SRY and Architectural Gene Regulation: The Kinetic Stability of a Bent Protein-DNA Complex Can Regulate Its Transcriptional Potency
Etsuji Ukiyama1,2,
Agnes Jancso-Radek2,
Biaoru Li,
Lukasz Milos,
Wei Zhang,
Nelson B. Phillips,
Nobuyuki Morikawa,
Chih-Yen King,
Ging Chan,
Christopher M. Haqq,
James T. Radek,
Francis Poulat,
Patricia K. Donahoe and
Michael A. Weiss
Pediatric Surgical Research Laboratories (E.U., C.M.H., N.M.,
P.K.D.) Massachusetts General Hospital Harvard Medical
School Boston, Massachusetts 02114
Department of
Biochemistry (A.J.R., B.L., L.M., N.B.P., C.-Y.K., G.C., J.T.R.,
M.A.W.) Case Western Reserve University Cleveland, Ohio
44106-4935
Institut de Genetique Humaine (F.P.) UPR
Centre Nationale de la Recherche Scientifique 1142
Montpellier 34396, France
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ABSTRACT
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Protein-directed DNA bending is proposed to
regulate assembly of higher-order DNA-multiprotein complexes
(enhanceosomes and repressosomes). Because transcriptional
initiation is a nonequilibrium process, gene expression may be
modulated by the lifetime of such complexes. The human
testis-determining factor SRY contains a specific DNA-bending motif,
the high-mobility group (HMG) box, and is thus proposed to
function as an architectural factor. Here, we test the hypothesis that
the kinetic stability of a bent HMG box-DNA complex can in itself
modulate transcriptional potency. Our studies employ a cotransfection
assay in a mammalian gonadal cell line as a model for SRY-dependent
transcriptional activation. Whereas sex-reversal mutations impair
SRY-dependent gene expression, an activating substitution is identified
that enhances SRYs potency by 4-fold. The substitution (I13F in the
HMG box; fortuitously occurring in chimpanzees) affects the motifs
cantilever side chain, which inserts between base pairs to disrupt base
pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA
complex to an extent similar to its enhanced function. By contrast,
equilibrium properties (specific DNA affinity, specificity, and
bending; thermodynamic stability and cellular expression) are
essentially unchanged. This correlation between potency and lifetime
suggests a mechanism of kinetic control. We propose that a locked DNA
bend enables multiple additional rounds of transcriptional initiation
per promoter. This model predicts the occurrence of a novel class of
clinical variants: bent but unlocked HMG box-DNA complexes with native
affinity and decreased lifetime. Aromatic DNA-intercalating agents
exhibit analogous kinetic control of transcriptional elongation whereby
chemotherapeutic potencies correlate with drug-DNA dissociation rates.
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INTRODUCTION
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Endocrine systems have long demonstrated the complementary
importance of kinetic and equilibrium principles. Gonadotropins, for
example, exist in the bloodstream as trapped heterodimers: despite weak
binding of
- and ß-subunits in vitro, dissociation of
the functional glycoprotein heterodimer is slow relative to its
physiological lifetime (1). Further, insulin analogs of similar
affinity for the insulin receptor have been observed to exhibit
different mitogenic potency. Although the postreceptor signaling
apparatus is incompletely understood, the overall strength of
insulins signal for DNA synthesis correlates with the lifetime
of the hormone-receptor complex rather than its fractional occupancy
(2, 3). In this paper we investigate whether the principle of kinetic
regulation can extend to the function of an endocrine transcription
factor. A model is provided by human SRY, the testis-determining factor
encoded by the Y chromosome (4, 5). Our strategy, uncovered in the
course of investigating mutations in SRY (6, 7), is similar to that
employed in studies of insulin (2, 3). We compare SRY analogs with
similar specific DNA affinities but differing kinetics of DNA
association and dissociation. Prolonging the lifetime of a bent
protein-DNA complex is shown to correlate with enhanced potency in a
cotransfection model of SRY-dependent transcriptional activation (6, 8). Our results suggest that the kinetic stability of component DNA
bends can regulate the ability of an enhanceosome to support multiple
rounds of transcriptional initiation (Fig. 1
A). To our knowledge, this is the first
report of possible kinetic regulation by a specific transcription
factor.

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Figure 1. Structure and Proposed Function of SRY as an
Architectural Transcription Factor
A, Enhancesome model of architectural gene regulation by SRY.
Right, activated transcription occurs in the presence of
bound SRY as specific DNA bend permits assembly of stable
activator-coactivator-basal preinitiation complex (enhanceosome; Ref.
27). Asterisk (red) indicates site of cantilever
insertion. Left, activated transcription is off in the
absence of bound SRY due to disassembly of DNA-multiprotein complex and
dissociation of activator-coactivator complex. We postulate that the
kinetic lifetime of the SRY-DNA complex regulates the overall lifetime
of the enhanceosome and thus its ability to support multiple rounds of
transcriptional initiation. B, Ribbon model of complex between SRY HMG
box (magenta) and 8-bp DNA site (green; Ref. 10).
The four hydrophobic wedge side chains (10 ) are also shown in
red (top to bottom: W43, F12, I13, and M9).
Asterisk indicates site of I13 insertion. C, Ribbon model of
SRY HMG showing selected side chains and sites of
sex-reversal-associated mutations (red). The residue
numbering scheme refers to intact human SRY (41 ) and not the HMG-box
consensus (10 ). Asterisks indicate F12 and I13 (F67 and I68 in the intact human sequence). D,
Sequences of HMG boxes (HMG-box residues 646) highlight conservation
of wedge side chains in helix 1 (boxes). First group
represents SRY and Sox alleles; middle group, other sequence-specific
domains; and bottom group, non-sequence-specific domains. Residue
numbers at bottom refer to HMG box consensus (10 ). Conserved
wedge side chains are shown in red, and substitutions in
blue. E, F12 and I13 (yellow) contact DNA between
successive AT bp (5'-ATTGTT; underlined).
Coordinates obtained from Ref. 10. F, Crystal structure of TBP (16 17 ). Paired Phe-Phe elements are shown in red and
yellow. Whereas the TBP saddle inserts two Phe side chains
(one in each lobe as shown in yellow in Fig. 1 E; Refs. 16
and 17), human SRY employs a single Ile (I13).
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SRY contains a high-mobility group (HMG) box (9), a
conserved motif of minor-groove DNA recognition. The structure of a
specific DNA complex exhibits a sharp DNA bend (Fig. 1
B; Refs. 10, 11, 12).
Mutations in the HMG box are associated with 46,XY pure gonadal
dysgenesis and sex reversal (sites highlighted in Fig. 1
C;
Refs. 6, 10, 13, 14, 15). The bound DNA structure, in part resembling
A-DNA, is similar to that induced by the TATA-binding protein TBP (Fig. 1
F; Refs. 16, 17). DNA bending is in each case associated with
insertion of a nonpolar cantilever side chain between base pairs (Fig. 1
E). Insertion disrupts base stacking but not base pairing (18). Human
SRY contains Ile at the cantilever position, residue 13 of the HMG box
consensus sequence. Cantilever side chains otherwise conserved among
SRY and related SOX sequences are Phe or Met (Fig. 1
B). Met occurs in
Lef1/Tcf1 HMG boxes (12) where its role is similar to that of I13 in
SRY (10). The inserted side chain is in each case flanked by a Phe,
which abuts the DNA backbone (Fig. 1
E). Substitution of either side
chain in SRY by Ala abolishes specific DNA-binding activity (19). A
mutation in SRY leading to a foreshortened and polar cantilever (I13T)
is associated with XY sex reversal and impairs specific DNA binding
(6). The variant cantilever accelerates dissociation of the I13T
protein-DNA complex by at least 50-fold leading to a proportionate
reduction in its association constant. These impaired kinetic and
equilibrium properties may each contribute to the failure of testicular
differentiation. Analogous partial intercalation occurs among diverse
protein-DNA complexes (20, 21, 22, 23, 24, 25, 26). Protein-directed DNA bending is
proposed to facilitate assembly of DNA-multiprotein preinitiation
complexes [enhanceosomes and repressosomes (27, 28)] giving rise to
architectural gene regulation (29, 30).
Experimental distinction between kinetic and equilibrium mechanisms
requires identification of amino-acid substitutions that affect one
property but not the other. Such substitutions are rare because the two
properties are linked: the equilibrium association constant
(Ka) is equal to the quotient between rates of
association and dissociation
(kon/koff). Studies of
insulin signaling thus used substitutions that confer
compensating changes in rates of receptor binding and
release (2, 3). To find such a compensating substitution in SRY, we
were guided by an analogy between the HMG box and the chemistry of DNA
intercalating agents (31). The latter are organic molecules containing
aromatic moieties; examples include daunorubicin, doxorubicin, and
adriamycin, agents widely employed in cancer chemotherapy (32, 33, 34, 35, 36, 37, 38, 39, 40).
Three considerations informed our structural intuition. First, these
drugs insert into DNA to disrupt base stacking but not base pairing, a
process reminiscent of insertion of a cantilever side chain (31, 39, 40); second, intercalative agents exhibit a wide range of rate- and
association constants, modulated in part by the structure and number of
aromatic intercalating moieties (32, 33, 34, 35); third, and most important,
transcriptional blockade in vitro and cytotoxicity in
vivo correlate with dissociation rates of drug-DNA complexes
(36, 37, 38). Kinetic regulation of transcriptional elongation
by a DNA-binding agent suggests the possibility of analogous kinetic
regulation of transcriptional initiation.
Would an aromatic cantilever in a bent SRY-DNA complex, like that in a
chemotherapeutic agent, modulate its kinetic and functional properties?
To test this possibility, we exploit a fortuitous experiment of nature:
the only difference between the SRY HMG boxes of human beings and
chimpanzees is the cantilever substitution I13F (41, 42). Comparison of
these HMG boxes demonstrates that the I13F substitution enhances
SRY-directed expression of a reporter gene; such expression is absent
in control studies of I13T and I13A variants. Despite the I13F
variants enhanced activity, the substitution appears not to alter the
structure, DNA-binding, or DNA-bending properties of the SRY HMG box in
accord with a previous study (42). However,
1H-nuclear magnetic resonance (NMR) studies of
dissociation kinetics indicate that the lifetime of an F13 complex is
prolonged relative to that of an I13 complex. Consistent with studies
of transcriptional blockage by DNA intercalative drugs, the extent of
kinetic stabilization is similar to the extent of transcriptional
enhancement. Accordingly, we suggest that an aromatic cantilever more
effectively "locks" the bent SRY-DNA complex in a specific
transcriptional preinitiation complex (Fig. 1
A). This model predicts
the occurrence of a novel class of clinical variants: bent but unlocked
HMG box-DNA complexes with native affinity but decreased lifetime.
Kinetic control of the assembly or disassembly of macromolecular
complexes may be a general feature of regulation in diverse biological
settings.
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RESULTS
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Transcriptional Activation Assay
Because genetic targets of SRY are unknown, we developed a
transcriptional assay in a cell line derived from the 14-day rat
urogenital ridge [i.e. the waning edge of SRYs site and
stage of expression, 1214 days post conception (6)]. This assay
employs full-length human SRY and uses the Müllerian inhibiting
substance (MIS) promoter (43) as a heterologous reporter for
transcriptional activation. Cotransfection with a plasmid expressing
wild-type SRY leads to a 11-fold induction of transcriptional activity
as monitored by a luciferase reporter (Table 1
; lane k in Fig. 2
A). Such activation is specific to SRY
and its consensus target sequences [5'-ATTGTT-3' (44, 45, 46, 47)]
rather than to a structure-specific DNA-binding activity as indicated
by the inactivity of human HMG2; further, architectural transcription
factors with different DNA sequence specificities [Lef-1 and IRE-ABP
with consensus sequences 5'-TTTGAA-3' (12, 44)] are only
weakly active in this assay (Fig. 2
A). Analysis of deletions and
base substitutions in the MIS promoters HMG-box binding
site [5'-TTTGTG; base pairs 48 through 56 relative to the start
site of transcription (47)] has shown that activation by SRY persists,
presumably through multiple 5'-ATTGTT-3' sites in the vector and
reporter.

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Figure 2. Cotransfection Assay for Transcriptional Activation
A, Fold-induction in luciferase activity is shown relative to a
control: lane a, cotransfection of the expression plasmid with an empty
vector (1 µg); lanes b and c, 0. 5 and 1.0 µg Sox-2; lanes d and g,
1 µg CDM8; lanes e and f , 0.5 and 1 µg IRE-ABP; lanes h and i, 0.5
and 1 µg Lef-1; and lane j, empty-vector control (1 µg). Ten-fold
enhancement is observed after cotransfection with wild-type SRY (lane
k; ref.). Error bars represent 2 SD
(n = 6). B, Comparison of wild-type, M9I, and I13F SRY variants
reveals attenuation of activation by M9I and enhancement of activation
by I13F. Error bars indicate 1 SD
(n = 6). See also Table 1 .
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Although the cotransfection assay does not reflect a physiological
effect of SRY on MIS regulation, it nonetheless provides a
model of SRY-dependent transcriptional activation. To correlate
structure and function, six substitutions in the SRY HMG box were
tested (Table 1
); assays were repeated at least three times to assess
statistical significance. Substitutions that reduce specific DNA
binding by 10-fold or more in vitro (M9A, F12A, I13A, and
I13T) exhibit no detectable transcriptional activation. A substitution
that impairs specific DNA binding by 2.5-fold (M9I; Ref. 15) exhibits
low but significant activity at high concentrations of vector DNA
(1.02.0 µg) whereas at lower concentrations any differences from
wild type are not significant. Because M9I and I13T are associated with
gonadal dysgenesis (10), human phenotypes (male or female) correlate
with maintenance or attenuation of transcriptional activity and,
in turn, with changes in specific DNA affinity (high or low).
Remarkably, the chimpanzee substitution I13F enhances transcriptional
activation by 40-fold, which is about 4-fold greater than that
stimulated by native SRY (Fig. 2
). No differences in protein expression
were detected in the transfected cells by Western blot using a murine
polyclonal antiserum specific for human SRY (Fig. 3
C). To investigate the origins of the
I13F variants enhanced activity, native and variant HMG boxes were
purified and characterized in vitro. Assays for DNA binding,
bending, structure, stability, and kinetics are described in turn
below.

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Figure 3. I13F Substitution Does Not Alter Specific DNA
Affinity or Base Specificity at Insertion Site
A, GMSA at 4 C. Complex 1 represents specific 1:1 complex with 36-bp
probe containing 5'-TGATTGTTCAG target site; complex 2
reflects DNA-dependent protein-protein association. Lanes ag show
binding of I13 SRY domain; lanes io show binding of I13F variant. The
free DNA is shown in lane h. Successive protein concentrations are as
follows: a and i, 2.5 nM; b and j, 5
nM; c and k, 10 nM; d and l, 20
nM; e and l, 40 nM; f and n, 80
nM; and g and o, 160 nM. B,
Comparison of variant 15-bp probes containing sequences
5'-ATTGTT, 5'-ATGGTT, 5'-ATAGTT, and
5'-ATCGTT (left panel). GMSA (right
panel) indicates that the I13 and F13 domains prefer T at this
position, tolerate A, and discriminate to a similar extent against G
and C at this position. The protein concentration is 75
nM. "X" indicates faster migrating complex
containing a proteolytic contaminant of F13 domain. C, Western blot
demonstrates that human SRY (lane 2) and I68F SRY (lane 3) are
expressed to similar extents on transfection in CH34 cells (upper
panel). Although the antibody may in principle cross-react with
rat SRY or other Sox domains, in the Western blot negligible
immunoreactive SRY or other proteins less than 40 kDa in molecular mass
are detected in an empty-vector transfection control (lane 1).
Lower panel shows GH control. In upper and
lower panels higher-molecular mass immunoreactive proteins
(>40 kDa) document that equal amounts of cell extract were loaded in
each lane.
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DNA-Binding Properties Are Similar
Human and chimpanzee domains exhibit indistinguishable
specific DNA binding by gel mobility-shift assay (GMSA) to a 36-base
pair (bp) duplex containing a central 5'-ATTGTT-3' consensus site (Fig. 3
A). In each case a higher-order complex is formed with increasing
protein concentration, previously shown to reflect a DNA-dependent
protein-protein interaction (48). The two domains exhibit similar GMSA
affinities at 4 and 20 C. Sequence specificity was investigated by
systematic GMSA shift of variant 15-bp probes (18) containing the four
possible base pairs 5' to the insertion site (5'-AXTGTT-3';
X = A, T, C, or G) or 3' to the insertion site
(5'-ATXGTT-3'; X = A, T, C or G). These studies
(illustrated in Fig. 3
B for the 3'-X site) demonstrate that
the I13F substitution does not alter nucleotide preference at either
DNA position. The I13F substitution likewise conferred no detectable
change in binding to a four-way DNA junction (data not shown). These
results strongly suggest that the enhanced transcriptional
activity of the I13F variant is not due to enhanced affinity for the
consensus SRY target sequence, variant sequences, or nonspecific
prebent DNA.
DNA-Bending Properties Are Similar
Because altered DNA bending may in principle be associated with
enhanced or decreased gene-regulatory activity independently of DNA
binding affinities (15), SRY-induced DNA bending was measured by
permutation gel electrophoresis (PGE) (Ref. 49 ; Fig. 4
). The DNA site was
5'-CCCATTGTTCTCT-3'. Human and chimpanzee domains exhibit
almost identical patterns of position-dependent electrophoretic
mobilities. Because interpretation can depend on gel composition,
experiments were performed with both 8% and 10% polyacrylamide.
Estimated DNA bends in the native complex are 80o
and 85o, respectively (inequivalent estimates
presumably reflect limitations of the PGE method). Such sharp bending
is consistent with prior biochemical studies (45). Binding of human and
chimpanzee domains yields similar patterns of flexure-dependent
electrophoretic mobilities.

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Figure 4. PGE DNA-Bending Assays Using SRY Target Site
5'-GAGCGCATTGTTATCA Using 8% Gel at 4 C
Lanes 16 contain native SRY domain and lanes 712 contain I13F
variant domain. Experiments were performed using 5'-fluorescein-labeled
probes for fluorescent scanning as described in Materials and
Methods.
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Quantitative interpretation of PGE mobilities suggests that I13F
substitution implies a small decrement (
2o) in DNA bend angle; this estimate is
unaffected by gel composition. Because PGE can be associated with
technical artifacts (50), however, SRY-induced DNA bending was also
evaluated by phase-modulation fluorescence resonance energy transfer
(FRET) (51). This technique employs a DNA probe containing a
fluorescent donor (fluorescein) at the 5'-end of one strand and
an acceptor (tetramethylrhodamine; TAMRA) at the 5'-end of the other,
each tethered by a flexible hexanyl linker. Bending is detected as
enhanced FRET efficiency due to a decreased end-to-end distance. The
frequency dependence of fluorescence phase and modulation enables donor
lifetimes to be obtained in the presence and absence of the acceptor
(Table 2
A). Such lifetimes permit
assessment of mean distance changes and modeling of distance
distributions (see Discussion).
Binding of the SRY HMG box causes marked changes in phase- and
modulation curves (Fig. 5
A). Similar
changes occur on binding of TBP to its DNA site (51). Data may be
fitted to a single mean lifetime or with improved fit to two discrete
lifetimes (Table 2
A). In the free DNA the mean lifetime of the donor in
the absence of acceptor is 4.44 nsec and is essentially unchanged on
protein binding. The presence of the acceptor leads to a reduction in
the donors mean lifetime to 2.56 nsec in the free DNA. Enhanced FRET
on protein binding yields mean lifetimes of 1.79 nsec in the native
complex and 1.81 nsec in the I13F complex. The difference (
0.02
ns) is in the same direction as implied by PGE but is within
experimental error. Corresponding FRET efficiencies are 0.42 (free DNA)
and 0.59 (each complex). Similar FRET efficiencies in the two complexes
imply similar mean induced DNA bends and similar distributions of DNA
bend angles. Although the extent of FRET enhancement is consistent with
a bend angle of 8085o (51), quantitative
interpretation is limited by possible confounding effects of DNA
unwinding.

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Figure 5. Phase-Modulation FRET Analysis of DNA Bending
A, Phase- and modulation curves for free DNA (red) and
native SRY HMG-box complex (blue). Phase curves are
indicated by crosses and modulation curves by
circles. B, Gaussian modeling of distance distributions
in free DNA (red) and native complex
(blue). Lifetimes are given in Table 2 A and gaussian
parameters in Table 2 B.
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Structures of Free and Bound Domains Are Similar
A combination of spectroscopic methods [circular dichroism
(CD), fluorescence, and 1H-NMR] was
employed to test whether the I13F substitution alters structure or
stability. The domains exhibit indistinguishable CD spectra in the far
ultraviolet, indicating corresponding secondary structures. Intrinsic
Trp fluorescence emission spectra likewise exhibit similar quantum
yields and emission maxima, probes of tertiary structure.
1H-NMR spectra of free domains are essentially
identical. In particular, aromatic and aliphatic spectral regions,
which contain sensitive probes for tertiary structure, exhibit
corresponding dispersion of chemical shifts and patterns of
differential resonance broadening. These residue- specific
similarities suggest that the domains structure and millisecond
dynamics are unaffected by the substitution.
Comparison of two-dimensional (2D) NMR spectra reveals the new F13
aromatic spin system, which exhibit properties expected of an
unhindered side chain at a protein surface: negligible secondary
shifts, motional narrowing, and absence of nonlocal nuclear Overhauser
effects (NOEs). Specific DNA complexes exhibit a similar correspondence
of NMR spectra (Fig. 6
). The
upfield methyl resonances of V5 and L46 provide probes for the
DNA-bound structure of the minor and major wings, respectively (10). As
expected, the unusual aliphatic cantilever resonances of I13 [shifted
upfield by the ring currents of flanking DNA base pairs (18)] are
absent in the variant complex. Together, the spectroscopic results
suggest that the enhanced activity of the I13F protein is not due to a
nonlocal change in the structure of the free or bound HMG box.

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Figure 6. I13 and F13 SRY-DNA Complexes Exhibit Similar 1D
1H-NMR Spectra at 500 MHz and 37 C
Upfield aliphatic spectra regions of native complex (A) and I13F
variant complex (B). Native and variant complexes exhibit similar
upfield shifts of the methyl resonances of V5 (induced fit of minor
wing; Refs. 68 and 69) and L46 (major wing). L46 is also shifted
upfield in free domains, indicating preformed structure of major wing.
Asterisk indicates anomalous upfield ß methylene
resonance of L46 due to ring current of W43 in core. I13 in native
complex is shifted far upfield due to ring currents of flanking base
pairs; this spin system is absent in spectrum of I13F variant.
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Stabilities of Free and Bound Domains Are Similar
Thermodynamic stabilities of native and I13F domains were
investigated by titration with denaturant guanidine hydrochloride. The
extent of unfolding was monitored by Trp fluorescence. The two domains
exhibit similar thermodynamic parameters as inferred from a two-state
model of an equilibrium between native and unfolded states (Table 3
).
Respective concentrations of guanidine at which 50% of the domain is
unfolded (Cmid; column 2 of Table 3
) are essentially identical. A slight
difference in free energies (
G
; column 4)
is suggested, but inferred differences are within experimental error
(
G
0.25 ± 0.26 kcal/mol). Thermal
stabilities, evaluated by measuring the temperature dependence of both
CD ellipticity (at helix-sensitive wavelength 222 nm) and Trp
fluorescence, are also similar (unfolding midpoints 40 ± 1 C and
42 ± 1 C, respectively; Table 1
). Because of the breadth of the
unfolding transition in the range 3050 C (47), fractional unfolding
at physiological temperature (37 C) would not differ significantly.
These in vitro results are in accord with the similar
biological expression stabilities of native and I13F variant SRY in
cell culture (see above).
I13 and F13 Complexes Exhibit Different Kinetic Lifetimes
NMR spectroscopy enables kinetic lifetimes of native and I13F
variant complexes to be estimated. The assay exploits the observation
that resolved DNA imino resonances exhibit large complexation shifts on
drug- or protein binding. Spectral changes can be slow, intermediate,
or fast on the time scale of 1H-NMR chemical
shifts (33, 34).
Imino resonances of a 15-bp DNA duplex containing a central
5'-ATTGTT-3' target site exhibit large changes in chemical shift on
specific binding of the SRY domain. Titration with the I13F domain
(spectra ce in Fig. 7
A) reveals that
free and bound DNA are in slow exchange on the
1H-NMR chemical-shift time scale as previously
described in the native complex (18). Arrows in Fig. 7
Ac
indicate discrete DNA imino resonances from a specific complex in
equilibrium with free DNA. Prominent is the anomalous position of the
T8 imino resonance, which is upfield among the guanine imino resonances
(Fig. 7
Ae). I13 and F13 complexes exhibit significant differences in
imino 1H-NMR signatures (Fig. 7
). These
differences are consistent with the ring current of an aromatic
cantilever: i.e. upfield differences are observed at
flanking thymidine imino and adenine H2 positions (hence above and
below the F13 aromatic ring), whereas smaller downfield differences
occur at the edge of the insertion site flanking (side-chain protons of
F12 and W43). This pattern implies planar (rather than perpendicular)
insertion of F13 between base pairs, consistent with the shape of the
cavity induced in the DNA and analogous to planar intercalative agents
(34, 39, 40). The slow-exchange condition provides a lower bound
on the lifetime of the bound state: slow exchange between resonances
separated by 0.1 ppm (40 Hz at a 1H-NMR resonance
frequency of 400 MHz) implies a lifetime greater than 25 msec.

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Figure 7. DNA Imino Resonances and Kinetics of Protein-DNA
Dissociation
A, 1H-NMR titration of 15-bp DNA duplex by I13F domain at
400 MHz; a and b, spectra of free DNA and native SRY-DNA complex; c and
d, addition of successive aliquots of I13F domain leads to appearance
of new set of imino resonances (arrows in c) in slow
exchange; and d, spectrum of 1:1 I13F variant complex. Assignments are
as indicated. The three indole NH resonances of tryptophan are as
labeled between 10 and 11 ppm. Asterisks in panel e
indicate insertion site of cantilever side chain (AT base pairs 8 and
9); asterisk in panel b indicates perturbation in W43 at
protein-DNA interface. Imino resonances of base pairs 1 and 15 are not
seen due to fraying; the resonances of base pairs 2 and 14 are
broadened. B and C, Chemical-exchange cross-peaks of equimolar mixtures
of free and bound DNA at 40 C at 500 MHz. A representative trace is
shown in each case to illustrate the ratio of diagonal resonance
intensity to cross-peak intensity; asterisks indicate
cross-peak. Smaller resonances in trace in panel B indicate neighboring
base pairs 11 and 9 in the bound state. Assignments of exchange
cross-peaks are as indicated. The DNA concentration in (A) was 2
mM in panel A and in (B) was 0.5 mM
in panel B.
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Slow exchange gives rise to cross-peaks in 2D NOESY spectra of
equimolar mixtures of free and bound DNA. Cross-peaks (obtained with a
mixing time of 200 msec as shown in Fig. 7
, B and C, for native and
variant complexes) connect the diagonal resonances of a given DNA
proton in the free and bound states. Representative traces
(inset in Fig. 7
, B and C) enable comparison of the relative
extent of exchange. Whereas in the wild-type complex the cross-peak
intensities are
65% of those of diagonal resonances, in the
variant complex their intensities are significantly lower (
1520%;
asterisks in Fig. 7
, B and C). Native exchange cross-peaks
are consistent with a lifetime similar to the NOESY mixing time whereas
the lifetime of the chimpanzee complex is prolonged 3- to 4-fold.
Gel-shift studies of the 33P-labeled 15-bp duplex
in NMR buffer indicate similar apparent binding of the two domains at
4, 20, and 37 C. In each case apparent binding is substantially weaker
than that observed using a 36-bp probe (see Fig. 3
, A and B). At 37
C and 100 nM protein concentrations, for example,
the percent shift is only 4 ± 1%. It is likely that the
intensity of the shifted bands is attenuated by rapid dissociation of
native and variant complexes on the time scale of electrophoresis.
A dissociation constant is the ratio of the rates of association and
dissociation. Because apparent affinities are similar, the prolonged
lifetime of the I13F domain indicates a similarly prolonged association
rate. Comparison with kinetic studies of a non-sequence-specific HMG
box [HMG1-A recognition of a cisplatin-DNA adduct (52)] implies that
sequence-specific DNA binding is significantly slower (both in
association and dissociation) than structure-specific DNA binding.
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DISCUSSION
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DNA-bending proteins containing an HMG box have attracted broad
interest due to their roles in mammalian organogenesis (53, 54, 55).
Because these proteins may lack discrete domains of transcriptional
activation or repression, such architectural transcription
factors are proposed to regulate gene expression in part through
changes in the structure of DNA or chromatin (29, 30). A model in which
introduction of a specific DNA bend enables cooperative assembly of a
DNA-multiprotein preinitiation complex is shown in schematic form in
Fig. 1
A. In this article we have compared SRY analogs to assess a
proposed chemical and functional analogy between the HMG box and
aromatic DNA-binding drugs (31). On intercalative DNA binding, such
drugs disrupt base stacking but seldom base pairing and often induce
features of A-DNA. Our strategy exploits a fortuitous experiment of
nature, the aromatic cantilever of chimpanzee SRY (F13; Refs. 41, 42). Although the equilibrium properties of I13 and F13 domains are
similar, an aromatic cantilever enhances potency in a model
transcriptional assay in association with enhanced kinetic
stability.
Phase-Modulation FRET Assay Implies Well Defined Bend Angle
Electrophoretic anomalies have been widely used as probes of DNA
bending. Interpretation of PGE may be confounded, however, by geometric
features of a protein-DNA complex (50). As a control for such
artifacts, we have employed a complementary FRET assay. FRET efficiency
reflects the distance between 5'-ends of the DNA, which is a function
of both DNA bending and DNA unwinding. A similar assay has been
described in studies of TBP (51). The present studies are in accord
with this and previous PGE studies of HMG-box complexes (15, 42, 45).
Electrophoretic- and FRET methods both indicate that the I13F
substitution has at most a small effect on induced DNA bending; a
decrement of 
2o as estimated by PGE is
within the error of the FRET measurements. Assuming no change in
orientation factor
2, corresponding mean FRET
distances are 58.9 Å ± 1 Å in the free DNA (using a measured
Ro value of 55.9 Å) and 51.1 Å ± 1 Å in the
complexes (using a measured Ro value of 54.3 Å).
The reduction in mean distance is hence about 8 ± 2 Å, which is
consistent with a bend angle of 8085o. The
single mean-lifetime and single mean-distance approximations neglect
the actual distribution of distances in solution that arise from
motions of the linkers, dyes, and DNA. These approximations are
nonetheless of qualitative value and have been successfully applied in
FRET studies of HMG-1a (52). Although the relationship between
end-to-end distance and bend angle is not straightforward (due to
variable DNA unwinding), the FRET observations corroborate PGE results
to demonstrate that the DNA-bending properties of the two proteins are
similar and so unlikely to be the source of the variants enhanced
transcriptional activity.
Neither PGE nor single-lifetime FRET analysis distinguishes between
static and dynamic changes in DNA bending. A mutation in a protein may
change the mean DNA bend angle, for example, without change in
fluctuations. Alternatively, a mutation may induce dynamic instability
or imprecision in DNA bending, leading to a larger distribution of
populated angles. By enabling an estimate of distance distributions,
FRET techniques can, in principle, provide insight into this
distinction (56). Thus, the present frequency-domain data of
donor-acceptor samples are much better fit by two discrete lifetimes
than by a single lifetime (Table 2
A).
These lifetimes can be interpreted according to a gaussian distribution
of distances (51, 56). This analysis, which also assumes that isotropic
orientation factor
2 equals 2/3, in each case,
suggests that binding of the I13 and F13 domains induces a 9 Å ± 1 Å
decrease in end-to-end distance (difference between <r> values for
free and bound DNA) with no significant change in widths of the
distributions (Fig. 5
B and Table 2
B). The widths are due predominantly
to reorientation of the linkers. These results are comparable to two
prior FRET studies of DNA bending induced by nonspecific HMG boxes (52, 57).
In the absence of an adequate cantilever side chain, would a variant
SRY-DNA complex be sharply bent and if so, would significant
fluctuations occur in bend angle? In the future this question may be
addressed through studies of additional analogs. It would be of
interest to find a variant cantilever sufficient to provide near-native
specific binding affinity (or equivalently, free energy
G) but
associated with nonlocal dynamic instability. The plausible existence
of such analogs arises from consideration of enthalpy-entropy
compensation (58): reduced enthalpy of side-chain insertion (
H) may
be compensated by the enhanced entropy of a fluctuating complex (
S).
Such analogs would enable the regulatory importance of precise and
stable DNA bending to be tested. A signature of such a variant complex
would be an anomalously broad distribution of end-to-end distances.
Mutation Enhances Transcriptional Potency and Kinetic Stability
The present study extends previous studies of SRY (13, 14, 15, 42) by correlating phenotypes and biochemical activities with
transcriptional regulation. Although the mechanism of SRY-dependent
transcriptional activation in our assay is unknown and not specific to
MIS, our vector-reporter system nonetheless provides a model
of ATTGTT-dependent transcriptional activation. Our study has
compared functional proteins (human SRY and I13F chimpanzee variant)
and nonfunctional proteins (clinical variants M9I and I13T and control
mutations F12A and I13A). Whereas residues I13 and F13 are each
compatible with the male phenotype in a primate, a polar and
foreshortened cantilever (I13T) or altered hydrophobic wedge (M9I)
blocks testicular differentiation (6, 10, 13, 14, 15). These and other
substitutions with decreased or undetectable specific DNA binding
exhibit decreased or undetectable SRY-dependent transcriptional
activation (Table 2
).
Because the chimpanzee SRY HMG box exhibits native DNA affinity,
bending, and nucleotide specificity at the insertion site, we
hypothesize that the variants enhanced activity in the cotransfection
assay is due to enhanced kinetic stability: the bent protein-DNA
complex is more stably maintained once formed in a preinitiation
complex. Physical evidence for slower dissociation (relative
to the native complex) is provided by 1H-NMR.
Quantitative implications for the kinetics of protein binding and
release may be estimated as follows. The change in barrier height
corresponding to a factor of 4 in on- or off rates
(
G+) would be modest (0.8 kcal/mol). A
lifetime of 200 msec at 40 C (consistent with the native NMR data)
would imply a dissociation rate of 5
sec-1. Assuming an
equilibrium constant of 50 nM under these
conditions, this would imply an association rate of
108
M-1sec-1.
This is 10-fold slower than the rate of association of HMG1 to a
prebent DNA cisplatin adduct at 25 C (koff 30
sec-1 and
kon 109
M-1sec-1;
Ref. 52). We propose that the rate-determining step in SRY binding
occurs in the pathway of HMG box-directed DNA reorganization and that
the protein cantilever participates in this step. The analogous kinetic
barrier in the adducted HMG1 domain complex is presumably reduced by
distortion of the free platinated DNA. Stopped-flow kinetic studies of
the binding of HMG-1a to cisplatin-modified DNA has recently shown that
although the association rate is near the diffusion limit, significant
differences in association rates (but not dissociation rates) are
observed when the DNA sequences are varied at the site of platination
(59). The present study, by contrast, highlights a difference in
dissociation rates. The slower dissociation rate for specific SRY-DNA
complexes is consistent with studies of lymphoid enhancer factor 1
(LEF-1) ( by competition kinetic GMSA (estimated lifetime 120 min or
koff 1.1 x
10-4
sec-1 at 20 C; 60).
Surface plasmon resonance studies of HMG1 and HMG2 (which each contain
two HMG box domains) indicate that bidentate binding to a 30-bp duplex
exhibits kinetic constants of 2 x 104
M-1
sec-1
(kon) and 67 x
10-2
sec1 (koff) (61).
Intriguingly, the prolongation of I13F variants lifetime is similar
to its extent of enhanced transcriptional activation. Although this
could be a coincidence, the absence of a plausible alternative
mechanism suggests a model of kinetic regulation in which the net
accumulation of mRNA is controlled not by the equilibrium constant of
the SRY-DNA complex but by its rate of dissociation from an activated
preinitiation complex. This model further posits that, of the many
protein-DNA interactions in a preinitiation complex, the lifetime of
the SRY-DNA complex can be the limiting parameter. We imagine that once
SRY dissociates and the DNA unbends, multiple other protein-DNA and
protein-protein interactions are weakened (Fig. 1
A). Accordingly, we
propose that kinetic stability of the bent SRY-DNA complex enables
maintenance of the preinitiation complex through multiple rounds of
transcription. Although the physiological significance of this
observation is not clear (as the present cotransfection assay does not
use a bona fide SRY target promoter and, in any case, both I13 and F13
ultimately results in male differentiation in the appropriate primate),
it is possible that SRY-responsive genes in vivo will be
found to exhibit diverse promoter/enhancer structures with distinct
rate-limiting steps in respective pathways of preinitiation complex
assembly and disassembly.
We caution that the present data are only correlative. Confounding
effects in the intracellular milieu of our gonadal cotransfection assay
(such as differential nuclear targeting, and differential binding to
bystander proteins in the cell) cannot be excluded. Because the
substitutions at position 13 are distant from possible phosphorylation
sites and nuclear localization signals, however, such effects seem
unlikely. It is also possible that the I13 and F13 domains have
different affinities for a 5'-ATTGTT-3' site within a nucleosome, a
structural target not tested in the present study. Despite these
caveats, a kinetic constraint on SRY activity would be broadly
consistent with kinetic control in other nonequilibrium systems as
discussed below.
Is Kinetic Control a General Biological Phenomenon?
The signature of kinetic control is a correlation between rates
(rather than equilibrium constants) and a functional endpoint. Examples
in endocrinology include the kinetic trapping of gonadotropin
heterodimers (1) and kinetic control of mitogenic signaling by insulin
(2, 3). Kinetic control has also been established among serpin
proteases and has physiological relevance (62). The misfolding of
proteins and subsequent trapping of non-ground-state forms within
aggregates are proposed as pathogenic steps in prion-related
encephalopathies (63). Kinetic control of a processive enzymatic
process may likewise be effected by rates as exemplified by
transcriptional elongation: rapidly dissociating drug-DNA complexes do
not hinder elongation even if compensated by rapid association rates
leading to similar equilibrium constants. Model studies have used T7
RNA polymerase (a small single-subunit polymerase) and the multisubunit
bacterial RNA polymerase of Escherichia coli (36, 37, 38).
Slowly dissociating intercalative agents cause prolonged pausing of the
polymerase preceding the site of intercalation, in turn enhancing the
probability of transcriptional termination with release of the enzyme
from the transcription bubble. The faster and less stable T7 enzyme
terminates at each slowly dissociating ligand complex. Actinomycin D,
echinomycin, mithracycin, and noglamycin provide examples of slowly
dissociating drugs (t1/2 > 5 min) whereas
adriamycin provides an example of a rapidly dissociating drug
(t1/2 < 1 sec). A metaphor of a train (the
processive RNA polymerase) being derailed by a car on the tracks (the
intercalative complex) is supported by the correlation between
diminished bioactivity and creep rate or lateral diffusion of the drug
along the DNA. Enhanced lateral diffusion by an intercalative agent
(visualized as a car outracing the train) correlates with impaired
bioactivity even if the overall rate of drug-DNA dissociation is slow
(35).
Summary
Structure-function relationships in the SRY HMG box have been
inferred from comparative studies of clinical variants (6, 10, 13, 14, 15, 44, 45). By correlating phenotype with biochemical activities, such
studies have suggested that SRYs genetic function requires specific
DNA binding (44, 45) and DNA bending (15). The present study has
focused on comparison of primate SRY HMG boxes (42), which differ only
by the presence of an aliphatic- (human, gorilla, orangutan, baboon,
and marmoset) or aromatic (common and pygmy chimpanzees) cantilever.
This comparison was motivated by an analogy proposed between
DNA-bending proteins and intercalative DNA-binding drugs (31). The
results highlight the importance of the cantilever mechanism and
suggest a new avenue of investigation, kinetic control of
transcription. In the future it would be of interest to extend this
analysis to M13 (the most common cantilever among mammalian Sry and SOX
sequences; Fig. 1
D). Characterization of this and diverse side chains
of chemical interest may further test the proposed relationship between
kinetic stability and transcriptional activation.
Future dissection of the structural, kinetic, and thermodynamic factors
underlying SRY-dependent gene regulation will require identification of
physiological target genes and biochemical reconstitution of sex- and
tissue-specific preinitiation complexes. It is likely that variant HMG
boxes with well defined biochemical properties will provide valuable
tools to uncover target genes and explore mechanisms of regulation. Our
hypothesis of kinetic control predicts the occurrence of novel clinical
mutations in HMG boxes that accelerate rates of specific DNA
association and dissociation but do not impair DNA affinity. The SRY
system may provide an opportunity to investigate a nonequilibrium
mechanism of transcriptional regulation.
 |
MATERIALS AND METHODS
|
---|
Protein Purification
The SRY HMG box (85 residues) was expressed in E.
coli as a thrombin-cleavable fusion protein as described (18).
Final purification was accomplished by FPLC using a MonoS column
(Pharmacia Biotech, Piscataway, NJ). HMG box fragments
were obtained both with and without C-terminal
His6 tag. The tag does not affect protein folding
(as determined by CD and 1H-NMR) or specific DNA
binding (as determined by GMSA). Purity was more than 98% as
assessed by SDS-PAGE.
Site-Directed Mutagenesis
Substitutions were introduced in phage M13 mp19RF by
oligonucleotide-directed mutagenesis (6) and recloned by PCR into an
expression plasmid (18). Constructions were verified by DNA
sequencing.
Four-Way DNA Junctions
Four 30-bp oligonucleotides J1 were prepared as described
(7). One strand was labeled with 32P, annealed
overnight with the other three strands from 80 C to 4 C in 50
mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 10
mM MgCl2, and 50 mg/ml BSA.
Efficiency of junction formation was more than 80% as estimated by
12% PAGE.
Cell Culture and Western Blot
Cell line CH34 (6) was cultured in DMEM containing 5%
heat-inactivated FBS and 1x penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD) at 37 C under 5%
CO2. Cotransfections were performed using the
lipofectAMINE 2000 kit (Life Technologies, Inc.). Briefly,
after cells were washed twice with 1x PBS, SRY expression plasmids
were cotransfected with pXGH5 (6) in 1:1 ratio as an internal control
for transfection efficiency and expression level. After overnight
cotransfection, fresh medium was added. For Western blots, identical
aliquots of cell extracts were electrophoresed on 10% SDS-PAGE. The
gel was blotted on nitrocellulose membrane (Bio-Rad Laboratories, Inc. Hercules, CA). Hybridization solutions containing anti-HMG
domain antibody SE75 at a concentration of 1:100 or antihuman-GH
antibody (obtained from Sigma, St. Louis, MO) at a
concentration of 1:100, respectively, were hybridized onto the
membrane. After goat antirabbit IgG was incubated for 1 h, a
chemiluminescent detection system (Bio-Rad Laboratories, Inc.) was used for specific protein detection. Antibody SE75 is
specific for the HMG box of human SRY and was raised as described (64).
Cross-reactivity to SOX HMG box of a 67 kDa can occur (P. Berta,
personal communication). The present Western blots contained no bands
less than 40 kDa in the control transfection (thus excluding detection
of rat Sry) and only a single band of size appropriate to human SRY in
the SRY- and I68F-SRY-transfected lanes.
DNA-Binding Assay
Oligonucleotides were purchased from Oligos, Etc. (Wilsonville,
OR). 1) The duplex probe was labeled with 32P or
33P, annealed, and analyzed using the
gel-retardation assay (18). Each reaction contained 2.5160
nM protein (see caption to Fig. 4
A) and less than 1
nM labeled DNA in 10 mM potassium phosphate (pH
7.0), 50 ng/ml BSA, 50 mM KCl, 4 mM
dithiothreitol, and 2.5 mM MgCl2; the
reaction was incubated for 1 h on ice. Only specific binding is
observed under these conditions. 2) For analysis of four-way DNA
junctions, reactions contained 25250 nM protein and 4
nM labeled junction in the same buffer. The 36-bp probe
contains the DNA sequence
5'-CATACTGCGGGGGTGATTGTTCAGGATCATACTGCG-3' and complement
(target site underlined). The parent 15-bp probe (also used
for NMR studies) contains the sequence 5'-GTGATTGTTCAG-3' and
complement. To assess sequence specificity, variant duplexes
5'-GTGAXTGTTCAG-3' and 5'-GTGATXGTTCAG-3' (X
= other base pairs) with respective complements were similarly
prepared.
Electrophoretic DNA-Bending Assay
Double-stranded oligonucleotides containing consensus SRY
binding site 5'-CCCATTGTTCTCT-3' and complement were cloned
between XbaI and SalI sites of vector pBend2
(65). Probes of equal length (147 bp) with the binding site at varying
distance from ends (distances of bend center from 5'-end are 120, 95,
79, 51, 47, and 27 bp) were generated by PCR, and 5'-labeled with
33P-ATP using T4 polynucleotide kinase. Ten
microliter binding reactions contained 50 mM KCl,
20 mM Tris-HCl, pH 7.4, 5
mM MgCl2, 50 ng
poly(dI-dC), 10% glycerol, approximately 1 nM
33P-labeled DNA probe, and 60
nM SRY-HMG box domain. After 12 h incubation on
ice, samples were run on polyacrylamide gels in 0.5x (0.045
M) Tris-borate buffer containing EDTA (TBE)
at
10 V/cm.
Steady-State Tryptophan Fluorescence and Stability
Measurements
Steady-state fluorescence spectra and automated guanidine
titrations were obtained using an Aviv spectrofluorimeter (model
ATF105, Aviv Instruments, Lakewood, NJ). Intrinsic Trp fluorescence was
measured at an emission frequency of 390 nm (slit width 5 nm) after
excitation at 270 nm (slit width 2 nm) at 4 C. Denaturation studies of
the human domain as monitored by CD at 222 nm yielded similar results.
Human and chimpanzee SRY domains were made 1 µM in 140
mM KCl, 10 mM potassium phosphate (pH 7.4) in
the titrating cuvette. The same concentration of SRY was used in the
titrant reservoir containing 7.2 M guanidine-HCl in the
above buffer. Experimental curves were fitted by nonlinear least
squares to the equations:
G =
Gu +
m x [guanidine] = RT x ln K, where
Gu is the free energy of unfolding
extrapolated to zero denaturant concentration and K is the equilibrium
constant between native and unfolded states of the protein (66).
Phase-Modulation Fluorescence and FRET
Time-resolved measurement were performed on a SPEX Fluorolog
-2 Lifetime Spectrofluorometer (Hoboken, NJ). The light
source was a xenon lamp with excitation wavelength of 490 nm. The
frequency of the excitation beam was modulated in a Pockel cell between
20 and 200 MHz. Sample response was monitored at the emission
wavelength of fluorescein (518 nm) by a photomultiplier (PMT) through a
Corion 520 band-pass filter (ThermoCorion Inc., Franklin, MA) at
magic angle. Time-resolved measurement were performed on 15
µM samples. The reference was LUDOX TM-50 colloidal
silica suspension (DuPont Merck Pharmaceutical Co.,
Wilmington, DE) obtained from Aldrich Chemical Co., Inc.
(Milwaukee, WI). Measurements were performed at 4 C. Fitting analysis,
performed on Globals Unlimited software (Laboratory for Fluorescence
Dynamics, University of Illinois, Urbana-Champaign, IL),
indicated that fluoresceins spectral response could be best described
with double-exponential curve with two distinct lifetimes contributing
to phase-response (51). Fitting parameter space was changed to
target parameters representing physical invariant of system, after
which a complete system could be specified. For fluorescein-rhodamine
pair energy transfer was assumed to give rise to two lifetimes.
The Förster distances for free and bound DNA were measured to be
56 Å and 54 Å, respectively, and the distance between chromophores
was assumed to vary according to normal distribution. Parameter
optimization was performed to minimize overall
2. Error levels of 0.2 (phase) and 0.01
(modulation) are assumed for analysis. FRET measurements were obtained
in 50 mM KCl and 50 mM Tris-borate (pH
7.4).
CD Spectroscopy
Spectra were obtained using an Aviv spectropolarimeter equipped
with thermister temperature control for automated analysis of thermal
melting. Samples were observed in a 1-mm pathlength quartz cuvette in a
buffer consisting of 10 mM potassium phosphate (pH 7.4) and
50 mM KCl.
NMR Spectroscopy
Spectra were observed at 400 and 500 MHz using Varian
spectrometers (Varian Instruments Inc., Palo Alto, CA) at
Harvard Medical School and The University of Chicago. Spectra of
exchangeable resonances were obtained in H2O
using laminar-shifted shaped pulses in the absence of solvent
presaturation as described (18). Spectra of free domains were obtained
at 25 C in 10 mM deuterated acetic acid (pH 4.5) and 140
mM KCl; such spectra are similar in the pH range 48.
Spectra of complexes were obtained at 40 C in 10 mM
potassium phosphate (pH 6.0) and 50 mM KCl.
 |
ACKNOWLEDGMENTS
|
---|
Acknowledgements
We thank E. Haas and V. Itah (Bar Ilan University, Ramat Gan,
Israel) for measurements of fluorescent Ro values
and advice regarding FRET methods; W. Olson and A. R. Srinivasan
(Rutgers University, Newark, NJ) for discussion of FRET design and
linker dynamics; P. Berta (Institut de Genetique Humaine, Montpellier,
France) for anti-SRY antisera; D. Love and P. E. Wright (Scripps
Research Institute, La Jolla, CA) for CURVES analysis of Lef1-DNA and
SRY-DNA structures; W. Jia for assistance with NMR measurements and
processing; G. L. Waneck for advice regarding site-directed
mutagenesis; and L. Labeots and M. Mellody for assistance with DNA
binding studies.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael A. Weiss, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935. E-mail: maw21{at}po.cwru.edu or Patricia K. Donahoe, Pediatric
This work was supported in part by grants from the NIH to P.K.D. (Grant
CA-17393) and M.A.W. (Grant CA-63485).
1 Present Address: Urawa Municipal Hospital, Department of Pediatric
Surgery, 2460 Mimuro Yrawa-shi, Saitama 336-8522 Japan. 
2 These authors contributed equally. 
Received for publication September 13, 2000.
Revision received January 2, 2001.
Accepted for publication January 4, 2001.
 |
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