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Molecular Endocrinology 11 (10): 1467-1475
Copyright © 1997 by The Endocrine Society

Involvement of the Transcription Factor IID Protein Complex in Gene Activation by the N-Terminal Transactivation Domain of the Glucocorticoid Receptor in Vitro

Jacqueline Ford, Iain J. McEwan, Anthony P. H. Wright and Jan-Åke Gustafsson

Department of Biosciences at Novum, Karolinska Institute, S-141 57 Huddinge, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HeLa cell nuclear extracts were used to study the mechanism of activation of RNA polymerase II-mediated transcription by the N-terminal transactivation domain ({tau}1) of the glucocorticoid receptor in vitro. When fused to the Gal4 DNA-binding domain, the {tau}1 domain activated transcription approximately 9-fold in HeLa nuclear extracts. Using heat treatment to inactivate transcription factor IID (TFIID) in the extract, it was shown that the addition of purified TFIID complex, but not the TATA-binding protein alone, was sufficient to restore this level of activation. The {tau}1 domain was shown to interact directly with the TFIID complex. This interaction was markedly reduced by a mutation in the {tau}1 domain that reduces its activity. Furthermore, the interaction was specific for the TFIID complex, since no interaction was seen with TFIIIB, an analogous protein complex involved in RNA polymerase III transcription. The {tau}1 domain was further shown to interact with the TATA-binding protein subunit of the TFIID complex. These results suggest a mechanism by which the GR {tau}1 domain might contribute to gene activation by recruitment of the TFIID complex to target promoters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors that function by modulating the activity of target genes (for reviews see Refs. 1 and 2). The amino terminus of GR contains a domain that is essential for full transactivation activity (3, 4, 5, 6) and constitutes amino acids 77–262 of the human GR (hGR) (4). When fused to a DNA binding domain, this domain ({tau}1) is constitutively active for transcription both in vivo in yeast (7) and in vitro in yeast extracts (8). The {tau}1 domain has been further dissected, and a single core region containing transactivation activity has been localized ({tau}1 core, residues 187–244 of hGR). Particular segments within this region have a propensity for {alpha}-helical conformation, and hydrophobic amino acids that would form patches on these putative {alpha}-helices appear to be important for the molecular mechanism by which {tau}1 transactivates (9, 10, 11).

Stimulation of eukaryotic transcription by RNA polymerase II requires removal or restructuring of chromatin and the ordered assembly of a preinitiation complex at the promoter of regulated genes (12). The preinitiation complex contains basal transcription factors, RNA polymerase II (see Refs. 13 and 14), and additional accessory factors such as coactivators. In vivo, in yeast and mammalian cells, it has been suggested that this complex may be largely preassembled in the form of a holoenzyme (15, 16, 17).

Transcriptional activators may function at one or more steps in the processes of transcriptional initiation and elongation. However, one important way in which many activators are thought to operate is by interacting with component(s) of the transcription machinery to recruit them to the promoter and so promote or stabilize the binding of the general transcription factors to the DNA (see Refs. 12 and 18). Interactions may occur directly between basal transcription factors and the activator and/or may occur between the activator and coactivators or adaptors, which form a bridge between the activator and the basal factors (for review, see 19 .

There is a growing number of reports concerning interactions between nuclear receptors and coactivator proteins, and for some, but not all, direct interactions have been shown with GR. Thus the mouse protein GRIP1 (20) and the human proteins GRIP170 (21) and RAP46 (22) interact directly with GR and may act as coactivators. GR transactivation can also be modestly stimulated by SRC-1 (23), which possibly occurs in a tertiary complex involving CBP (CREB binding protein) and GR (24). Additional proteins that can coactivate with GR include hbrm (a human homolog of the yeast SWI/SNF proteins), which is thought to be involved in chromatin structure modulation (25), and the yeast proteins SPT3 and RSP5 (26). A factor RAF has also been isolated that potentiates DNA binding by GR (27).

In contrast to the identification of these potential coactivators for GR, there are still no reported interactions between GR and specific basal transcription factors. In addition, no factors are reported to bind directly to the {tau}1 domain of GR despite the fact that 1) it is constitutively active when fused to a DNA-binding domain (DBD) and therefore must make sufficient interactions to activate transcription; 2) it is the domain of GR that contributes most to the receptor’s transactivation activity (4), and 3) studies on GR (and specifically {tau}1) suggest that it can act by recruitment and stabilization of the preinitiation complex at the promoter (28, 29, 30), possibly via some direct interaction(s) with component(s) of the basal transcription machinery (8).

In recent years, much attention has been focused on interactions between activators and TFIID. TFIID is a multiprotein complex containing TATA box-binding protein (TBP) and tightly associated factors termed TBP-associated factors (TAFs). TBP is a target for many activators (for examples, see Refs. 31 and 32), but the TAFs are necessary for activation in vitro and are also direct targets for many transactivators (for reviews, see Refs. 33–35). In this study we investigate whether TFIID might play a role in gene activation by the GR {tau}1 domain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TFIID Is Required for Activation by {tau}1 in HeLa Nuclear Extracts
We wished to determine whether {tau}1 requires TFIID for activation in vitro. It has been reported previously that {tau}1, fused to a heterologous DBD, activates transcription in vitro in yeast nuclear extracts (8). In this study we used a HeLa system because it has been reported that TBP and TFIID are heat labile in HeLa extracts (36). We constructed plasmids for the expression and purification of amino acids 1–100 of Gal4 and a fusion protein in which {tau}1 is fused to Gal(1–100). The Gal4 sequence is preceded by a histidine tag, which simplified purification of the proteins (Fig. 1AGo). In vitro transcription experiments were performed in HeLa nuclear extracts with template pG5HMC2AT (37) in which five Gal4-binding sites are situated upstream of the HIV TATA box. A titration of Gal4-{tau}1 protein gave a dose-dependent increase in transcription products that required the presence of the {tau}1 activation domain (Fig. 1BGo). The addition of 8 pmol Gal4-{tau}1 consistently produced 7- to 10-fold activation in our experiments (see Fig. 2BGo, lanes 1 and 2).



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  		Figure 1. Gal(1–100)-{tau}1 Activates Transcription in HeLa Nuclear Extracts

A, Coomassie-stained gel showing purified Gal(1–100) and Gal(1–100)-{tau}1 proteins. B, In vitro transcription experiment in HeLa nuclear extracts using template pG5HMC2AT (37). Gal(1–100) or Gal(1–100)-{tau}1 was added to the reactions as indicated. Transcripts from the Gal template are marked with an arrowhead.

 


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  		Figure 2. Purified TFIID Efficiently Restores {tau}1-Dependent Activation to Heat-Treated HeLa Nuclear Extracts

A, Western blot using an anti-TBP antibody and showing the relative intensities of TBP contained in 1 U crude TFIID (from 1 M P11 fraction; labeled 1 M P11), 1 U affinity-purified TFIID (labeled TFIID), 1 U rhTBP, and 10 U rhTBP. B, In vitro transcription from HeLa nuclear extracts. Extracts were heated at 46 C or 47.5 C as shown. After cooling they were either not supplemented or supplemented with 1 U TFIID obtained from the 1 M P11 fraction of HeLa nuclear extracts or with 1 U affinity-purified TFIID or with 1 or 10 U of rhTBP. Template DNA, nucleotide triphosphates, and Gal(1–100)-{tau}1 (8 pmol, where indicated) were then added, and the transcription reactions were allowed to proceed. Transcripts from the Gal responsive template pG5HMC2AT in the presence (+) or absence (-) of Gal(1–100)-{tau}1 (8 pmol), under different supplementation conditions, are marked (G). Fold induction (+{tau}1/-{tau}1) mediated by the addition of Gal(1–100)-{tau}1 is shown for the different supplementation conditions. Experiments have also been performed using a basal template as an internal standard for the level of {tau}1-dependent activation in reconstitution reactions (data not shown). The levels of activation under the different reconstitution conditions in these experiments were very similar to those shown for parallel reactions, ± {tau}1.

 
To test the functional requirement of {tau}1 for TBP or TFIID, HeLa nuclear extracts were heat treated, recooled, and then supplemented with either recombinant human TBP (rhTBP) or TFIID. TFIID was either supplied as the crude 1 M KCl phosphocellulose (1 M P11) fraction, or alternatively it was immunoaffinity purified from the 1 M P11 fraction of LTR{alpha}3 cells, which express HA-tagged TBP (38). The relative amounts of TBP contained in the crude 1 M fraction and affinity-purified material were estimated relative to rhTBP by semiquantitative Western blotting using an antibody against hTBP (data not shown). Approximately equal molar amounts of TFIID and rhTBP (Fig. 2AGo; arbitrarily given the value of 1 U and approximately equal to 2.5 ng TBP) were used to supplement the heat-treated nuclear extracts. In addition, 10-fold higher amounts of rhTBP were used (Fig. 2AGo). Thus, in Fig. 2BGo, {tau}1 activated transcription in untreated HeLa nuclear extracts by a factor of 9-fold (Fig. 2BGo, lanes 1–2). After the crude HeLa nuclear extracts had been heat-treated in a 46 C water bath, {tau}1-dependent transactivation was lost, consistent with the destruction of an essential component of the transcription machinery (Fig. 2BGo, lane 3). Transcriptional activity was restored by addition of both TFIID and rhTBP, but only TFIID restored {tau}1-dependent stimulation of transcription to the starting level (Fig. 2BGo, lanes 4–7). rhTBP restored basal transcription but had very little effect on {tau}1-activated levels (Fig. 2BGo, lanes 8–11). At 47.5 C the distinction between TFIID and TBP is even clearer such that even a 10-fold excess of TBP does not restore detectable transcription levels while TFIID still reconstitutes {tau}1-dependent activated transcription (Fig. 2BGo, lanes 12–16). The level of reconstituted transcription at 47.5 C is somewhat lower than at 46 C, and it is possible that the higher temperature affects the activity of other transcriptional components.

{tau}1 Interacts with TFIID Complexes Present in Cell Extracts
We next wished to determine whether or not {tau}1 would interact physically with TFIID. As a control, we wanted to test the ability of {tau}1 to discriminate between TFIID and TFIIIB (the analogous complex specific for transcription by RNA polymerase III). Using yeast strains that express epitope-tagged TAFs (39), we prepared cell extracts containing either hemagglutinin (HA)-tagged yTAFII130 or Brf1 (a polymerase III-specific TAF). When these were incubated with the glutathione-S-transferase (GST) activation domain constructs (Fig. 3AGo), both wild type GST-{tau}1 and wild type GST-{tau}1core clearly interacted with TFIID as indicated by coprecipitation of yTAFII130 (Fig. 3BGo). The interactions were severely weakened when reduced activity mutant (H1A) {tau}1 or {tau}1core fusion proteins were used (Fig. 3BGo). In the H1A mutant proteins, five hydrophobic residues in {tau}1core are substituted by alanine, causing reduced transactivation activity in yeast and mammalian cells (11). Under identical conditions, negligible interaction was observed with TFIIIB (Brf1, Fig. 3CGo). Thus, the interaction was selective for the TFIID complex over TFIIIB and could be disrupted by reduced activity mutations within {tau}1core.



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  		Figure 3. {tau}1 Interacts with TFIID Complexes

A, Coomassie-stained gel of GST activation domain proteins used in panel B. {tau}1c refers to {tau}1core, and H1A indicates a mutated protein in which five hydrophobic amino acids have been changed to alanine. B, GST-fusion proteins (shown in Fig. 3AGo) were incubated with yeast extracts expressing HA-tagged yTAFII130. The protein in the load (L) and supernatants (S) were in each case in 6 times the volume of the washed bead pellets (P), and therefore the P lanes are 6-fold concentrated relative to the L and S lanes. The fractions were analyzed by SDS-PAGE and Western blotting using monoclonal antibody 12CA5 against the HA tag of yTAFII130. C, A control experiment was performed as described for panel B except that yeast extracts expressing HA-tagged Brf1 were used. D, GST-{tau}1 was tested for binding to TBP-containing complexes in fractionated HeLa nuclear extracts. The protein in the load (L) and supernatants (S) were in each case in 6 times the volume of the washed bead pellets (P). The fractions were analyzed by SDS-PAGE and Western blotting using a monoclonal antibody against hTBP. Double bands are observed since the extracts were prepared from LTR{alpha}3 cells that express approximately 70% HA-tagged TBP and 30% nontagged TBP (38).

 
We next wanted to investigate the interaction of {tau}1 with mammalian complexes, and thus HeLa nuclear extracts were prepared. To partially separate different complexes, the nuclear extracts were fractionated on phosphocellulose (P11) (40), and 0.3 M and 1 M KCl fractions were collected. The 0.3 M fraction contains TFIIIB, but also additional complexes that have been reported to include B-TFIID (41) and a subpopulation of TFIID complexes, termed TFIID{alpha} (42). B-TFIID is not thought to contribute to polymerase II transcription (see 41 , and TFIID{alpha} has only been reported to have a stimulatory effect on the activator TEF-1 (43). The 1 M KCl fraction contains additional TFIID complexes including TFIIDß (42). TFIID complexes from the 1 M fraction have been described to be involved in transactivation by a number of activation domains including VP16, the estrogen receptor activation domains, the AH synthetic activator, and activation domains from Zta, E1A, and Sp1 (37, 38, 43). The fractions were incubated with GST-{tau}1, and the supernatants and pellets were probed for TBP using Western blotting and an anti-TBP antibody. While the interactions detected in these experiments were weak, we observed preferential binding of GST-{tau}1 to TBP-containing complexes from the 1 M P11 fraction, and no interactions were detected with TFIIIB complexes in the 0.3 M P11 fraction under the same conditions (Fig. 3DGo). Taken together, these results suggest that the {tau}1 domain of GR can interact specifically with TFIID complexes involved in transcription by RNA polymerase II.

The {tau}1 Domain of GR Can Interact with Recombinant Human TBP
Transactivation domains from a number of activator proteins have been reported to interact with TFIID, either via interactions with the TBP subunit or with the TAF subunits (see Introduction). Although we have not so far seen reproducible interactions between {tau}1 and TAF subunits (see Discussion), we have observed interactions between {tau}1 and TBP. Similar to the interactions with TFIID shown in Fig. 3BGo, TBP interacts with the GST-{tau}1 and GST-{tau}1core fusion proteins but not with GST alone (Fig. 4AGo). Interaction of both proteins with TBP is severely reduced by the H1A mutation that substantially reduces the activity of the {tau}1 and {tau}1core domains in vivo (11). As a further control, the binding of GST-hTBP to a series of LexA DBD fusion proteins was tested since it is known that the Lex-{tau}1c fusion protein is transcriptionally active in yeast both in vivo (9) and in vitro (I. J. McEwan, unpublished results). Purified GST-hTBP or GST alone (Fig. 4BGo) was immobilized on glutathione-agarose beads, and equivalent amounts of beads were incubated with recombinant purified Lex proteins (Fig. 4CGo). Binding of the Lex fusion proteins was then detected by Western blot using an antibody to LexA DBD. The results showed that there was a strong interaction between Lex-{tau}1core and GST-TBP that was strongly reduced by the H1A mutant (Fig. 4DGo, upper panel). There was no interaction between LexA DBD alone and GST-TBP (Fig. 4DGo, upper panel) or between GST and any of the LexA fusion proteins tested (Fig. 4DGo, lower panel).



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  		Figure 4. {tau}1 Interacts with TBP

GST proteins were immobilized on glutathione agarose and tested for binding to recombinant purified hTBP (A) or recombinant purified LexA fusion proteins (D). The protein remaining in the supernatants (S) after incubation and the protein pelleted with the beads (P) after extensive washing were analyzed by SDS-PAGE and Western blotting. In each case, the pellet fractions were 8-fold concentrated relative to the load and supernatant fractions. A, Western blot using an antibody against hTBP and showing the interactions of hTBP with the GST activation domain proteins shown in Fig. 3AGo. The diluted hTBP loaded onto the beads is marked L. B, Coomassie-stained gel of GST and GST-TBP proteins used in panel D. C, Western blot using an antibody against LexA DBD showing the Lex fusion proteins used in panel D. D, Western blots using an antibody against LexA DBD and showing the results of incubation of the Lex fusion proteins with GST-TBP-bound beads (upper panel) or GST-bound beads (lower panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study we show that the TFIID complex is necessary for the transactivation activity of the GR {tau}1 domain in vitro. The {tau}1 domain can interact with TFIID directly, and at least part of the interaction appears to involve direct contact between {tau}1 and TBP. The physical interactions observed are specific for the TFIID complex since no interaction was observed with the analogous complex (TFIIIB) involved in transcription of RNA polymerase III genes. Furthermore, a reduced activity mutant form of {tau}1 was severely reduced in its ability to interact with both the intact TFIID complex and the TBP subunit. This mutant also reduces hormone-dependent gene activation by the intact GR in transient transfection experiments in mammalian cells (11).

These findings are significant because no target factors have previously been identified that might account for the activity of the GR N-terminal transactivation domain. Early studies showed that the N terminus of the GR (3, 4, 5, 6), and the {tau}1 domain in particular (4), is critical for glucocorticoid signaling such that a receptor derivative lacking {tau}1 was reported to have only 5–10% of wild type activity (4). The TFIID complex has been shown to be important for the activity of a number of activator proteins in vitro (see Introduction). Evidence from these studies suggests that interaction with the activator protein helps to recruit TFIID to the promoter and thus facilitates binding of TBP to DNA and subsequent assembly of the transcription complex. The importance of recruiting TBP is also suggested by in vivo experiments in yeast, which show that artificial recruitment of TBP alleviates the requirement for activator proteins (45, 46). The TFIID recruitment mechanism could help to explain previous reports in which both the intact GR and the isolated N-terminal transactivation domain have been shown to facilitate transcription complex formation in vitro (28, 29, 30).

Recent evidence from studies in yeast suggests that TFIID may not be important for activation of all genes in vivo (47, 48). The data show that while TBP was essential for activation of all the genes tested, mutations that drastically reduced the levels of the TAF subunits had no effect on the activation of most of the genes studied. However, the TAFs are essential proteins, and it appears that they are absolutely required for in vivo activation of some genes in both yeast and Drosophila (47, 48, 49). Thus recruitment of TFIID by the {tau}1 domain is likely to play a role in the activation of at least some GR-responsive genes in vivo. The yeast results suggest that there may be other strategies to ensure the recruitment of TBP that do not involve the TAF subunits. One possibility is that activator proteins interact directly with TBP (as we have shown for {tau}1) and thus recruit it to transcription complexes independently of the TAF subunits. While the simplicity of this model is attractive, the interaction between activators (including {tau}1) and TBP is relatively weak, and so far there is no evidence to suggest that it would be sufficient to recruit TBP independently in vivo. The relative weakness of interactions between activation domains and TBP may explain why the {tau}1-TBP interaction was not detected previously using less sensitive methods (8). Interestingly, it has been reported that interaction of the VP16 activation domain with endogenous TBP in yeast nuclear extracts requires an accessory protein called Ada2 (50), which has also been identified in human cells (51). Ada2 is part of a transcriptional adaptor complex, one function of which appears to be to facilitate the recruitment of TBP by activator proteins. Thus the Ada adaptor could represent an alternative to TFIID and facilitate recruitment of TBP to TFIID-independent promoters. Interestingly, we have shown in a parallel study that the Ada adaptor also plays a role in the function of the GR {tau}1 domain (52).

As stated in the Introduction, many activators that function via TFIID have been shown to make contacts with the TAF subunits of the TFIID complex. Within the family of nuclear receptors, the estrogen receptor (ER) has been shown to interact with human TAFII30{alpha} (42) while the progesterone (PR), retinoid X (RXR), and thyroid hormone (TR) receptors interact with Drosophila TAFII110 (53, 54, 55). The interaction of TAFII110 with the PR is with the DBD and thus although it may be important it does not explain the function of the activation domains. TAFII110 was recently shown to interact with the ligand-binding domain of the TR and although the interaction does not require the AF-2 activation function, TAFII110 did enhance transactivation activity substantially. In the case of RXR, TBP interacted more strongly than TAFII110, and the TBP interaction required the AF-2 transactivation domain while the TAFII110 did not. In similar studies to those above, we have tried to identify interactions with a range of Drosophila and human TAFs, but no reproducible interactions have been observed. Thus, while we cannot rule out that {tau}1 interacts with TFIID via TAFs, our present results indicate a role for interactions between {tau}1 and the TBP subunit of TFIID, as has been described for the RXR AF-2 activation domain.

It could be suggested that our observation of direct interactions between {tau}1 and TBP is contradictory to our demonstration that the whole TFIID complex is required for {tau}1-mediated transcriptional activation because {tau}1 should be able to recruit TBP in the absence of the TAFs. However, there are two complementary explanations that can account for our observations: 1) The interaction with TBP could be only one of several interactions required to recruit TFIID to promoters such that as yet unidentified interactions with the TAF subunits could also be important; 2) The TAF subunits of TFIID could play an essential role in {tau}1-mediated activation other than recruitment of the complex to the promoter. Consistent with the latter suggestion, protein kinase and histone acetylase enzyme activities have recently been associated with TAFII250 (56, 57).

In conclusion, we suggest that recruitment of TFIID can account for part of the mechanism by which the GR activates at least some target genes. Previous studies, using cell-free transcription systems, have suggested that the GR {tau}1 domain may also function at one or more steps in transcriptional activation that follow transcription complex formation (30). That activator proteins can facilitate transcriptional activation by several mechanisms is supported by the variety of interactions that they make with other proteins. Thus, for VP16 interactions with TBP (58), TFIIB (59), TFIIA (60), TFIIH (61), Drosophila TAFII40 (62), and its human homolog hTAFII32 (63), Drosophila TAFII60 (64), the yeast coactivator Ada2 (50, 65), and the human coactivator PC4 (66) have been reported. It remains to be determined to what extent interactions with different targets represent independent alternative activation strategies as opposed to constituent parts of a single concerted mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
HeLa Nuclear Extract Preparation and Chromatography
Cell line LTR{alpha}3 (38), which expresses HA-tagged TBP, was obtained as a kind gift from A. J. Berk (University of California, Los Angeles, CA). Nuclear extract was prepared from 6l suspension cultures as described by Dignam et al. (40), except that extraction of nuclear proteins was performed using KCl at a final concentration of 300 mM. Nuclear extract was dialyzed against buffer D [20 mM HEPES-KOH, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% (vol/vol) glycerol, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonylfluoride (PMSF)] for direct use in in vitro transcription assays or for fractionation. LTR{alpha}3 nuclear extracts were fractionated by chromatography on phosphocellulose (Whatman, Clifton, NJ) as described (40), and the fractions were dialyzed against buffer D, except for the 1 M fractions that were to be used for subsequent TFIID affinity purification. Affinity purification was performed as described (38) using protein-A Sepharose 4FF (Pharmacia, Piscataway, NJ) that had been covalently coupled to monoclonal antibody 12CA5 (Boehringer Mannheim, Indianapolis, IN) at a concentration of 1 mg monoclonal antibody /ml beads. Elution of affinity- purified TFIID was performed in 2 x 50 µl using peptide YPYDVPDYA (synthesized by Neosystem Laboratoire, Strasbourg, France) at a concentration of 1 mg/ml in buffer D containing 200 mM KCl, 50 µg/ml BSA, 0.1% NP-40.

Yeast Whole Cell Extract and Chromatography
Yeast strains, derived from the diploid strain SEY6210.5 and expressing HA-tagged yTAFII130 or HA-tagged Brf1 (39) were obtained as a kind gift from P. A. Weil (Vanderbilt University School of Medicine, Memphis, TN). Whole cell extracts were prepared from 4l cultures according to Woontner et al. (67) and were fractionated by Bio-Rex 70 (Bio-Rad, Richmond, CA) chromatography as described (39, 68) to obtain 600 mM KAc fractions containing high-salt, TBP-containing complexes.

Overexpression and Purification of Recombinant Proteins
Plasmids expressing GST-TBP (pGEX-KG-TBP) and GST-VP16 (pGEX-CS-VP16AD; 67) were kindly provided by A. Kouzarides (Cambridge, UK) and S. A. Johnston (University of Texas, Dallas, TX) respectively. Plasmid GST-{tau}1 was constructed by subcloning an EcoRI-XbaI (blunted) fragment (containing hGR residues 77–262 and additional N-terminal residues NSSSVPG) from plasmid pEhGR770 (8) in-frame into pGEX-4T-3 (Pharmacia). GST-{tau}1(H1A), containing mutations F191A, I193A, L194A, L197A, F199A, was constructed by swapping the wild type HindIII-StyI fragment (from GST-{tau}1) encoding hGR amino acids 120–218 for a mutated fragment from plasmid pRS315{tau}1(H1Ala) (gift from T. Almlöf, Stockholm, Sweden). pRS315{tau}1(H1Ala) contains the full-length {tau}1 sequence mutated at the required positions (11). GST-{tau}1core ({tau}1c) was constructed by subcloning of hGR residues 187–244 from plasmid YEp{Delta}2-{tau}1c as an in-frame fragment into the EcoRI site of pGEX-5X-2 (Pharmacia). GST-{tau}1c(H1A) was constructed identically to the wild type except that cDNA containing the five alanine mutations was subcloned from YEp{Delta}2-{tau}1c(H1Ala) (cDNA obtained as a gift from T. Almlöf). The YEp{Delta}2-{tau}1c plasmids contain the {tau}1core sequence flanked by EcoRI (5') and BamHI (3') sites and will be described elsewhere. Both GST-{tau}1c and GST-{tau}1c(H1A) were sequenced from upstream of the fusion junctions with GST until downstream of the stop codons because it was observed on SDS PAGE that they ran with apparently different molecular masses. The only differences between the sequences were due to the described mutations. Proteins were expressed and purified according to the manufacturer’s instructions (Pharmacia). Production of the LexA DBD (Lex), Lex-{tau}1core ({tau}1c), and Lex-{tau}1c(H1A) will be described elsewhere. The plasmid expressing Gal(1–100) was created by introducing Gal4(94–100) and a linker containing 3' EcoRI and BamHI sites into pRJR1 (70) by PCR (pRJRI was a kind gift from R. J. Reece, University of Manchester, Manchester, UK). The sequence after Gal(100) was CTTAAGTTCCTAGG. The sequence of the PCR fragment was confirmed by sequencing. Gal(1–100)-{tau}1 was created by subcloning of hGR residues 77–262 into the in-frame BamHI site of Gal(1–100). The histidine-tagged Gal4 proteins were expressed and purified as previously described (70). Plasmid pET-6HishTBP, for the expression of hTBP, was obtained as a gift from M. Meyer (EMBL, Heidelberg, Germany). Human TBP was prepared from the vector in accordance with the manufacturer’s recommendations for the pET series (Novagen, Madison, WI).

Cell-Free Transcription Assay
In vitro transcription reactions were performed in 25 µl reaction volumes that contained 100 ng (0.04 pmol) template pG5HMC2AT (37) (a gift from R. G. Roeder, Rockefeller University, New York, NY) along with approximately 60 µg HeLa nuclear extract, 0–50 pmol activator proteins, 80 µg/ml BSA, 12 mM HEPES-KOH, pH 7.9, 6 mM MgCl2, 12% (vol/vol) glycerol, 60 mM KCl, 0.1 mM EDTA, 0.4 U Prime RNase Inhibitor (5 Prime to 3 Prime, Inc), 5 mM DTT, 0.4 mM each of ATP and CTP, 4 µM UTP, 5 µCi {alpha}-32P-UTP (>400 Ci/mmol, Amersham, Arlington Heights, IL), 0.2 mM 3'-O-methyl-GTP (Pharmacia), 4 mM phosphoenyl pyruvate (Boehringer Mannheim). Reactions were allowed to proceed for 55 min at 30 C, after which they were stopped by incubation at room temperature for 15 min with 200 µl stop buffer [10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA, 0.5% glycogen, 0.4 U RNase T1 (Boehringer Mannheim)], extracted with phenol-chloroform, and precipitated with ethanol. Transcription products were resolved on 7% (29:1) polyacrylamide-7 M urea gels and quantified by PhosphorImager analysis using a BAS2000 bioimaging analyzer (Fuji Film, Tokyo, Japan). Where indicated, HeLa nuclear extract was heat treated in a water bath at 46 C or 47.5 C for 15 min and then recooled before its addition to the transcription reaction (36). Reactions containing heat-treated extracts were supplemented with approximately 2.5 ng TBP in the form of purified rhTBP, TFIID from 1 M phosphocellulose fractionated HeLa nuclear extract, or affinity-purified TFIID. rhTBP was also added at 10- fold higher amounts (~25 ng). TBP concentrations in TFIID fractions were estimated by semiquantitative Western blotting using the ECL system (Amersham) employing rhTBP as a standard and detecting TBP with a primary monoclonal anti-TBP antibody (Promega, Madison, WI).

Protein-Protein Interaction Assays
GST fusion proteins were bound to glutathione agarose beads (Sigma, St. Louis, MO) at an approximate concentration of 1 mg protein/ml beads. The packed volume of beads was 25 µl per experiment. Purified proteins [LexA DBD, Lex-{tau}1c, Lex-{tau}1c(H1A) each at approximately 20 µg/ml; rhTBP at approximately 2 µg/ml], were added to the beads in eight-bead volumes of IP buffer [20 mM HEPES-KOH, pH 7.9, 10% (vol/vol) glycerol, 100 mM KCl, 0.2 mM EDTA, 5 mM MgCl2, 1 mM DTT, 0.2 mM PMSF] supplemented with 0.01% NP-40 and 1 mg/ml BSA. Incubation with rotation was carried out overnight at 4 C, and the supernatants were recovered. Alternatively, 600 mM KAc fractions from Bio-Rex 70 chromatography of yeast whole cell extracts were added at approximate protein concentrations of 1.5 mg/ml in six-bead volumes of YB [20 mM HEPES-KOH pH 7.6, 150 mM KAc, 10% (vol/vol) glycerol, 5 mM Mg(Ac)2, 1 mM EDTA, 0.02% NP-40, 1 mg/ml BSA, 1 mM DTT, 0.2 mM PMSF]. Incubation with rotation was carried out for 2 h at 4 C, and the supernatants were recovered. Alternatively HeLa nuclear extracts fractionated on phosphocellulose were added to the beads at approximate concentrations of 0.5 mg/ml (1 M P11) or 1.3 mg/ml (0.3 M P11) in six-bead volumes of IP buffer. Incubation with rotation was carried out overnight at 4 C, and the supernatants were recovered. In all cases, the beads were then washed four times with 20 vol of the corresponding incubation buffer, and the beads or initial supernatants were boiled with SDS sample buffer. The proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the appropriate antibody. Blots were developed using the enhanced chemiluminescence system (Amersham).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge A. J. Berk (UCLA, Los Angeles, CA) for cell line LTR{alpha}3, P. A. Weil (Vanderbilt University School of Medicine, Memphis, TN) for yeast strains, T. Almlöf (Karolinska Institute, Stockholm, Sweden), S. A. Johnston (University of Texas, Dallas, TX), A. Kouzarides (Cambridge University, Cambridge, UK), M. Meyer (EMBL, Heidelberg, Germany), R. J. Reece (University of Manchester, Manchester, UK), R. G. Roeder (Rockefeller University, New York, NY), and R. Tjian (University of California, Berkeley, CA) for plasmids. We are very grateful to T. Almlöf and A. Henriksson for communicating results before publication. We thank A. J. Berk, Q. Zhou (MIT, Boston, MA) and R. J. Reece for helpful advice during this project and E. Treuter for interesting discussions.


   FOOTNOTES
 
Address requests for reprints to: Anthony P. H. Wright, Department of Biosciences at Novum, S-141 57 Huddinge, Sweden.

This work was supported by Swedish Medical Research Council Grant 13x-2819 and Swedish Natural Science Research Council Grant K-KV9756–301. J. F. was supported by a Travelling Research Fellowship from The Wellcome Trust, UK (Grant reference number: 040181/Z/93/Z/LEC/cg).

Received for publication March 13, 1997. Revision received June 5, 1997. Accepted for publication June 18, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ribeiro RC, Kushner PJ, Baxter JD 1995 The nuclear hormone receptor gene superfamily. Annu Rev Med 46:443–453[CrossRef][Medline]
  2. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
  3. Giguère V, Hollenberg SM, Rosenfeld MG, Evans RM 1986 Functional domains of the human glucocorticoid receptor. Cell 46:645–652[CrossRef][Medline]
  4. Hollenberg SM, Evans RM 1988 Multiple and cooperative trans-activation domains of the human glucocorticoid receptor. Cell 55:899–906[CrossRef][Medline]
  5. Danielsen M, Northrop JP, Jonklaas J, Ringold GM 1987 Domains of the glucocorticoid receptor involved in specific and nonspecific deoxyribonucleic acid binding, hormone activation, and transcriptional enhancement. Mol Endocrinol 1:816–822[Abstract]
  6. Godowski PJ, Picard D, Yamamoto KR 1988 Signal transduction and transcriptional regulation by glucocorticoid receptor-LexA fusion proteins. Science 241:812–816[Abstract/Free Full Text]
  7. Wright APH, McEwan IJ, Dahlman-Wright K, Gustafsson J-Å 1991 High level expression of the major transactivation domain of the human glucocorticoid receptor in yeast cells inhibits endogenous gene expression and cell growth. Mol Endocrinol 5:1366–1372[Abstract]
  8. McEwan IJ, Wright APH, Dahlman-Wright K, Carlstedt-Duke J, Gustafsson J-Å 1993 Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery. Mol Cell Biol 13:399–407[Abstract/Free Full Text]
  9. Dahlman-Wright K, Baumann H, McEwan IJ, Almlöf T, Wright APH, Gustafsson J-Å, Härd T 1995 Structural characterisation of a minimal functional transactivation domain from the human glucocorticoid receptor. Proc Natl Acad Sci USA 92:1699–1703[Abstract/Free Full Text]
  10. Dahlman-Wright K, McEwan IJ 1996 Structural studies of mutant glucocorticoid receptor transactivation domains establish a link between transactivation activity in vivo and alpha-helix-forming potential in vitro. Biochemistry 35:1323–1327[CrossRef][Medline]
  11. Almlöf T, Gustafsson J-Å, Wright APH 1997 Role of hydrophobic amino acid clusters in the transactivation activity of the human glucocorticoid receptor. Mol Cell Biol 17:934–945[Abstract]
  12. Zawel L, Reinberg D 1995 Common themes in assembly and function of eukaryotic transcription complexes. Annu Rev Biochem 64:533–561[CrossRef][Medline]
  13. Conaway RC, Conaway JW 1993 General initiation factors for RNA polymerase II. Annu Rev Biochem 62:161–190[CrossRef][Medline]
  14. Orphanides G, Lagrange T, Reinberg D 1996 The general transcription factors of RNA polymerase II. Genes Dev 10:2657–2683[Free Full Text]
  15. Koleske AJ, Young RA 1994 An RNA polymerase II holoenzyme responsive to activators. Nature 368:466–469[CrossRef][Medline]
  16. Kim YJ, Bjorklund S, Li Y, Sayre MH, Kornberg RD 1994 A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77:599–608[CrossRef][Medline]
  17. Ossipow V, Tassan JP, Nigg EA, Schibler U 1995 A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation. Cell 83:137–146[CrossRef][Medline]
  18. Triezenberg SJ 1995 Structure and function of transcriptional activation domains. Curr Opin Genet Dev 5:190–196[CrossRef][Medline]
  19. Guarente L 1995 Transcriptional coactivators in yeast and beyond. Trends Biochem Sci 20:517–521[CrossRef][Medline]
  20. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  21. Eggert M, Mows CC, Tripier D, Arnold R, Michel J, Nickel J, Schmidt S, Beato M, Renkawitz R 1995 A fraction enriched in a novel glucocorticoid receptor-interacting protein stimulates receptor-dependent transcription in vitro. J Biol Chem 270:30755–30759[Abstract/Free Full Text]
  22. Zeiner M, Gehring U 1995 A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning. Proc Natl Acad Sci USA 92:11465–11469[Abstract/Free Full Text]
  23. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  24. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[CrossRef][Medline]
  25. Muchardt C, Yaniv M 1993 A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor. EMBO J 12:4279–4290[Medline]
  26. Imhof MO, McDonnell DP 1996 Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors. Mol Cell Biol 16:2594–2605[Abstract]
  27. Kupfer SR, Marschke KB, Wilson EM, French FS 1993 Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors. J Biol Chem 268:17519–17527[Abstract/Free Full Text]
  28. Freedman LP, Yoshinaga SK, Vanderbilt JN, Yamamoto KR 1989 In vitro transcription enhancement by purified derivatives of the glucocorticoid receptor. Science 245:298–301[Abstract/Free Full Text]
  29. Tsai SY, Srinivasan G, Allan GF, Thompson EB, O’Malley BW, Tsai MJ 1990 Recombinant human glucocorticoid receptor induces transcription of hormone response genes in vitro. J Biol Chem 265:17055–17061[Abstract/Free Full Text]
  30. McEwan IJ, Almlöf T, Wikström AC, Dahlman-Wright K, Wright APH, Gustafsson J-Å 1994 The glucocorticoid receptor functions at multiple steps during transcription initiation by RNA polymerase II. J Biol Chem 269:25629–25636[Abstract/Free Full Text]
  31. Nikolov DB, Burley SK 1994 2.1 A resolution refined structure of a TATA box-binding protein (TBP). Nature Struct Biol 1:621–637[CrossRef][Medline]
  32. Hernandez N 1993 TBP, a universal eukaryotic transcription factor?. Genes Dev 7:1291–1308[Free Full Text]
  33. Goodrich JA, Tjian R 1994 TBP-TAF complexes: selectivity factors for eukaryotic transcription. Curr Opin Cell Biol 6:403–409[CrossRef][Medline]
  34. Tjian R 1996 The biochemistry of transcription in eukaryotes - a paradigm for multisubunit regulatory complexes. Philos Trans R Soc Lond [Biol] 351:491–499[Medline]
  35. Burley SK, Roeder RG 1996 Biochemistry and structural biology of transcription factor IID (TFIID). Annu Rev Biochem 65:769–799[CrossRef][Medline]
  36. White JH, Brou C, Wu J, Burton N, Egly JM, Chambon P 1991 Evidence for a factor required for transcriptional stimulation by the chimeric acidic activator GAL-VP16 in HeLa cell extracts. Proc Natl Acad Sci USA 88:7674–7678[Abstract/Free Full Text]
  37. Chiang CM, Ge H, Wang Z, Hoffmann A, Roeder RG 1993 Unique TATA-binding protein-containing complexes and cofactors involved in transcription by RNA polymerases II and III. EMBO J 12:2749–2762[Medline]
  38. Zhou Q, Lieberman PM, Boyer TG, Berk AJ 1992 Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter. Genes Dev 6:1964–1974[Abstract/Free Full Text]
  39. Poon D, Bai Y, Campbell AM, Bjorklund S, Kim YJ, Zhou S, Kornberg RD, Weil PA 1995 Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae. Proc Natl Acad Sci USA 92:8224–8228[Abstract/Free Full Text]
  40. Dignam JD, Martin PL, Shastry BS, Roeder RG 1983 Eukaryotic gene transcription with purified components. Methods Enzymol 101:582–598[Medline]
  41. Rigby PW 1993 Three in one and one in three: it all depends on TBP. Cell 72:7–10[CrossRef][Medline]
  42. Jacq X, Brou C, Lutz Y, Davidson I, Chambon P, Tora L 1994 Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor. Cell 79:107–117[CrossRef][Medline]
  43. Brou C, Chaudhary S, Davidson I, Lutz Y, Wu J, Egly JM, Tora L, Chambon P 1993 Distinct TFIID complexes mediate the effect of different transcriptional activators. EMBO J 12:489–499[Medline]
  44. Deleted in proof
  45. Chatterjee S, Struhl K 1995 Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain. Nature 374:820–822[CrossRef][Medline]
  46. Klages N, Strubin M 1995 Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo. Nature 374:822–823[CrossRef][Medline]
  47. Walker SS, Reese JC, Apone LM, Green MR 1996 Transcription activation in cells lacking TAFIIS. Nature 383:185–188[CrossRef][Medline]
  48. Moqtaderi Z, Bai Y, Poon D, Weil PA, Struhl K 1996 TBP-associated factors are not generally required for transcriptional activation in yeast. Nature 383:188–191[CrossRef][Medline]
  49. Sauer F, Wassarman DA, Rubin GM, Tjian R 1996 Taf(Ii)S mediate activation of transcription in the Drosophila embryo. Cell 87:1271–1284[CrossRef][Medline]
  50. Barlev NA, Candau R, Wang L, Darpino P, Silverman N, Berger SL 1995 Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. J Biol Chem 270:19337–19344[Abstract/Free Full Text]
  51. Candau R, Moore PA, Wang L, Barlev N, Ying CY, Rosen CA, Berger SL 1996 Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5. Mol Cell Biol 16:593–602[Abstract]
  52. Henriksson A, Almlöf T, Ford J, McEwan IJ, Gustafsson J-Å, Wright APH 1997 Role of the Ada adaptor protein complex in gene activation by the glucocorticoid receptor. Mol Cell Biol 17:3065–3073[Abstract]
  53. Schwerk C, Klotzbucher M, Sachs M, Ulber V, Klein-Hitpass L 1995 Identification of a transactivation function in the progesterone receptor that interacts with the TAFII110 subunit of the TFIID complex. J Biol Chem 270:21331–21338[Abstract/Free Full Text]
  54. Schulman IG, Chakravarti D, Juguilon H, Romo A, Evans RM 1995 Interactions between the retinoid x receptor and a conserved region of the TATA binding protein mediate hormone dependent transactivation. Proc Natl Acad Sci USA 92:8288–8292[Abstract/Free Full Text]
  55. Petty KJ, Krimkevich YI, Thomas D 1996 A Tata binding protein associated factor functions as a coactivator for thyroid hormone receptors. Mol Endocrinol 10:1632–1645[Abstract]
  56. Dikstein R, Ruppert S, Tjian R 1996 TAFII250 is a bipartite protein kinase that phosphorylates the base transcription factor RAP74. Cell 84:781–790[CrossRef][Medline]
  57. Mizzen CA, Yang XJ, Kokubo T, Brownell JE, Bannister AJ, Owen-Hughes T, Workman J, Wang L, Berger SL, Kouzarides T, Nakatani Y, Allis CD 1996 The TAF(II)250 subunit of TFIID has histone acetyltransferase activity. Cell 87:1261–1270[CrossRef][Medline]
  58. Stringer KF, Ingles CJ, Greenblatt J 1990 Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature 345:783–786[CrossRef][Medline]
  59. Lin YS, Ha I, Maldonado E, Reinberg D, Green MR 1991 Binding of general transcription factor TFIIB to an acidic activating region. Nature 353:569–571[CrossRef][Medline]
  60. Kobayashi N, Boyer TG, Berk AJ 1995 A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly. Mol Cell Biol 15:6465–6473[Abstract]
  61. Xiao H, Pearson A, Coulombe B, Truant R, Zhang S, Regier JL, Triezenberg SJ, Reinberg D, Flores O, Ingles CJ, Greenblatt J 1994 Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol 14:7013–7024[Abstract/Free Full Text]
  62. Goodrich JA, Hoey T, Thut CJ, Admon A, Tjian R 1993 Drosophila TAFII40 interacts with both a VP16 activation domain and the basal transcription factor TFIIB. Cell 75:519–530[CrossRef][Medline]
  63. Klemm RD, Goodrich JA, Zhou S, Tjian R 1995 Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation. Proc Natl Acad Sci USA 92:5788–5792[Abstract/Free Full Text]
  64. Thut CJ, Chen JL, Klemm R, Tjian R 1995 p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60. Science 267:100–104[Abstract/Free Full Text]
  65. Silverman N, Agapite J, Guarente L 1994 Yeast ADA2 protein binds to the VP16 protein activation domain and activates transcription. Proc Natl Acad Sci USA 91:11665–11668[Abstract/Free Full Text]
  66. Ge H, Roeder RG 1994 Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes. Cell 78:513–523[CrossRef][Medline]
  67. Woontner M, Wade PA, Bonner J, Jaehning JA 1991 Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae. Mol Cell Biol 11:4555–4560[Abstract/Free Full Text]
  68. Poon D, Weil PA 1993 Immunopurification of yeast TATA-binding protein and associated factors. Presence of transcription factor IIIB transcriptional activity. J Biol Chem 268:15325–15328[Abstract/Free Full Text]
  69. Melcher K, Johnston SA 1995 GAL4 interacts with TATA-binding protein and coactivators. Mol Cell Biol 15:2839–2848[Abstract]
  70. Reece RJ, Rickles RJ, Ptashne M 1993 Overproduction and single-step purification of GAL4 fusion proteins from Escherichia coli. Gene 126:105–107[CrossRef][Medline]



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