©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Promoter-dependent Photocross-linking of the Acidic Transcriptional Activator E2F-1 to the TATA-binding Protein (*)

Andrew Emili , C. James Ingles (§)

From the (1) Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

RNA polymerase II (pol II)() 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.

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 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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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.

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 (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.

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.


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).




DISCUSSION

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 for activator-TBP interactions estimated previously (2 10M) (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.

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.


FOOTNOTES

The abbreviations used are: pol II, RNA polymerase II; TBP, TATA-binding protein; PAGE, polyacrylamide gel electrophoresis; Ad2ML, adenovirus 2 major late; DTT, dithiothreitol.

A. Pearson, J. Archambault, and J. Greenblatt, personal communication.

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: C. H. Best Institute, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada. Tel.: 416-978-7400; Fax: 416-978-8528.


ACKNOWLEDGEMENTS

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.


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