From the
Sequence-specific transcriptional activators, such as the human
factor E2F-1, increase the rate of initiation of transcription by RNA
polymerase II, possibly by contacting one or more of the RNA polymerase
II-associated general initiation factors. One candidate target of
transactivators is the TATA-binding protein (TBP), which, when bound to
a promoter, nucleates the formation of a preinitiation complex.
Previous studies using affinity chromatography techniques have shown
that the activation domains of certain activators, including the acidic
activation domain of E2F-1, can interact with TBP in the absence of
DNA. Using a site-directed photoaffinity cross-linking approach, we
demonstrate here that the activation domain of the chimeric activator
LexA-E2F-1 can be cross-linked to TBP when both factors are bound to a
transcriptionally responsive RNA polymerase II promoter. Mutations
within the activation domain of LexA-E2F-1 that impaired its ability to
activate transcription in vitro were found to reduce
cross-linking of LexA-E2F-1 to TBP. The association of initiation
factor TFIIB with the TBP-promoter complex did not preclude this
promoter-dependent cross-linking of LexA-E2F-1 to TBP. TFIIB itself
could also be cross-linked to LexA-E2F-1; however, this cross-linking
was promoter-independent. In contrast, TFIIA strongly inhibited the
promoter-dependent cross-linking of LexA-E2F-1 to TBP. These results
directly demonstrate that acidic activators such as E2F-1 can interact
with TBP during the earliest stages in the assembly of an RNA
polymerase II preinitiation complex.
RNA polymerase II (pol II)(
In a recent study of the process of transcriptional activation in
prokaryotes, photochemical cross-linking of proteins has been used to
characterize a promoter-dependent interaction between the bacterial
activator protein CAP and the
As shown in lane2 of
Fig. 2A, two closely spaced bands that had a mobility
consistent with the formation of a complex between LexA-E2F-1 and TBP
(i.e. 60-64 kDa) were observed. Both of these complexes
represented a covalent heterodimer of LexA-E2F-1 with TBP as they could
each be specifically immunoprecipitated with antisera against TBP
(Fig. 2B, lanes 2-4) and LexA (lane6), but not with antisera against an unrelated protein
(lane5). As the mobility of TBP on denaturing gels
is particularly sensitive to structural alteration
(26) , the
formation of two activator-TBP complexes may be due to the covalent
attachment of LexA-E2F-1 to different residues on TBP. Importantly,
these same complexes were absent in control reactions that did not
contain the activator (Fig. 2A, lane1) and were significantly reduced (
The recombinant TFIIA
and TFIIB used in our study (Fig. 1A) readily formed
characteristic complexes with TBP that could be visualized using an
electrophoretic mobility shift assay (Fig. 6A).
Surprisingly, preincubation of yeast TBP with an equimolar amount of
yeast TFIIA resulted in a nearly complete inhibition of cross-linking
of LexA-E2F-1 to TBP whether using the yeast CYC1 promoter
(Fig. 6B) or the mammalian Ad2ML promoter
(Fig. 6C) as template. We also found that yeast TFIIA
could inhibit cross-linking of LexA-E2F-1 to human TBP (Fig. 6D),
a result consistent with the ability of yeast TFIIA to bind human
TBP
(28) . In contrast, preincubation of yeast TFIIB with yeast
TBP (Fig. 6, B and C) or human TFIIB with human
TBP (Fig. 6D) resulted in each case in only a modest
reduction in the level of cross-linking of LexA-E2F-1 to the TBP
target. These results suggest that the activation domain of E2F-1 can
bind to TBP that is complexed with TFIIB but cannot do so in the
presence of TFIIA.
The experiments reported here provide direct biochemical
evidence that a transcriptional activator can interact with TBP when
bound upstream of a pol II promoter. Although it had been shown
previously that acidic activators including E2F-1 can bind to TBP in
solution (for example see Refs. 4, 10, and 16), our study also provides
the first indication that this interaction can occur when TBP is itself
bound to the TATA-element. The previous studies that implicated TBP as
a target for transactivators have largely relied on affinity
chromatographic techniques, which typically employ high concentrations
of activator as ligand. In the photochemical cross-linking approach
described here, we have used a markedly lower concentration of
activator more similar to that of activators in eukaryotic cells, a
concentration that is also sufficient for robust transcriptional
activation in vitro. The ability of LexA-E2F-1 to be
cross-linked specifically to TBP at the low concentration (170
nM) we have used is consistent with the
K
Although the interaction of an
activator with TBP may facilitate the binding of TBP to a promoter
in vivo(29) , our results are consistent with the
notion that TBP remains an important target of transactivators even
after it has been recruited to a promoter. By directly contacting TBP
at a promoter, an activator like LexA-E2F-1 or GAL4-VP16 may displace
inhibitors of transcription associated with TBP in vivo, which
impede the formation a productive preinitiation complex
(31) .
Alternatively, the activator might confer a conformational change in
the promoter-bound TBP in a manner that facilitates the subsequent
recruitment of other general initiation factors, such as TFIIB, to the
promoter
(2, 30, 32) . Consistent with this
latter possibility, specific point mutations in TBP, which both reduce
its ability to bind to the activation domain of VP16 and result in a
defective transcriptional response to GAL4-VP16, hinder
GAL4-VP16-mediated recruitment of TFIIB to the initiation
complex
(30) . Although the recruitment of TFIIB to the promoter
can be a rate-limiting step in the initiation of transcription, it need
not be the only step accelerated by transactivators. For example, the
ability of LexA-E2F-1 (this study) and GAL4-VP16
(5) to interact
directly with TFIIB may, in turn, facilitate the association of pol II
and TFIIF with the preinitiation complex.
TFIIA is required for
efficient transactivator function under certain conditions in
vitro(28, 33, 34, 35) , and the
formation of a preinitiation complex containing TFIIA is thought to be
an important step in the transactivation process
(36) .
Unexpectedly, however, we found that TFIIA could largely inhibit the
cross-linking of LexA-E2F-1 to TBP at both yeast and mammalian
promoters. This result suggests that the acidic activation domain of
E2F-1 binds to an overlapping region or surface of TBP that is also
contacted by TFIIA. Alternatively, TFIIA may alter the conformation of
the TBP-promoter complex in a way that precludes the subsequent
association of an activator with TBP. The interaction of activators
with TBP may even assist in the recruitment of TFIIA, as well as TFIIB,
to the promoter by displacing inhibitors of transcription that bind to
TBP and block the association of TFIIA with the TBP-promoter
complex
(31) . Although we have only used yeast TFIIA in this
study, we expect that human TFIIA will also display a similar ability
to inhibit the cross-linking of E2F-1 to human TBP since both homologs
are structurally and functionally conserved
(28) . In contrast,
neither yeast TFIIB or human TFIIB appears to block the cross-linking
of LexA-E2F-1 to TBP. Thus, TBP can be a target of an activator even
after the association of TFIIB with the preinitiation complex.
Following the association of TFIIA with the TBP-promoter complex, the
activator may become displaced from its contact with TBP and would then
be free to interact with other components of the transcription
apparatus, including those that function at a later stage in the
initiation process such as TFIIH
(6) . The experiments reported
here have demonstrated the utility of a photocross-linking approach to
address the important and now controversial question of the identity of
the targets of eukaryotic activators of transcription. It may be
anticipated that cross-linking experiments, similar to those reported
here but performed in the context of a complete activator responsive
system, should help identify critical interactions mediated by
transcriptional activators with the pol II transcriptional machinery.
We thank D. Cress, C. Hagemeier, E. Harlow, W. Kaelin,
and T. Kouzarides for generously providing E2F-1 cDNA derivatives. We
also thank J. Brickman and M. Ptashne for the LexA expression vector,
R. Brent for antibody to LexA, and R. Ebright and J. Greenblatt for
helpful advice.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
requires a
number of accessory protein factors in order to initiate transcription
accurately from a promoter. These general initiation factors associate
through extensive protein-protein interactions and recruit pol II to
the promoter to form a preinitiation complex (reviewed in Ref. 1).
Sequence-specific transcriptional activators can stimulate
transcriptional initiation by pol II, at least in part, by facilitating
the assembly of a productive preinitiation complex (reviewed in Ref.
2). Indeed, transcriptional activators might function at more than one
step during this process
(3) . Consistent with this possibility,
the activation domains of some transactivators have been found to bind
directly to more than one component of the pol II preinitiation
complex. For example, the acidic activation domain of herpes simplex
protein VP16 has been reported to bind independently to the
TATA-binding subunit (TBP) of the general initiation factor
TFIID
(4) , to the general initiation factors TFIIB
(5) and TFIIH
(6) , as well as to the transcriptional
coactivators TAF40
(7) and PC4
(8) , which associate with
the preinitiation complex. Similarly, the glutamine-rich activation
domains of the human transcription factor Sp1 have been shown to bind
both to the TBP-associated factor TAF110
(9) as well as to TBP
itself
(10) . The ability of the activation domains of
transactivators to interact with several different protein targets in
the pol II initiation machinery may account for the transcriptional
synergy that is observed with multiple promoter-bound transactivators.
Nevertheless, given the multiplicity of activator targets thus far
proposed, the identity of biologically relevant transactivator
target(s) has remained somewhat controversial. In this respect, it is
noteworthy that most of the studies reporting the direct binding of an
activator to a target initiation factor have not been performed in the
context of functional promoter and, therefore, it remains to be
demonstrated if and at what stage such interactions occur during
preinitiation complex assembly or the initiation of transcription.
subunit of Escherichia coli RNA polymerase
(11) . As this methodology may be of similar
use in understanding transactivation mechanisms in eukaryotes, we have
developed a related strategy to study protein-protein interactions
mediated by a eukaryotic transcriptional activator bound to a cognate
DNA element upstream of a pol II promoter. Our approach involved the
generation of a chimeric transcriptional activator containing the
acidic activation domain of human activator E2F-1, which could then be
derivatized selectively with a photoreactive cross-linking moiety.
Using this method, we show that this activator can be cross-linked
directly to TBP when bound upstream of a variety of pol II promoters.
Our results are consistent with the notion that TBP is an important
target of transcriptional activators during early preinitiation complex
assembly and establish the usefulness of the cross-linking approach to
study the mechanisms of transcriptional activation in eukaryotes.
Protein Expression Vectors
Bacterial expression
vectors for LexA and LexA-E2F-1, each containing an N-terminal
polyhistidine tag, were prepared respectively by subcloning an
oligonucleotide linker encoding an translational stop codon and a DNA
fragment encoding amino acids 368-437 of E2F-1
(13) into
the bacterial expression vector pJB07 (12). Truncated wild type (amino
acids 400-437) and mutant E2F-1 activation domain
derivatives
(13, 14, 15, 16, 17) were amplified by polymerase chain reaction and subcloned
for expression as LexA fusions into pJB07. The LexA constructs were
transformed for expression into the E. coli cell line DH5
or JM107. DNA fragments encoding full-length yeast TBP, human TBP,
yeast TFIIB (Sua7), and human TFIIB were subcloned into the bacterial
expression vector pET19b (Novagen) or a pET19b derivative encoding the
recognition sequence for heart muscle kinase
(18) adjacent to
the N-terminal polyhistidine tag and were transformed for expression
into the E. coli strain BL21(DE3).
Promoter Constructs
An XbaI DNA fragment
from pG5Lx2E4
(12) containing two LexA binding sites was
subcloned into the XbaI site upstream of the Ad2ML promoter in
pAd2ML(-50) (19) and upstream of the CYC1 promoter using
an NheI site introduced in pGALCG-
(20) . The same LexA
binding sites were situated upstream of the HIS3 gene T
promoter by subcloning a BamHI to HindIII DNA
fragment from pG5Lx2E4 into BamHI- and
HindIII-digested pGCG17, pGC204, and pGC205
(21) . The
distance from the 3`-most LexA binding site to the (nearest) TATA
element is 41, 36, and 41 base pairs for the Ad2ML, CYC1, and
HIS3 T
promoter constructs respectively. Template
DNA was purified by cesium chloride density gradient centrifugation.
Protein Expression and Purification
Overnight
cultures were diluted 1:15 into fresh LB media, grown at 30 °C (for
pET-19 derivatives) or 37 °C (for LexA derivatives) to OD of
1.0, and induced with
300 µM
isopropyl-1-thio-
-D-galactopyranoside for 2-3 h.
The bacterial pellets were resuspended in buffer A (5 mM
imidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.9, 5
mM
-mercaptoethanol) containing 1 mM
phenylmethylsulfonyl fluoride and 5 mM benzamidine
hydrochloride. The cells were sonicated on ice and the debris pelleted
by centrifugation (20,000
g for 30 min at 4 °C).
Soluble extract was loaded at 8 °C onto columns containing
Ni
-NTA-agarose (400-500-µl bed volume;
Qiagen) pre-equilibrated with buffer A. The columns were washed
successively with five column volumes each of buffer A, buffer A
containing 45 mM imidazole, and buffer B (20 mM
HEPES-NaOH, pH 7.9, 100 mM NaCl, 20% glycerol, 0.2 mM
EDTA), and were eluted with buffer B containing 1 mM DTT and
0.5 M imidazole (pH 7.9). Human TBP was further purified by
chromatography on heparin-Sepharose (Pharmacia Biotech Inc.) as
described
(10) . The protein eluates were dialyzed extensively
against buffer B containing 1 mM DTT and stored at -70
°C. Recombinant yeast TFIIA subunits TOA1 and TOA2 were each
expressed in the strain BL21(DE3) and purified as recommended by the
authors
(22) . The TFIIA heterodimer was further purified by
affinity chromatography on a column containing immobilized recombinant
yeast TBP coupled to Affi-Gel 10 resin (3 mg/ml; Bio-Rad). Bound TFIIA
was eluted with buffer B containing 500 mM NaCl and dialyzed
extensively against buffer B containing 1 mM DTT.
Protein Derivatization with the
Photocross-linker
5-10 µg of LexA-E2F-1 fusion protein
was diluted with buffer C (20% glycerol, 100 mM NaCl, 20
mM HEPES-KOH, pH 7.0) containing 5 mM
-mercaptoethanol and incubated with 50 µl (bed volume) of
buffer C equilibrated Ni
-NTA-agarose beads for 30 min
at room temperature. The beads were then washed three times with 1 ml
of degassed buffer C, and, under reduced lighting,
maleimide-4-benzophenone (Sigma) was then added from a freshly made 20
mM stock solution in dimethyl formamide to a 10-fold molar
excess relative to protein. After incubation in the dark for 4 h at
room temperature, the beads were washed once with buffer C containing 5
mM
-mercaptoethanol. and the bound protein eluted with
buffer B containing 1 mM DTT and 0.5 M imidazole (pH
7.9). The solvent-accessible thiol (i.e. cysteine) residues
were derivatized to
90%, as determined by titration with
Ellman's reagent as recommended by the manufacturer (Pierce). The
photoreactive protein was stored before use in amber microtubes at
-70 °C.
Protein Radiolabeling
10-20 µg of kinase
sequence-tagged TBP or TFIIB was treated with 20-40 units of
heart muscle kinase (catalytic subunit; Sigma) and 20-40 µCi
of [P]ATP (6000 Ci/mmol) (DuPont NEN) in
buffer B containing 1 mM DTT and 10 mM
MgCl
. After a 90-min incubation at 30 °C, the mixture
was loaded onto a NAP-5 gel filtration column (Pharmacia)
pre-equilibrated with buffer B containing 1 mM DTT. The column
was washed continuously with equilibration buffer and the excluded
volume containing the labeled protein collected. Control reactions
using non-sequence-tagged TBP and TFIIB confirmed that the kinase
labeled the recognition sequence tag site-specifically. The labeled TBP
was also found to support both basal and activated transcription in
vitro when added to a TBP-depleted yeast whole cell extract (data
not shown).
Photocross-linking
Template DNA (approximately 0.5
pmol), P-labeled TBP or TFIIB, and, where appropriate,
unlabeled transcription initiation factors were added to 35 µl of
buffer D (12 mM HEPES-NaOH, pH 7.9, 60 mM KCl, 12%
glycerol, 5 mM MgCl
, 1 mM EDTA, 0.6
mM DTT) or yeast transcription buffer (Fig. 5) (23)
contained in the wells of a microtiter plate that had been preblocked
overnight with 10 mM Tris-HCl, pH 7.9, 100 M NaCl,
0.05% (v/v) Tween 20, 0.5% (w/v) gelatin. After incubation for 10 min
at 23 °C, photoreactive LexA-E2F-1 fusion protein was added under
reduced light and the incubation continued for an additional 30 min in
the dark. The plates were then placed on a UV-transilluminator
(Fotodyne; model 3-3100) and irradiated for 5 min to initiate
photolysis. Following irradiation, SDS-PAGE sample buffer was added and
the reaction mixtures transferred into microtubes and boiled.
Cross-linked products were separated by electrophoresis on 10%
polyacrylamide gels containing SDS. The gels were dried and exposed to
film with a single intensifying screen for 12-24 h at room
temperature.
Figure 5:
The
degree of cross-linking correlates with transcriptional activation.
A, top panel, purified recombinant LexA fusion
proteins with either a wild type or mutant truncated E2F-1 activation
domain; the identity of each fusion protein is shown at the top of the
gel. Middle panel, transcriptional activation of the CYC1
reporter template. Bottom panel, UV-induced cross-linking to
P-labeled yeast TBP exhibited by photoreactive derivatives
of each of the fusion proteins shown above and in the presence of the
CYC1 promoter template. The transcription and cross-linking
reactions contained 3 and 9 pmol of the LexA-E2F-1 fusion proteins,
respectively. B, quantitative analysis of the relative
efficiencies of transcriptional activation (lightbars) and cross-linking to TBP (darkbars) exhibited by each of the E2F-1 derivatives shown
above. The derivatives have been aligned arbitrarily to highlight the
correlation between effects of the mutations on transactivation and on
cross-linking.
Immunoprecipitation and DNA Mobility Shift
Assay
For the immunoprecipitation analysis, 25 µl of a
standard cross-linking reaction was diluted with 500 µl of TTBS
(0.05% Tween 20, 10 mM Tris-HCl, pH 7.9, 0.5 NaCl) and
incubated with rabbit antisera (2 µl) for 4 h on ice. Protein
A-Sepharose beads (20 µl; Sigma) were then added and the incubation
continued with rotation for 6 h at 8 °C. The beads were
subsequently washed five times with TTBS and boiled in SDS-PAGE sample
buffer. The bead supernatant was analyzed by electrophoresis on a 10%
polyacrylamide gel containing SDS followed by autoradiography. Addition
of ethidium bromide to 400 µg/ml in the incubation buffer to ensure
a complete inhibition of DNA binding by the proteins did not affect the
precipitation efficiency (data not shown). For the electrophoretic
mobility shift assay, the proteins were assembled in a 20-µl volume
of cross-linking buffer (buffer D) and incubated for 20 min at room
temperature. The reactions were then run on a 5% polyacrylamide native
gel (40:1 mono:bis ratio, 2.5% glycerol) in TGE buffer (25 mM
Tris base, 190 mM glycine, 1 mM EDTA, final pH 8.5).
Following electrophoresis for 2 h at room temperature, the gel was
dried and exposed to film.
In Vitro Transcription
In vitro transcription of the G-less cassette reporter templates was
performed essentially as described
(23) with the following
modifications. Reactions (30 µl) contained 4.5 µl of yeast
whole cell extract (80 mg/ml) prepared as described
(25) from
the strain BJ2168 (a prb1-1122, pep4-3,
prc1-407), as well as 100 µM
3`-O-methyl GTP (Pharmacia), 600 µM each of ATP
and UTP, 20 µM CTP, 20 units of RNase Block I
(Stratagene), 10 units of RNaseT1 (Boehringer), 2.5 µCi of
[-
P]CTP (3000 Ci/mmol) (DuPont NEN), the
appropriate template DNA (15-30 µg/ml), and carrier DNA (10
µg/ml). Reactions were assembled on ice and supplemented with
recombinant transcription factors as required. Transcription was
initiated by the addition of NTPs and allowed to proceed at 23 °C
for 45 min. The reactions were terminated by the addition of 10 µl
of stop buffer (80 mM EDTA, 200 mM NaCl, 2% SDS)
containing 100 µg of proteinase K followed by incubation at 37
°C for 20 min. The nucleic acids were precipitated with carrier
tRNA and isopropanol, boiled in deionized formamide, and separated on
6% polyacrylamide gels containing urea. The gels were dried and exposed
to film with a single intensifying screen overnight at -70
°C.
Generation of the Transactivator
To position a
photoreactive cross-linking reagent uniquely within the activation
domain of a pol II-specific transcriptional activator, we generated a
chimeric activator consisting of the C-terminal acidic activation
domain of the human transcription factor E2F-1 (amino acids
368-437)
(13, 15) fused to the bacterial
sequence-specific DNA-binding protein LexA (amino acids
1-202)
(24) . The resulting fusion protein contained only a
single cysteine residue (Cys-427 in E2F-1), which allowed for the
site-directed introduction of a thiol-reactive cross-linking reagent at
a defined position within the transactivator. We reasoned that the
Cys-427 residue would be exposed on the surface of the E2F-1 activation
domain likely to interact with the pol II transcription apparatus,
since it is located within the core of the E2F-1 activation domain and
is immediately adjacent to the binding site for the retinoblastoma gene
product, a repressor of activation by E2F-1
(15) . As expected,
purified recombinant LexA-E2F-1 (Fig. 1A, lane2) was found to be a potent sequence-specific
transcriptional activator of the yeast CYC1 promoter in in
vitro reactions using a transcription competent yeast whole cell
extract (Fig. 1B, compare top and bottompanels). Activation was fully attributed to the E2F-1
activation domain since LexA alone did not stimulate transcription in
this system (Fig. 1B). The LexA-E2F-1 fusion protein was
then derivatized with the thiol-specific heterobifunctional
photocross-linking reagent maleimide-4-benzophenone, which has been
used extensively to study protein-protein interactions in vitro (see Ref. 25, and references therein). This permitted UV-induced
covalent cross-linking of LexA-E2F-1 to proteins that are in close
proximity to the E2F-1 activation domain (i.e. within a
10-Å radius from Cys-427 of E2F-1; Ref. 25). Importantly,
introduction of this cross-linking reagent onto >90% of the
LexA-E2F-1 molecules (see ``Experimental Procedures'') did
not impair the ability of the LexA-E2F-1 preparation to activate in
vitro transcription (Fig. 1C).
Figure 1:
Purified transcription factors.
A, purified recombinant proteins (2 µg of each) used in
these studies. Lane 1, LexA; lane 2, LexA-E2F-1
(full-length activation domain); lanes 3-5, kinase
recognition sequence-tagged yeast TBP, human TBP, and yeast TFIIB,
respectively; lanes 6-8, yeast TFIIA, human TFIIB, and
yeast TFIIB used in the competition assays. The proteins were run on a
12.5% polyacrylamide gel containing SDS and stained with Coomassie
Blue. The sizes of the protein markers (M) are given to the
right in kDa. B, sequence-specific transcriptional
activation by the LexA-E2F-1 fusion protein. RNA transcripts
(arrowheads) produced by in vitro transcription from
the yeast CYC1 promoter, with (upper panel) or
without (lower panel) two upstream LexA-binding sites, in
reactions containing yeast whole cell extract supplemented with either
buffer alone (-), LexA or LexA-E2F-1 (5 pmol of each).
C, in vitro transcription driven from the CYC1 promoter in reactions supplemented with buffer alone or with
LexA-E2F-1 protein (5 pmol) that had either been derivatized with the
cross-linker (MBP) or had been mock-treated
(MOCK).
Promoter-dependent Cross-linking of LexA-E2F-1 to
TBP
Since the activation domain of E2F-1 had been shown to
interact with TBP in solution
(16) ,()
we
assessed the ability of LexA-E2F-1 to interact with TBP when bound
upstream of a pol II promoter. To allow the detection of both free and
cross-linked TBP species to be readily followed, the TBP used in these
experiments was first radiolabeled with
P in vitro (see ``Experimental Procedures''). CYC1 promoter template DNA, which was responsive to LexA-E2F-1 in the
in vitro transcription system, was incubated with
P-labeled yeast TBP and with photoreactive LexA-E2F-1 at a
concentration (
170 nM) similar to that used in the in
vitro transcription analysis. The assembled ternary complexes were
UV-irradiated to initiate photolysis and the resulting cross-linked
protein complexes subsequently detected by SDS-PAGE and
autoradiography.
6-fold) when a
control template that lacked LexA binding sites was used instead
(lane3). The residual cross-linking that occurred in
the absence of the activator binding sites (lane3)
may reflect a less stable interaction of the activator with TBP in
solution
(16) . As described below, we also found that LexA-E2F-1
could be cross-linked to human TBP in a similar binding site-dependent
manner (see below), a result consistent with the evolutionary
conservation of the structure of TBP and the fact that E2F-1 is a
transactivator of human origin.
Figure 2:
Promoter-dependent cross-linking of an
activator to TBP. A, SDS-PAGE fractionation of UV-irradiated
mixtures that contained P-labeled yeast TBP (3 pmol) and,
as indicated, photoreactive LexA-E2F-1 (7.5 pmol) and CYC1 promoter DNA with or without two upstream LexA-binding sites. The
position of the cross-linked LexA-E2F-1-TBP heterodimer complexes
(bracket), free TBP (arrowhead), and an
activator-independent complex (*), possibly representing a TBP
homodimer, produced in the cross-linking reaction are indicated.
B, cross-linking reactions, as in lane 2 of
A, were precipitated with antisera specific to either yeast
TBP, LexA, or BRF1, an unrelated RNA polymerase III transcription
factor. Where shown, recombinant yeast TBP and TFIIB (30 µg of
each) were added as competitor just prior to immunoprecipitation to
characterize the specificity of the TBP
antisera.
Specificity of Covalent Cross-linking of E2F-1 to
TBP
The specificity of the photocross-linking reaction was
evaluated in a series of control experiments. As expected,
cross-linking of the activator to TBP was found to be highly contingent
upon both UV irradiation and derivatization of the activator with the
cross-linking reagent (Fig. 3A, compare lanes1 and 4 with lanes2 and
3) since only a low background level of cross-linking occurred
in their absence. Cross-linking could be specifically competed with an
excess of unlabeled TBP (Fig. 3B, lanes2 and 3) but not with the same quantity of the
similarly charged protein lysozyme (lanes4 and
5), indicating that the interaction was specific. The
C-terminal 37 amino residues of E2F-1, which contain Cys-427, were
sufficient for cross-linking to TBP (Fig. 3A, lane
5), consistent with the observations that this same region of
E2F-1 can function as an activation domain both in mammalian cells
in vivo(15) and in a yeast cell-derived extract in
vitro (Fig. 3C, lane 2). In this context,
mutation of the reactive cysteine residue to alanine (C427A) reduced
the level of cross-linking to TBP to a background level
(Fig. 3B, lane6), although it did not
noticeably affect transcriptional activity in vitro (Fig. 3C, lane3). Interestingly,
cross-linking could be restored only partially by the introduction of a
cysteine residue at position 420 in E2F-1 (G420C) and very poorly when
introduced at position 411 (Y411C) (Fig. 3A, lanes6 and 7), although both mutants strongly
activated transcription in vitro (Fig. 3C,
lanes4 and 5). These combined results
confirm the specificity of the original cross-linking protocol and
suggest that the naturally occurring cysteine in E2F-1 lies near or
within the activation domain surface of E2F-1 that contacts TBP,
although this residue is not essential per se for activation
domain function.
Figure 3:
Site-specificity of the
photocross-linking. A, SDS-PAGE analysis of cross-linking
reactions that contained CYC1 promoter DNA bearing two
upstream LexA-binding sites, P-labeled yeast TBP (3 pmol),
and LexA-E2F-1 fusion proteins (7.5 pmol). LexA fusion protein
containing a full-length (amino acids 368-437) E2F-1 activation
domain was treated with the cross-linking reagent (MBP) and
subjected to UV-irradiation as indicated (lanes 1-4).
The remaining reactions (lanes 5-8) contained, as
indicated above each lane, photoreactive LexA fusions to either a wild
type or mutant truncated (amino acids 400-437) E2F-1 activation
domain and were all subjected to UV-irradiation. B,
cross-linking reactions performed as in lane 1 of A,
both in the absence (lane 1) and presence of 30 or 60 pmol
each of unlabeled TBP (lanes 2 and 3) or lysozyme
control protein (lanes 4 and 5). C, in
vitro transcription of template DNA containing two LexA-binding
sites upstream of the CYC1 promoter in reactions supplemented
with the LexA-E2F-1 derivatives shown in lanes 5-8 of
A (4 pmol of each).
Cross-linking Requires a TATA Element
To test the
generality of the promoter-dependent interaction observed between
LexA-E2F-1 and TBP, we extended our analysis to the adenovirus major
late (Ad2ML) promoter, which has been used extensively in in vitro studies of transcription and which contains a single TATA-element,
unlike the CYC1 promoter, which has three distinct TATA elements. As
with the CYC1 promoter constructs, purified LexA-E2F-1 could
both strongly activate in vitro transcription
(Fig. 4A, compare top and bottompanels) and be cross-linked to TBP
(Fig. 4B) in a sequence-dependent manner at the Ad2ML
promoter. Quantitation of the data shown in Fig. 4B indicated that 0.15 pmol of TBP was cross-linked to the activator
in the presence of 0.5 pmol of Ad2ML promoter DNA. Since the majority
of the cross-linked complexes formed in a promoter-dependent manner, we
infer that a productive (i.e. cross-linkable) interaction
occurred between the activator and TBP at nearly 30% of the available
promoters. To confirm that the TBP that had been cross-linked to the
activator had itself been in contact with the promoter (i.e. bound to the TATA element), we compared the ability of LexA-E2F-1
to interact with TBP at a promoter containing either a wild type TATA
element (derived from the HIS3 gene T promoter; Ref. 21) or one of two mutant TATA elements previously
shown to be defective for binding to TBP
(21, 27) . As
seen in Fig. 4D, cross-linking of LexA-E2F-1 to TBP was
largely restricted to the promoter bearing a functional (i.e. wild type) TATA-element. Of the three templates studied, only this
same construct was transcriptionally responsive to LexA-E2F-1 in
vitro (Fig. 4C). Therefore, a physical and
functional interaction between LexA-E2F-1 and TBP occurred
preferentially when each of these factors was bound to their respective
promoter elements.
Figure 4:
Interaction of the activator with TBP at
other promoters. A, RNA transcripts produced by in vitro transcription of Ad2ML promoter template DNA with (upper
panel) or without (lower panel) two upstream LexA-binding
sites in reactions supplemented with buffer alone (-) or with
LexA or full-length LexA-E2F-1 (5 pmol of each). B, SDS-PAGE
fractionation of UV-irradiated mixtures that contained photoreactive
full-length LexA-E2F-1 (7.5 pmol), P-labeled yeast TBP (3
pmol), and Ad2ML promoter DNA with (lane 1) or without
(lane 2) two upstream LexA-binding sites. C,
transcriptional activation of the yeast HIS3 gene
T
promoter bearing a wild type (TATAA) or a mutant
(TGTAA, TCTAA) TATA element. D, SDS-PAGE fractionation of
UV-irradiated mixtures that contained
P-labeled yeast TBP
(3 pmol), photoreactive LexA-E2F-1 (7.5 pmol), and DNA bearing two
LexA-binding sites upstream of the HIS3 T
promoter
that had either a wild type or mutant TATA
element.
Cross-linking Correlates with Transactivation
To
ascertain the functional relevance of this promoter-dependent
interaction between LexA-E2F-1 and yeast TBP, we analyzed the effects
of a series of mutations in the E2F-1 activation domain on both in
vitro transcriptional activation and cross-linking by LexA-E2F-1.
A total of 10 mutant derivatives were expressed and purified as fusions
to LexA (Fig. 5A, toppanel), all of
which bound DNA with a similar efficiency relative to the wild type
LexA-E2F-1 fusion (data not shown). We first established the relative
strengths of the wild type and mutant LexA-E2F-1 derivatives to
activate in vitro transcription from the CYC1 reporter gene. Consistent with the results of transcriptional
studies performed in vivo (14-16), the different E2F-1
activation domain mutants exhibited a range of transcriptional activity
in vitro relative to the wild type construct
(Fig. 5A, middlepanel). The proteins
were each then derivatized with the cross-linking reagent and assessed
for their ability to interact with TBP at the same promoter
(Fig. 5A, bottompanel). To a large
extent, the degree of promoter-dependent cross-linking to TBP achieved
with each of the LexA-E2F-1 derivatives correlated directly with their
respective abilities to activate transcription in vitro (for a
quantitative comparison, see Fig. 5B). For example, the
mutations in the activation domain of E2F-1 that exhibited the greatest
reduction in cross-linking to TBP (e.g. L415P/L424P,
Y411A/F413A) were the same mutations that most dramatically impaired
transcriptional activation. On the other hand, mutants that displayed a
more modest reduction in cross-linking to TBP (e.g. F429P,
del420-422/Y411A) had a correspondingly less pronounced effect on
transactivation. While, in general, the mutations had a more pronounced
effect on transcriptional activation in vitro as compared to
their effect on transcriptional activation in
vivo(14, 15, 16) and on cross-linking to
yeast TBP, taken on the whole, the results of this analysis support the
notion that the promoter-dependent interaction of LexA-E2F-1 with TBP
plays an important role in the transactivation process. One mutant
(T433A/P434A) that was impaired in its ability to activate
transcription in vitro did not, however, display a noticeably
reduced ability to be cross-linked to TBP. This suggests that the
activation domain of E2F-1, like the activation domain of VP16, may
also interact with additional components of the pol II transcriptional
apparatus such as TFIIB
(5) , TFIIH
(6) , or TAFs
(7) during the transactivation process.
Effects of TFIIA and TFIIB on Cross-linking
The
transcription stimulatory factor TFIIA and the general initiation
factor TFIIB can each interact independently with TBP at a promoter
(reviewed in Ref. 1). To investigate whether LexA-E2F-1 could still
bind to TBP that was present in such early intermediates of
preinitiation complex assembly, we performed the cross-linking reaction
in the presence of purified TFIIA or TFIIB.
Figure 6:
Effects of TFIIA and TFIIB on the
activator-TBP interaction. A, nondenaturing polyacrylamide gel
electrophoresis showing binding of yeast TBP (25 ng) to
P-labeled Ad2ML promoter DNA both in the absence or
presence of yeast TFIIA, yeast TFIIB, and human TFIIB (50 ng of each).
DNA-bound complexes containing TBP (D), TFIIB (B),
and/or TFIIA (A) are indicated to the right.
B, SDS-PAGE fractionation of UV-irradiated mixtures that
contained
P-labeled yeast TBP (3 pmol), photoreactive
full-length LexA-E2F-1 (7.5 pmol), CYC1 promoter DNA with or
without two LexA binding sites, and yeast TFIIA (yIIA) or
TFIIB (yIIB) (3 pmol of each) as indicated. C,
SDS-PAGE fractionation of UV-irradiated mixtures that contained
P-labeled yeast TBP (3 pmol), photoreactive full-length
LexA-E2F-1 (7.5 pmol), and Ad2ML promoter DNA with or without two
upstream LexA-binding sites. The reactions were supplemented with yeast
TFIIA and yeast TFIIB (3 pmol of each) as indicated. D,
SDS-PAGE fractionation of UV-irradiated mixtures that contained
P-labeled human TBP (3 pmol), photoreactive full-length
LexA-E2F-1 (7.5 pmol), CYC1 promoter DNA with or without two
upstream LexA-binding sites, and either yeast TFIIA or human TFIIB
(huIIB) (3 pmol of each).
Since TFIIB has itself been reported to bind
directly with acidic transcriptional activators
(5) , we
performed an analogous series of cross-linking experiments using
P-labeled TFIIB, as well as TBP, as the target. As shown
in Fig. 7A, LexA-E2F-1 could be cross-linked to yeast
TFIIB (compare lanes4 and 7 to lane3) in a manner almost as efficient as it was to yeast TBP
(lanes2 and 5). This interaction, like that
of LexA-E2F-1 with TBP, appeared to be specific in that it could be
competed with an excess of unlabeled TFIIB (Fig. 7B,
lanes2 and 3) or TBP (lanes6 and 7) but not with a similar amount of the control
protein lysozyme (lanes4 and 5). While
these results are consistent with the notion that TFIIB might also be a
target of the activation domain of E2F-1, we note that an essentially
identical level of cross-linking of LexA-E2F-1 to TFIIB occurred both
in the presence (Fig. 7A, lanes4 and
5) or absence of TBP (lanes6 and
7) or activator binding sites in the template DNA (lanes6 and 4). Therefore, unlike its interaction with
TBP, the interaction of LexA-E2F-1 with TFIIB did not exhibit any
promoter dependence.
Figure 7:
Cross-linking of the activator with TFIIB.
A, SDS-PAGE fractionation of UV-irradiated mixtures that
contained, as indicated, P-labeled yeast TFIIB and/or
yeast TBP (3 pmol of each), photoreactive full-length LexA-E2F-1 (7.5
pmol), and CYC1 promoter DNA with or without upstream
LexA-binding sites. Cross-linked complexes containing LexA-E2F-1
covalently attached to either TFIIB or TBP are indicated by the
arrow and bracket, respectively. B,
cross-linking of LexA-E2F-1 to TFIIB in the absence (lane 1)
or presence of 30 or 60 pmol of unlabeled TFIIB (lanes 2 and
3), yeast TBP (lanes 6 and 7), or lysozyme
(lanes 4 and 5).
for activator-TBP interactions
estimated previously (2
10
M
)
(4) . Our experiments
provide two additional lines of evidence that the ability of an
activator to interact with promoter bound TBP is biologically relevant.
First, LexA-E2F-1 was found to interact preferentially with TBP at
three distinct pol II promoters that were responsive to this activator.
Second, we found that mutations that reduced the ability of LexA-E2F-1
to be cross-linked to TBP at the CYC1 promoter concomitantly
affected the ability of the activator to stimulate transcription in
vitro from this same promoter.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.