(Received for publication, September 26, 1995; and in revised form, December 22, 1995)
From the
Eukaryotic transcriptional regulatory proteins typically comprise both a DNA binding domain and a regulatory domain. Although the structures of many DNA binding domains have been elucidated, no detailed structures are yet available for transcriptional activation domains. The activation domain of the herpesvirus protein VP16 has been an important model in mutational and biochemical studies. Here, we characterize the VP16 activation domain using time-resolved and steady-state fluorescence. Unique intrinsic fluorescent probes were obtained by replacing phenylalanine residues with tryptophan at position 442 or 473 of the activation domain of VP16 (residues 413-490, or subdomains thereof), linked to the DNA binding domain of the yeast protein GAL4. Emission spectra and quenching properties of Trp at either position were characteristic of fully exposed Trp. Time-resolved anisotropy decay measurements suggested that both Trp residues were associated with substantial segmental motion. The Trp residues at either position showed nearly identical fluorescence properties in either the full-length activation domain or relevant subdomains, suggesting that the two subdomains are similarly unstructured and have little effect on each other. As this domain may directly interact with several target proteins, it is likely that a significant structural transition accompanies these interactions.
Transcription initiation by RNA polymerase II in eukaryotic cells requires the assembly of a basal transcription complex containing the polymerase and several general transcription factors(1) . The actual level of transcription, however, is regulated by gene-specific proteins termed transcriptional activators or repressors. These proteins usually contain two functional domains. One domain directs the gene-specific binding, and the other domain performs the transcription activation or repression function(2, 3, 4, 5) .
VP16 is a virion protein of herpes simplex virus that specifically activates viral immediate early gene expression(6, 7) . The amino-terminal region of this protein interacts with host DNA binding proteins to associate with the immediate early gene promoter sequences (8, 9) . The activation function resides within the carboxyl-terminal 78 amino acids(10, 11, 12) . As one of the most potent activators known, the VP16 activation domain has been studied widely in many systems and by various experimental designs. In light of these studies, several models have been proposed for the mechanisms of activation. Activators might function by relieving the repression effect of chromatin structure (13) . Alternatively, they may interact with components of the basal transcription complex, directly or indirectly, to either speed up or stabilize the formation of the preinitiation complex(14, 15, 16, 17, 18) . Some activators may affect initiation, promoter clearance, or transcriptional elongation(19, 20, 21) .
Despite their central importance in gene regulation, the structures
of the transcriptional activation domains remain a mystery. No
activation domain structure has yet been solved by x-ray
crystallographic analyses or NMR. Most clues to the structures of
activation domains come from mutational analyses. Many activation
domains are rich in acidic amino acids; in the case of VP16, 21 acidic
residues are found in the 78-amino acid domain. Initially, an
``acidic blob'' random coil model was suggested for these
acidic activation domains (AADs)()(22) . According
to this model, AADs would function primarily through electrostatic
interactions. Subsequent mutational analyses provided evidence against
this model(23, 24, 25) , in that no strict
correlation between negative charge and activity was observed. An
alternative, the so called amphipathic
-helix model (26) ,
was also refuted by mutational analyses of the VP16
AAD(23, 27) . No relation was observed between
predicted amphipathy and activity, and proline substitutions introduced
into the putative helix had no effect on activity. Instead, particular
aromatic and bulky hydrophobic residues were found important for
function. These and other studies also suggested that VP16 AAD had two
subdomains, namely, the N-terminal subdomain (residues 413-456)
and the C-terminal subdomain (residues
453-490)(17, 27, 28) . Phe
was deemed the most critical residue in the N-subdomain, and its
aromaticity was the most important feature. Although the pattern of
amino acids surrounding Phe
resembled that surrounding
Phe
, Phe
was not as sensitive to mutations.
Thus, these two subdomains apparently depend on different patterns of
residues and might function through different mechanisms.
Few
biophysical studies of transcriptional domains have been reported. Both
one- and two-dimensional NMR of the isolated VP16 AAD demonstrated that
this domain lacked stable secondary and tertiary
structure(29) . Similarly, circular dichroism (CD) experiments
indicated that this isolated domain was devoid of any stable
-helical or
-strand structure(30) , although more
-helical structure was induced under hydrophobic conditions or at
low pH. Parallel studies by CD spectroscopy revealed that the AADs of
yeast activators GAL4 and GCN4 were conformationally mobile at neutral
pH and underwent a transition to
-sheet in acidic
solution(31) . Taken together, the limited biophysical studies
have not detected the secondary structure of AADs under physiological
conditions.
Fluorescence spectroscopy can provide a rich variety of information about protein conformation, including the local environment of specific residues, populations of protein conformers, and dynamics(32) . Here, we describe a fluorescence analysis employing chimeric proteins comprising the DNA-binding domain of yeast protein GAL4 fused to the AAD of VP16. Trp residues were substituted for Phe (at either position 442 or 473) to provide unique intrinsic probes within each subdomain. The results of fluorescence quenching, time-resolved intensity decay and time-resolved anisotropy decay studies show that the VP16 AAD is largely unstructured. Moreover, the structure of either subdomain seems unaffected by the presence or absence of the counterpart subdomain, reinforcing the concept that the two subdomains have independent structures and activities.
A Trp codon within
the GAL4 DNA binding domain was changed to a Val codon using
oligonucleotide-directed mutagenesis(34) . The altered DNA
fragment was then subcloned into each of the three E. coli expression vectors, pFS12182, pAC-del456, and pLA31Sma.
Previously mutated VP16 activation domains (F442W or F473W) were
further subcloned into these E. coli expression plasmids from
mammalian VP16 expression vectors derived from
pMSVP16(23, 27) .
The steady-state fluorescence spectra were obtained on a SLM 8000 spectrofluorometer operated in a ratio mode. ``Magic angle'' configuration was used to avoid rotational artifacts(38) . The bandwidths for excitation and emission slits were 4 nm. The excitation wavelength was 297 nm.
Quenching experiments were performed at an excitation wavelength of 297 nm. Aliquots of stock quenching solutions (4 M KI, 4 M CsCl, and 8 M acrylamide) were added to 1.4-ml protein samples. The values of fluorescence emission intensity at 350 nm were corrected for dilution prior to data analysis. Quenching data were analyzed by the Stern-Volmer equation for dynamic quenching,
or by the single species dynamic-static quenching equation,
where F and F are the fluorescence
intensity in the absence and presence of quencher, [Q] is the
quencher concentration, K
is the Stern-Volmer
dynamic quenching constant, and V is the static quenching
constant (or ``active volume''). K
and V values were determined using least-squares regression (IGOR,
Wavemetrix, Lake Oswego, OR). The bimolecular collisional quenching
constant k
was calculated from the
following,
where <> is the mean (intensity-weighted)
fluorescence lifetime obtained from time-resolved measurements.
Time-resolved fluorescence was measured on a single photon counting
fluorometer(39) . A synchronously pumped, mode-locked,
cavity-dumped dye laser (Spectra-Physics 3520) was used as the light
source, providing pulses of width <10 ps at 297 nm with a repetition
rate of 4 MHz and an average power of 200 µW. The vertically
polarized UV pulses were obtained by frequency doubling of horizontally
polarized dye laser pulses. The exciting light time profile was
obtained with a light-scattering suspension (Ludox, DuPont). The
intensity decay profiles were collected through an emission sheet
polarizer oriented 55° from the vertical symmetry
axis(38) . Emission was selected by computer-controlled JYH10
monochromator with the bandwidth set at 8 nm, and a glass slide was
added to further reject stray excitation as needed. Decay curves were
recorded at 5-nm intervals across the emission band (310-460 nm)
by using standard time-correlated single photon counting modules and an
Ortec ADCAM multichannel analyzer under computer control. The
decay-associated spectra were obtained from global
analysis(40, 41) . The fluorescence intensity decay,
I(,t), was fit to a sum of
exponentials,
where is the emission wavelength independent
decay time of the ith decay component, and
is its preexponential term at emission wavelength
. The
fractional fluorescence, f
(
), of the ith
component at wavelength
is defined by the following
equation(42) .
The mean lifetime <> is defined by the following
equation.
The confidence limits for decay-associated spectra (DAS) have been explored by Boens et al.(43) ; for our experimental conditions, we anticipate that these parameters will be recovered within 5%.
Anisotropy decay curves were obtained by alternatively recording emission oriented parallel and perpendicular to the plane of excitation at an emission wavelength of 350 nm. Time per channel was 90 ps, and 512 channels were recorded. Data were analyzed by the ``sum and difference'' method(44) . The anisotropy decay curve, r(t), was obtained from the difference curve and total intensity curve by the following,
where I and I
are
emission intensities measured parallel and perpendicular to the
excitation plane, respectively. Data were corrected for the G factor,
even though a depolarizer in our system makes the G factor very close
to unity. r(t) was modeled by a sum of
exponentials,
where is the rotational correlation time of
the jth component and
is its preexponential
term. A fixed 50-ps component was introduced to compensate for both
scattering and color shift artifacts. If one assumes segmental motion
can be reconciled with the ``wobbling in cone''
model(45, 46) , the cone semiangle,
, is given
by the following,
where is the preexponential term for the
global rotation of the macromolecule and r
is the
limiting (``time zero'') anisotropy.
The wild-type VP16 AAD has no indigenous tryptophan residues. To
obtain unique intrinsic fluorescence probes at key positions within the
VP16 AAD, Phe to Trp mutations were introduced at either position 442
or 473. These mutations had modest or no effects on transcriptional
activation when tested in transient transfection assays(27) .
For this study, these mutations were transferred to the expression
vector for the GAL4-VP16 fusion protein as both full-length AAD and as
relevant subdomains (413-456 or 453-490). These fusion
proteins (represented in Fig. 1) were purified to more than 95%
homogeneity as judged by SDS-polyacrylamide gel electrophoresis. These
proteins were transcriptionally active when tested by in vitro transcription assays (Fig. 2). The addition of
GAL4W36V-VP16, GAL4W36V-VP16F442W, and GAL4W36V-VP16F473W to the in
vitro transcription reactions strongly stimulated transcription
from the multiple start sites of the yeast CYC1 promoter (lanes 3-5), while the addition of no activator or the
addition of the GAL4 DNA binding domain alone resulted in basal level
transcription only (lanes 1 and 2). Thus, the
structural features revealed by Trp or Trp
should reflect those of the wild-type VP16 AAD.
Figure 1: Schematic representations of the various transactivators used in this study. All proteins contain the GAL4 DNA binding domain (residues 1-147) with a tryptophan to valine substitution at position 36, designated as GAL4W36V. All proteins also contain a 2- or 3-amino acid linker between GAL4 domain and VP16 domain. GAL4W36V-VP16-(413-490), GAL4W36V-VP16F442W, or GAL4W36V-VP16F473W contain the in-frame fused wild-type full-length VP16 activation domain (residues 413-490) or phenylalanine to tryptophan substitution at position 442 or 473, respectively. GAL4W36V-VP16N F442W contains the in-frame fused VP16 activation N subdomain (residues 411-456) with the tryptophan substitution at position 442. GAL4W36V-VP16C F473W contains the in-frame fused VP16 activation C subdomain(453-490) with the tryptophan substitution at position 473.
Figure 2: Autoradiogram of primer extension assay reflecting the transcriptional activities of the transactivators used in this study. No transactivator (lane 1), 2 pmol of GAL4-(1-147) (lane 2), 2 pmol of GAL4W36V-VP16-(413-490) (lane 3), 2 pmol of GAL4W36V-VP16F442W (lane 4), or 2 pmol of GAL4W36V-VP16F473W (lane 5) was added to the in vitro transcription reactions. Protein concentrations were determined by the Bradford assay.
Figure 3: Normalized emission spectra of the various transactivator proteins. Solid line represents GAL4W36V-VP16F442W, long dash-dotted line represents GAL4W36V-VP16N F442W, dotted line represents GAL4W36V-VP16F473W, and short dash-dotted line represents GAL4W36V-VP16C F473W.
To further assess the solvent access to the surroundings of
Trp and Trp
, quenching studies were
undertaken using anionic (iodide), cationic (cesium), and neutral polar
(acrylamide) quenching agents. The results of these studies are given
in Fig. 4, and the results of the analysis in terms of are given in Table 1. The
Stern-Volmer plot of acrylamide for GAL4W36V-VP16F442W was linear (Fig. 4A), giving a Stern-Volmer quenching constant (K
) of 15.7 M
. The
Stern-Volmer plot of acrylamide for GAL4W36V-VP16F473W showed upward
curvature (Fig. 4A); a single-species dynamic-static
model fit the data significantly better than did a pure dynamic model.
This analysis gave a K
of 6.6 M
and a static quenching constant (V) of 2.3 M
. V reflects
the strength of the ground state complex between the quencher and Trp.
The quenching rate constants for GAL4W36V-VP16F442W and
GAL4W36V-VP16F473W were 4.2 M
ns
and 2.0 M
ns
, respectively, within the range (2-4 M
ns
) typically seen for
exposed Trp residues in proteins with little secondary
structure(48) . These results suggest that both Trp residues
are highly exposed.
Figure 4: Stern-Volmer plots for the quenching of the fluorescence of the transactivator proteins by acrylamide (panel A), KI (panel B), or CsCl (panel C). Closed circles are for the GAL4W36V-VP16F442W; open circles are for the GAL4W36V-VP16N F442W; closed triangles are for the GAL4W36V-VP16F473W; open triangles are for the GAL4W36V-VP16C F473W.
Using iodide as a quenching agent (Fig. 4B), the Stern-Volmer constants K for GAL4W36V-VP16F442W and GAL4W36V-VP16F473W were 3.1 M
and 2.2 M
,
respectively, significantly lower than K
values
for acrylamide. Acrylamide and iodide typically yield similar K
results when shielding by the protein matrix is
determined by steric effects only(32) . The lower K
values seen for iodide quenching in the present
experiments presumably reflect the fact that both probes (at 442 and
473) are surrounded by numerous acidic residues. The Stern-Volmer
constants K
of KI for the two VP16 subdomains
(GAL4W36V-VP16NF442W and GAL4W36V-VP16C F473W) were 2.9 M
and 2.1 M
,
respectively. These constants are very close to those observed for the
full-length activators. Thus, truncation of the activator has no effect
on the extent of exposure of Trp
and Trp
.
Analysis of cesium quenching (Fig. 4C) for the
full-length proteins with Trp or Trp
using
the dynamic quenching model gave K
values of 2.3 M
and 1.2 M
,
respectively. The quenching efficiency of cesium for an indole ring is
much lower than that of iodide(48) . The similar values of K
of both quenchers for both proteins again
indicate that the microenvironments surrounding both tryptophans are
negatively charged. Additional studies of the ionic strength dependence
and pH dependence of the quenching reactions would be needed to further
characterize the local electric potentials in the vicinity of these
tryptophans(49) .
Figure 5:
Resolution of the total fluorescence
spectrum into the DAS. Total, 1,
2, and
3
indicate the total spectrum, the spectrum associated with the short
lifetime component, the spectrum associated with the middle lifetime
component, and the spectrum associated with the long lifetime
component, respectively. In panel A, the spectra of
GAL4W36V-VP16F442W are denoted by the solid lines, and the
spectra of GAL4W36V-VP16F473W are denoted by the dotted lines.
In panel B, the spectra of GAL4W36V-VP16F442W are denoted by
the solid lines, and the spectra of GAL4W36V-VP16N F442W are
denoted by the dotted lines.
Figure 6: Time-resolved anisotropy decay curves. In panel A, the decay curve of GAL4W36V-VP16F442W is denoted by the solid line, and the decay curve of GAL4W36V-VP16F473W is denoted by the dotted line. In panel B, the decay curve of GAL4W36V-VP16F442W is denoted by the solid line, and the decay curve of GAL4W36V-VP16N F442W is denoted by the dotted line. Smoothed curves of the raw data are shown. A scaled lamp curve is given for reference (dashed line).
The fluorescence studies of the VP16 AAD described here
showed that this domain was highly flexible and mobile, suggesting that
it is poorly structured. The intrinsic probes placed at either of the
two subdomains had very similar properties, suggesting that both
subdomains are similarly unstructured. Each showed the characteristic
``exposed'' fluorescence spectrum with around 350 nm, consistent with highly exposed Trp. Rate constants
for quenching by acrylamide for both probes were comparable with those
of proteins with exposed Trp residues and little secondary structure.
KI quenching for the two probes indicated that their microenvironments
were negatively charged, consistent with the primary structures of
these subdomains. Time-resolved intensity decay yielded similar
lifetime species with similar contributions for these two Trp residues.
Anisotropy decay measurements suggested that both Trp residues were
associated with highly flexible, disordered segments. Noteworthy is
that each probe experienced the same environment whether in the
full-length context or in truncated subdomains. We infer that deletion
of either subdomain had no gross structural effect on the other
subdomain. The fact that individual DAS components for each protein
were not identical but were distinguishable in these proteins shows
that some structures persist, at least on nanosecond
timescales(39) . On the other hand, the Trp multiexponentiality
and anisotropy results point toward multiple conformers that intermix;
no evidence for rapid (ns) exchange is seen, however. At this juncture,
the most attractive view is one of a flexible but ``lumpy''
structure whose features switch and vary in microseconds. In summary,
these fluorescence properties closely resemble those of the well
characterized class of polypeptides such as adrenocorticotropin,
bombesin, and glucagon that have little persistent three-dimensional
structure and behave nearly as flexible coils (50, 51, 52) . Recent mutational analyses of
this domain further suggested the importance of residues Phe
and Phe
. (
)Trp substitution mutants at
these positions can be subjected to the same kinds of studies. We
expect similar results will be obtained to illustrate the disordered
structure of this AAD.
These results are consistent with results from the previous CD and NMR studies of the isolated VP16 AAD(29, 30) , in which no significant secondary structure was detected. In those studies, an isolated AAD peptide fragment was used. Structural analyses of the GAL4, GCN4, and glucocorticoid receptor AADs also employed peptide fragments(31, 53) . In the present work, we used the chimeric GAL4-VP16 proteins and determined the transcription activities of these proteins. The concordant results suggest that the presence of the GAL4 DNA binding domain does not induce or confer any specific structure in the VP16 AAD.
Our results indicate that the VP16 AAD is largely disordered in solution, and the two aromatic amino acids at positions 442 and 473 are solvent-exposed. In the primary structure of the VP16 AAD, abundant acidic residues are found near these aromatic residues. These acidic residues may increase the solubility of this domain.
The disordered structure of the VP16 AAD may be fundamental to the nature of the activation process. Eukaryotic transcriptional activation is tremendously complicated, involving a large number of protein-protein interactions. Biochemical and genetic studies have suggested multiple target proteins of activators(14, 15, 16, 17, 18) , and many activators enhance transcription synergistically(54, 55) . To promote such complicated macromolecular associations in vivo, an unstructured polymeric domain may have many advantages over a specific structured domain(56) . For example, flexible, weakly interacting, relatively unstructured polymeric domains can promote the rapid renaturation of complementary DNA strands(57, 58) . In such weak interactions, charged groups and hydrophobic residues in an unstructured polymeric domain have been thought to provide a suitable interaction force in the promotion of macromolecular associations(56) . For many AADs, transcriptional activities generally correlate with the number of acidic residues and are also dependent on the bulky hydrophobic residues(5, 23) . According to this model, these residues in the AAD are important to enhance the large number of macromolecular associations in many steps of the transcription process, mainly through nonspecific interactions. Relatively unstructured domains in activators may permit interaction with any of several different target proteins and thus may function at several steps in transcription activation (15) .
Recent studies show that distinct regions of the large subunit of RNA polymerase II share features in common with either acidic activators or a proline-rich activator(59, 60) . On the basis of the present and other structural studies, we believe these shared domains are relatively unstructured. These domains in the polymerase may interact with the same target proteins as those of activators. A tether-and-competition model for activation has been proposed(59, 60) , in which the dynamic exchange of numerous protein-protein interactions allows the assembly and the disassembly of the transcription complex. Thus, these unstructured domains may facilitate the dynamic exchange interactions in the activation process.
The lack of structure of the VP16 AAD inferred
from biophysical studies seems contradictory to the mutational analyses
of the VP16 AAD, which showed that its activity is critically dependent
on certain types of hydrophobic residues in certain positions. A
hypothesis to explain this paradox is that whatever structural element
is needed for this specificity is formed during interaction with in
vivo targets and that certain hydrophobic residues in the AAD are
critical for this transition. The -helix structure in the VP16 AAD
observed under more hydrophobic and low pH conditions and the
-sheet structure induced in the AADs of GAL4 and GCN4 in acidic
solution support this
hypothesis(29, 30, 31) . This hypothesis can
be further tested by studying the biophysical properties of an AAD in
the presence of its putative target proteins. To this end, we have
examined the fluorescence properties of the VP16 AAD, labeled with Trp
analogs as intrinsic probes, in the presence of various general
transcription factors(61) . These experiments provide
biophysical evidence for ``target-induced structure,'' and
important qualitative and quantitative insights have been gained from
this approach. Thus, time-resolved fluorescence may cast new light on
mechanisms of transcriptional activation.