA sequence double-labeled with 5'-TAMRA and
3'-fluorescein; TAMRA, carboxytetramethylrhodamine; dpx, duplex.
2 M. Daugherty, manuscript in preparation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. L. Williams, L. K. Parkhurst, and L. J. Parkhurst Changes in DNA bending and flexing due to tethered cations detected by fluorescence resonance energy transfer Nucleic Acids Res., February 14, 2006; 34(3): 1028 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gilfillan, G. Stelzer, E. Piaia, M. G. Hofmann, and M. Meisterernst Efficient Binding of NC2{middle dot}TATA-binding Protein to DNA in the Absence of TATA J. Biol. Chem., February 18, 2005; 280(7): 6222 - 6230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. B. Hopkins and N. O. Reich Simultaneous DNA Binding, Bending, and Base Flipping: EVIDENCE FOR A NOVEL M.EcoRI METHYLTRANSFERASE-DNA COMPLEX J. Biol. Chem., August 27, 2004; 279(35): 37049 - 37060. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
