THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1994 by The American Society for Bioehemiatry and Molecular Biology, Inc.

Vol. 269, No. 5 , Issue of February 4, pp. 3489-3497, 1994 Printed in U S A .

Role of Transcription Factor TFIIF in Serum Response Factor-activated Transcription"

(Received for publication, June 14, 1993, and in revised form, October 12, 1993)

Hua ZhuS, Vhronique JoliotSl, and Ron Prywea From the Department of Biological Sciences, Columbia University, New York, New York 10027

We have found that the general transcription factor TFIIF has an important role in serum response factor (SRF)-activated transcription in u i h . A low amount of TFIIF was sufficient for basal transcription, whereas higher amounts were required for SRF, but not Spl, ac- tivation. High TFIIF levels also increased activation by GAIA-VP16, whereas none of the other general tran- scription factors had these properties. TFIIF could also relieve squelching by SRF in vitro, suggesting that SRF may directly bind TFIIF. We found more direct evidence for SRF-TFIIF interaction by DNA binding assays where the RAP74 subunit of TFIIF bound DNA in coqjunction with SRF, but not alone. RAP74 also bound DNA with GAIA-VP16, but not with Spl or the DNA binding do- main of GAIA. These results suggest that the mechanism of transcriptional activation by SRF, and perhaps some other activators, involves their interaction with TFIIF.

Transcriptional regulation is controlled by sequence-specific transcriptional activators and repressors. How activators stimulate transcription is still unclear. Activators generally contain distinct DNA binding and activation domains (re- viewed in Mitchell and Tjian, 1989). Activation domains have been divided into different groups based on their amino acid sequence: acidic (GAL4 and VP16), glutamine-rich (Spl and Oct2), and proline-rich domains (CTF and AP2) (for review see Mitchell and Tjian, 1989). The activation domains of many factors, such as serum response factor (SRF),l do not clearly fall into any of these classifications. Many activators have been found to function at the level of transcriptional initiation (Kata- gin et al . , 1990; Lin and Green, 1991; Zhu et al., 1991). Thus, activation domains must communicate with the general tran- scription apparatus to stimulate initiation. The general appa- ratus consists of RNA polymerase I1 and TFIIA, -B, -D, -E, -F, and -H (reviewed in Zawel and Reinberg, 1992). Specific acti- vators may interact with one of these factors directly or through an intermediary factor.

Great efforts have been made in the last few years to find the targets of transcriptional activators and some potential targets have been reported. For example, VP16, Ela , Zta, and p53 directly bind the TATA-binding subunit of TFIID (TBP)

* This work was supported by Grant CA 50329-01 from the National Cancer Institute and by support from the Searle Scholars Programfl'he Chicago Community Trust (to R. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

sidered co-first authors. $ The first two authors made equal contributions and should be con-

$ Recipient of a fellowship from the Ligue Nationale Contre Le Can- cer, France.

lumbia University, 116th and Broadway, New York, NY 10027. Tel.: 1 To whom correspondence should be addressed: Fairchild 813B, Co-

212-854-8281; Fax: 212-865-8246. The abbreviation used is: SRF, serum response factor.

(Stringer et al., 1990; Lee et al . , 1991; Lieberman and Berk, 1991; Seto et al., 1992) and VP16, and several members of the steroid receptor family can directly bind TFIIB (Lin and Green, 1991; Lin et al . , 1991; Ing et al., 1992). In addition, an acidic activator, G U - A H , can recruit TFIIB to a preinitiation tran- scription complex (Lin and Green, 1991).

Coactivators are factors that are required for gene-specific factor-activated transcription but not basal transcription. Co- activators have been proposed to act as intermediary factors between transcriptional activators and the general transcrip- tion apparatus (reviewed in Gill and Tjian, 1992). One type of coactivator copurifies with TFIID activity and is tightly asso- ciated with TBP (Dynlacht et al., 1991; Pugh and Tjian, 1991; Tanese et al . , 1991). One such TBP-associated factor (TAF), TAF110, has recently been cloned. Interestingly, TAFllO spe- cifically binds the glutamine-rich activator Sp l (Hoey et al . , 1993). Other coactivator fractions have been identified in yeast and human cells, but it is unclear as yet whether they directly bind transcriptional activators (Flanagan et al . , 1991; Meister- ernst et al., 1991; White et al., 1991).

SRF is a 64-kDa transcription factor which binds to the se- rum response element in the c-fos proto-oncogene. It is an es- sential component for serum and growth factor induction of the c-fos gene (reviewed in Treisman, 1992). SRF has 508 amino acids and binds to DNA as a dimer (Norman et al., 1988). The DNA binding and dimerization domains of SRF are in the cen- ter of protein (amino acids 133-268) (Norman et al., 1988). SRF's transcriptional activation domain has been mapped to the C-terminal part of the protein in vitro and in vivo (Prywes and Zhu, 1992; Johansen and Prywes, 1993) and has no clear homology to known transcriptional activation domains.

We have studied how SRF activates transcription. Besides understanding how this activator affects transcription, it will also be interesting to know how its interaction with the general transcription machinery is regulated. We found previously that high amounts of SRF could inhibit (squelch) transcription ac- tivated by itself or several other factors in vitro, suggesting that SRF can titrate out a common coactivator required by these activators (Prywes and Zhu, 1992). We then identified and par- tially purified a fraction, COS, with coactivator-like activity, in that it was required for SRF-activated, but not basal, transcrip- tion (Zhu and Prywes, 1992). We describe here that we have found that this activity corresponds to the general transcrip- tion factor TFIIF and that TFIIF has a unique role in SRF- activated transcription.

EXPERIMENTAL PROCEDURES

stored in buffer BClOO (100 m KCl, 20% glycerol, 20 m Tris-HC1, pH Purification of Dunscription Factors-All transcription factors were

7.9, 0.2 m EDTA, 0.5 phenylmethylsulfonyl fluoride, 0.5 m dithio- threitol, 0.05% Nonidet P-40). The concentration oftotal protein is given below for each fraction. RNA polymerase I1 (0.08 mg/mV, TFIIA (2.9 mglml), and TFIIE (0.5 mg/ml) were purified from HeLa cells as de- scribed (Zhu and Prywes, 1992).

3489

3490 Role of TFIIF in SRF-activated Danscription TFIID (o fraction; 0.3 mg/ml) was first purified from HeLa nuclear

extracta on phosphocellulose (P11) and DEAE-5PW columns (Zhu and Prywes, 1992) and then further purified through an -amino octyl- agarose column with a 0.1-0.5 M KC1 linear gradient as described (Na- kajima et al., 1988). TFIID (SP-5PW fraction; 0.3 mg/ml) was similarly purified from a DEAE-5PW fraction on an SP-5PW column (8 x 80 mm, Waters) with a linear gradient of 0.1-0.5 M KCl.

TFIIF (COS; 0.035 mg/ml) was purified from HeLa nuclear extracts as described (Zhu and Prywes, 1992). For further purification, TFIIF (phe- nyldPW fraction) was precipitated by adding 3.8 M ammonium sulfate (pH 7.9) to a final concentration of 2.85 M. The precipitate was resus- pended in BC700 (same as BClOO but with 700 m KC1 and 10% glycerol) and loaded on a 300SW gel filtration column (8.0 mm x 30 cm, Waters). Fractions were dialyzed against BClOO and assayed for coac- tivator-like activity (Zhu and Prywes, 1992). TFIIF (0.030 mg/ml) was also purified from HeLa cytoplasmic extracts (Dignam et al., 1983) by the same procedure described above.

TFIIH (0.42 mg/ml) was purified as in the purification of the COS (TFIIF) fraction (Zhu and Prywes, 1992), except that TFIIH activity eluted from the phenyl-5PW column at about 0.4 M ammonium sulfate in a gradient of 0.9 M to 0 M ammonium sulfate.

Recombinant TFIIB, TBP, TFIIE,, RAP30, and W 7 4 were ex- pressed in bacteria and purified as described (Ha et al., 1991; HofFmann and Roeder, 1991; Sumimoto et al., 1991; and Finkelstein et al., 1992). We found that recombinant TFIIF (rTFIIF) was most active when we added 60% RAP74 and 40% RAP30 by weight. TFIIE, coding sequence from NdeI to BamHI (Ohkuma et al., 1991) was inserted into NdeI to BamHI sites of the 6HisT-pET11 vector (HofFmann and Roeder, 1991). Recombinant histidine-tagged TFIIE, was then made in bacteria and purified as described (HofFmann and Roeder, 1991).

Recombinant SRF was purified from Escherichia coli by electroelu- tion as described (Prywes and Zhu, 1992). In addition, SRF's coding region was cloned into 6HisT-pET11 and histidine-tagged SRF was purified on a Ni2+-nitrilotriacetic acid-agarose column (HofFmann and Roeder, 1991). SRF(141-244) was constructed in two steps. First, an amino acid 1-140 deletion mutant, pARSRF(141-508), was generated from pARSRF-Nde (containing wild type SRF Manak et al., 1990) by deleting a BamHI to SmaI fragment and inserting an 8-mer BamHI DNA linker (New England Biolabs) at the SmaI site (amino acids 141). Then, pARSRF(141-508) was digested with BglII, blunted with Klenow fragment of DNApolymerase, and an 8-mer BarnHI linker was added at the BglII site. A 310-base pair fragment containing the coding region for amino acids 141-244 of SRF was purified and inserted into the BamHI site of pAR3040. The plasmid was transformed into E. coli strain BL21(DE3) (Novagen), and the protein was expressed and purified as described (Prywes and Zhu, 1992).

GAIA-VP16 was purified from bacteria as described (Chasman et al., 1989) and was a kind g f t from Jerry Workman. Histidine-tagged GAIA- VP16 and histidine-tagged GAIA-SRF(245-508) were also made and purified on Ni2+ columns.2 Spl was purified from HeLa cell nuclear extracts by DNA affinity chromatography as described (Jackson and Tjian, 1989) and was a kind gift from Hui Ge and Robert Roeder. Spl was further immunodepleted with anti-RAP74 sera to remove the pos- sibility of contamination of TFIIF. GU-Spl(A+B) was expressed in bacteria and purified by DNA affinity chromatography and was a kind gift from Timothy Hoey and Robert Tjian.

Zn Vitro Thnscription Assays-Templates pFC53X and pFC53G5 contain a high affinity SRF binding site or five GAL4 binding sites, respectively, cloned into pFC53 upstream of -53 to +42 of the c-fos promoter followed by the bacterial CAT gene (Zhu and Prywes, 1992). pSVFC53.2 contains a fragment of the SV40 early gene (nucleotides 37-84 of SV40), including three Spl binding sites, cloned at -53 in pFC53 and was a kind gift of G. Farmer and C. Prives. This fragment also contains a p53 binding site as described (Bargonetti et al., 1992).

The following amounts of each of the transcription factor fractions were used in the transcription assays except where indicated (volumes are shown for fractions that were not purified close to homogeneity): 0.6 p1 of RNA polymerase II,0.2 pl of TFIIA, 5 ng of rTFIIB, 2 pl of TFIID (SP-5PW or -amino octyl fractions) or 6 ng of rTBP, 40 ng of rTFIIE (l:l, (I and p subunits), 0.2 pl of HeLa TFIIF (containing about 7 ng TFIIF) or 30 ng of rTFIIF (18 ng of RAP74 and 12 ng of WBO), 0.2-0.5 pl of TFIIH, 5 ng of SRF, 15 ng of GAIA-VP16, and 15 ng of Spl. The in vitro transcription assay and S1 nuclease hybridization method were as described (Prywes and Zhu, 1992). Single-round transcription assays were also as described (Zhu et al., 1991). The amounts of specific tran- scripts were quantitated using a PhosphorImager and ImageQuant

* V. Tirnanic, F.-E. Johansen, and R. Prywes, unpublished data.

Software data analysis (Molecular Dynamics). Zmmunodepletion and Zmmunoblotting-TFIIF and other transcrip-

tion factor preparations were depleted of TFIIF using antisera specific to RAP30 and RAP74. These sera were raised against bacterially made full-length proteins and were a kind gift of Kathy Wang, Philippe Pog- nonec, and Robert Roeder. Protein A-agarose (40 pl), as a 50% slurry in BC100, was mixed with 10 pl of both anti-RAP30 and anti-RAP74 sera or 20 pl of preimmune sera and rotated at 4 "C for 2-8 h. The protein A-agarose-antibody complexes were then washed four times with 1 ml of BC100. This material was then mixed with 40 pl of factor fractions and 40 pl of BClOOO (same as BClOO but with 1 M KCl). The mixture was rotated at 4 "C for 2-8 h and protein A-agarose, with bound com- ponents, was removed by centrifugation. The supernatants were dia- lyzed in BClOO at 4 "C and used in in vitro transcription assays. Im- munoblotting with anti-RAP30 and RAP74 sera was performed under standard conditions (Harlow and Lane, 1988) with a 1:lOOO dilution of sera and alkaline phosphatase-conjugated goat anti-rabbit IgG as a secondary antibody.

Gel Mobility Shift A s s a y s 4 1 mobility shift assays were carried out basically as described (Peterson et al., 1990). Probes were DNA frag- ments from transcription templates pFC53X, pFC53G1, and pSVFC53.2 containing fos promoter sequence from -53 to +42 and one protein binding site. They were isolated as approximately 140-base pair Hind111 to XbaI fragments of these plasmids. The probes were labeled with 32P by Klenow fragment of E. coli DNA polymerase or polynucleo- tide kinase. The reactions contained 4 pl of 5 x gel shift buffer (100 m Hepes, pH 7.9, 125 m KC1, 0.5 m EDTA, 50% glycerol, 10 m MgClZ), 1 pl of 40 m spermidine, 1 pl of 10 m dithiothreitol, 1 pl of 0.5% Nonidet P-40, 1 pl of 100 pg/ml poly(dG-dC), 1 pl of 2 mg/ml bovine serum albumin, 1 pl 100 pg/d herring sperm DNA (in some experi- ments as indicated), 1 ng of 32P-labeled probe DNA, 3 pl of protein samples (in BClOO), and HzO to 20 pl. The amounts of protein used are indicated in the figure legends. The reactions were carried out at room temperature for 40 min. For antibody supershiR assays, 0.1 pl of anti- SRF, 0.1-1 pl of anti-RAP74, anti-RAP30, and preimmune sera were incubated with protein fractions at room temperature for 40 min. The remaining gel mobility shift reagents were then added and incubated at room temperature for another 40 min. The reactions (10 pl) were then loaded on a native 4% polyacrylamide gel (except where indicated) containing 0.5 x TBE, 1 m EDTA, and 0.05% Nonidet P-40 and elec- trophoresed in 0.5 x "BE at 4 "C at 200-250 volts (not to exceed 40 mA) for 2 to 3 h. The anti-RAP74 and anti-RAP30 sera for these experiments were raised against full-length bacterially made proteins and were kindly provided by Zachary Burton (Wang et al., 1993). ht i -SRF sera were as described (Manak and Prywes, 1993). Competitor double- stranded oligonucleotides (25 ng) for the SRF gel mobility shift assays were XGL, containing a high affinity SRF binding site, or XGLM, with a double point mutation that completely abolishes SRF binding (Manak et al., 1990).

RESULTS

Addition of TFIIF Is Required for SRF-activated Dunscrip- tion-We have previously identified and partially purified from HeLa cell nuclear extracta a fraction with coactivator-like ac- tivity, termed COS, which could increase activation of transcrip- tion in vitro by SRF (Zhu and Prywes, 1992; Fig. 1, lanes 1 4 ) . In order to identify the polypeptides corresponding to this ac- tivity, we have purified this factor to near homogeneity. It con- tained 30- and 74-kDa polypeptides which coeluted with activ- ity (Fig. 2 and data not shown). The general transcription factor TFIIF also contains two subunits with these sizes, termed RAP30 and RAP74. We found by immunoblotting that the two proteins in the purified COS fraction were recognized by anti- sera raised against recombinant RAP30 and W 7 4 , suggest- ing that they are the two subunits of TFIIF (Fig. 2). In addition recombinant RAP30 and RAP74 comigrated in SDS-polyacryl- amide gels with these bands (data not shown).

As further evidence that the coactivator-like activity corre- sponds to TFIIF, we used the anti-RAP30 and -RAP74 sera to immunodeplete the COS fractions as described under "Experi- mental Procedures." These sera abolished the coactivator ac- tivity, whereas preimmune sera had no effect (data not shown). We next found that recombinant TFIIF (RAP30 and RAP741 could substitute for our COS fraction and stimulate SRF-acti-

Role of TFIIF in SRF-activated Danscription 3491

A TFIIF

Activator

Probe - fOSCAT Transcript -

Lane: Fold Activation:

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 2 3 4 1.1 3.9 0.9 55 2.3 5.3 7.8 6.7

TFIIF ( r ) was added to transcription reactions with SRF, GA"VF'16 (GVP), or Spl, as indicated. The fosCAT plasmid templates, pFC53X (lanes F I G . 1. Effect of recombinant TFIIF on basal and activated transcription. A, either HeLa cell-derived human TFIIF ( h ) or recombinant

la), pFC53G5 (lanes 9-12 ), or pSVFC53.2 (lanes 13-16), containing binding sites for SRF, GALA, and Spl, respectively, were used in the reactions. Transcripts were analyzed by the S1 nuclease method with a probe specific for fosCAT RNA. The positions of reanneled probe and specifically initiated transcripts are indicated to the left of the figure. The reactions contained transcription factors RNApolymerase 11, TFIIA, ffFIIB, ffFIIE, TFIIH, and TFIID ( w fraction). Single round initiation conditions were used for all the reactions. The -fold activations with uersus without activator, as quantitated with a Phosphorimager (Molecular Dynamics), are indicated below the lanes. B, TFIIF is required for basal transcription. In uitro transcription reactions were performed as in A with pFC53X as template, except that in lanes 2 4 recombinant TBP was used in place of TFIID, and the RNA polymerase 11, TFIIA, and TFIIH fractions were immunodepleted of TFIIF using anti-RAP30 and anti-RAP74 sera. In addition, standard (muciround) transcription conditions were used.

Silver Stain Immunoblot

93kD -

45

30

FIG. 2. COS is TFIIF. Purified COS (10 pl, left; 2 pl, right) was elec- trophoresed on a 10% SDS-polyacrylamide gel and visualized by silver staining or immunoblotting with anti-RAP74 and anti-RAP30 sera. The positions of molecular weight markers are indicated to the left and the positions of RAP74 and RAP30 to the right.

vated transcription (Fig. L4, lanes 7 and 8). No effect on basal transcription was observed (lanes 5 and 7). Both RAP30 and RAP74 were required for the increase in activated transcrip- tion (data not shown). Addition of up to a 4-fold excess of any of the other general transcription factors (TFIIA, -B, -D, -E, and -H or RNA polymerase 11) did not increase activation of tran- scription by SRF under these conditions (data not shown). The above evidence demonstrates that the coactivator-like activity is TFIIF, and the COS fractions will henceforth be referred to as TFIIF.

In the experiments described here, we have used RNA po-

lymerase I1 purified from HeLa cell nuclear pellets and TFIIA, TFIID, and TFIIH purified from HeLa cell nuclear extracts. TFIIB and TFIIE were made from clones expressed in bacteria and were subsequently purified to near homogeneity. When these factors were reconstituted in transcription assays, we found that the TFIIF fraction was not required for basal tran- scription, although it was still required for SRF-activated tran- scription (Fig. 1, lanes 1-4). The lack of requirement for TFIIF in basal transcription could be due to small amounts of TFIIF contaminating the other transcription factor fractions. A small amount of TFIIF could be detected in the TFIID fraction by immunoblotting, but not in the other fractions (data not shown). We used recombinant TFIID (the TATA-binding sub- unit, TBP) as a purer source of TFIID and still observed sig- nificant amounts of transcription without our TFIIF fraction (data not shown). In order to completely remove contaminating TFIIF from the other fractions (RNA polymerase 11, TFIIA, and TFIIH), we immunodepleted TFIIF from these fractions using anti-RAP30 and anti-RAP74 sera (see "Experimental Proce- dures"). After this treatment, we observed a clear requirement for TFIIF in basal transcription. TFIIF purified from HeLa cells or recombinant TFIIF reconstituted basal transcription (Fig. 1B). Both the RAP30 and RAP74 subunits of TFIIF were required for basal activity (data not shown).

Effect of TFIIF on Activation by Other Activators-We found previously that the coactivator-like activity could stimulate GALA-W16 activation 3-fold in vitro, although it was not ab- solutely required (Zhu and Prywes, 1992). We similarly found that recombinant TFIIF (rTFIIF) increased activation by GAL4-VP16. In several experiments, only a small increase in the GALA-W16-activated level was observed; however, since the basal level was reduced by addition of TFIIF, a 2-fold in- crease in activation was seen (Fig. 1, lanes 9-12). We also tested activation by the Spl transcription factor. Sp l activated transcription equally well with or without adding rTFIIF (Fig. 1, lanes 13-16). This suggests that for SRF activation TFIIF is not simply limiting for higher transcription levels and that Spl either requires only low levels of TFIIF for activation or uses a different mechanism for activating transcription than SRF. The more modest effects of TFIIF on GALA-VP16 activation may also be because GAL4-VP16 requires less TFIIF for activation or because it uses additional mechanisms. These transcription assays also indicate that recombinants TFIIB, TFIIE, and TFIIF have the ability to support activated transcription.

3492 Role of TFIIF in SRF-activated Danscription

Rc. 3. Limiting amounts of TFIIF, rn e & but not the other general transcrip- tion factors, affect SFW activation. The reactions were camed out under standard (multiround) conditions in di which increasing amounts of each tran- scription factor, as indicated, were used 8 pl of RNA polymerase 11, 0.2 pl of TFIIA, with constant amounts of the others (0.6

5 ng of rTFIIB, 2 pl of TFIID (SPdPW fraction), 40 ng of ffFIIE, 30 ng of ffFIIF, and 0.5 pl of TFIIH). The RNA polymer- :! ase 11, TFIIA, TFIID, and TFIIH fractions + used in panel 1 were immunodepleted of possible contaminating TFIIF using anti- RAP30 and RAP74 sera. HeLa-derived TFIIE was used in panel 5. The levels of Q)

specifically initiated transcripts, as de- > tected by S1 nuclease analysis, were 2 quantitated using a Phosphorimager (Mo- 2 lecular Dynamics).

r r p n s c r , 1w T

Effect of Limiting Amounts of the Other General Zkanscrip- tion Factors on SRFActivation-One possible reason that more TFIIF is required for SRF activation is that the limiting amounts of TFIIF are not sufficient to support the higher level of transcription. We believe that this is not the case. First, addition of TFIIF did not increase basal transcription, suggest- ing that TFIIF was not limiting in this reaction (Fig. 1). Second, Spl was capable of activating transcription without addition of TFIIF, indicating that transcription could reach higher levels under these conditions. Third, we tested whether using limit- ing levels of the other transcription factors would also affect activation. We titered down in turn the amount of each factor used, leaving the others constant. Representative experiments are shown in Fig. 3. For titration of TFIIF, the other transcrip- tion fractions were immunodepleted of RAP30 and RAP74 to remove the low levels of contaminating TFIIF. In this experi- ment, the immunodepletion reduced but did not completely abolish basal transcription levels (Fig. 3, panel 1 ). Addition of increasing TFIIF had only a small effect on basal transcription and plateaued quickly. Low amounts of TFIIF had little effect on SRF-activated transcription, but higher levels increased ac- tivation (panel 1). If TFIIF were simply limiting for higher transcription levels, increasing TFIIF should have resulted in a linear increase in SRF-activated transcription. This suggests that TFIIF is required in a different manner for SRF-activated transcription than for basal transcription. In contrast to TFIIF, limiting amounts of the other general transcription factors could support SRF-activated transcription (Fig. 3, panels 2-6). Although the basal and activated transcription levels were re- duced at low factor levels, the -fold activation by SRF was not

_.- 1 I

' -I

significantly affected. In addition, the levels of basal and acti- vated transcription increased relatively linearly with increas- ing amounts of each factor. Thus, under conditions where indi- vidual factors are limiting for basal transcription, good activation is observed with all the general factors except for TFIIF.

Since the TFIIF levels used are saturating for basal tran- scription, we tried to reach saturation with the other factors. As stated above, 4-fold excess of the general factors did not affect the -fold activation by SRF. This level of factors, however, was not clearly saturating for basal transcription. We were not able to define clear saturating levels, because addition of higher amounts of the factors resulted in inhibition of transcription, either due to contaminants or to direct inhibitory effects of the excess factor. Thus, we cannot conclude how saturating levels of these factors would affect activation. Nevertheless, a clear difference is seen at the limiting levels used in Fig. 3. It is surprising that levels of TFIIF that are saturating for basal transcription are limiting for SRF-activated transcription. However, this suggested a more direct role of TFIIF in SRF activation which will be explored later in the paper.

In our previous work (Zhu and Prywes, 1992) we concluded that the COS fraction had coactivator activity and was not a basal factor. This conclusion was based on the lack of require- ment of COS for basal transcription. We have found here, how- ever, that low levels of TFIIF were contaminating the other transcription factor fractions and that this low level was suffi- cient for basal-activated, but not SW-activated, transcription. We also found previously that a fraction we believed to contain TFIIF was required for basal transcription (Zhu and Prywes,

Role of TFIIF in SRF-activated Danscription 3493

0.3M KC1 rTFlIF

SRF

Probe -

fOSCAT Transcript -

Lane:

Fold Activation:

M.5min -0.5min +0.5min M.5 min -3Omin -30min -0.5min

””

1 2 3 4 5 6 7 8

3.9 15.1 2.0

Template GemralFactors

+/- SRF NTPS 4 4 stop

A ‘ \ limc(min) -30

“\ n f -0.5 0 4 . 5 +5

+/- TFIIF 0.3M KCI FIG. 4. Effect of TFIIF during preinitiation complex formation.

Single-round transcription reactions were performed by the addition of KC1 (0.3 M) 30 s after starting transcription with the addition of nucleo- tides. The order of addition of recombinant TFIIF (rTFIIF), other fac- tors, and KC1 are as indicated and diagrammed at the bottom of the figure. The template plasmid, pFC53X, and factors were used as de- scribed in the legend to Fig. IA. The -fold activations, with uersus without SRF for each condition, are indicated below the lanes.

1992). Our conclusion that this fraction was TFIIF was based on its fractionation properties, since antibodies were not avail- able at that time. I t is now apparent that the fraction we previously believed to contain TFIIF in fact contains another required activity, most probably TFIIH. TFIIH is the most re- cently identified general transcription factor and was described after submission of our previous work (Flores et al., 1992). TFIIH only separates from TFIIF upon extensive chromatog- raphy (Flores et al., 1992). Our TFIIH activity separated from TFIIF on the phenyl-5PW column (see “Experimental Proce- dures”), similar to the separation described by Flores et al. (1992), and does not contain detectable TFIIF reactivity upon immunoblotting (data not shown).

Addition of TFIIF Is Required during Preinitiation Complex Formation-TFIIF is not only an initiation factor, but also af- fects elongation (Flores et al., 1989; Price et al., 1989; Bengal et al., 1991). In order to determine whether the effect of TFIIF in SRF activation is during initiation or elongation, we performed transcription reactions under conditions where only one round of initiation occurs. A single round of transcription can be achieved by preincubating transcription factors and templates, adding nucleotides to start the reaction, and then blocking reinitiation by adding 0.3 M KC1 (Cai and Luse, 1989). Tran- scriptional elongation was allowed to proceed for 5 min. Longer periods for elongation did not affect transcript levels (data not shown). When 0.3 M KC1 was added just before the addition of nucleotides, no transcription was observed, demonstrating that this treatment blocked initiation (Fig. 4, lanes 3 and 4 ). Using this method, we have shown that SRF activates transcription by affecting preinitiation complex formation (Zhu et al., 1991). The same kind of experiment was performed by preincubating with or without rTFIIF. Addition of rTFIIF strongly increased activation when added during the preincubation compared with no addition or addition of TFIIF after the preincubation (compare lanes 5 and 6 with lanes 1,2, 7, and 8). These results suggest that TFIIF has its effect on SRF-activated transcrip- tion during preinitiation complex formation and thus affects

initiation. I t may still differentially affect elongation, but only if allowed to first affect (or join) the preinitiation complex.

We have observed some variability in activation by SRF without TFIIF. For instance, in lanes 1 and 2, significant acti- vation was observed, although this activation was strongly in- creased upon addition of TFIIF (lanes 5 and 6). In Fig. 1, however, which was also performed under single-round tran- scription conditions, only very low activation was observed without TFIIF. Although similar fractions were used in these experiments, they were derived from different preparations and may contain different levels of contaminating TFIIF which could account for the differences. Alternatively, the fractions may contain differing levels of factors that are not well under- stood, such as inhibitors of basal transcription, that can also affect the level of activation. In general, when we have observed some activation without adding TFIIF, the level was increased even further by addition of TFIIF, as in Fig. 4. The increase in SRF activation due to addition of TFIIF was quite reproducible. In 13 experiments, we found that TFIIF increased activation

Squelching by SRF Can Be Relieved by TFIIF-We have found previously that high amounts of SRF can inhibit (squelch) SRF-, GALA-VP16-, and CREB-activated transcrip- tion, but not basal transcription (Prywes and Zhu, 1992 and Fig. 5A, lanes 1-4). These results were interpreted as evidence for SRF binding and titrating out a common coactivator re- quired for activated transcription. If squelching is in fact due to binding of SRF to a required factor, addition of excess amounts of this factor should relieve squelching by SRF. We added ad- ditional amounts of TFIIF and each of the general transcription factors to test whether any of them would affect squelching by SRF. HeLa cell-derived TFIIF was used because excess rTFIIF inhibited transcription, perhaps due to contaminants in the preparation. We estimate from comparisons by immunoblotting that roughly a %fold excess of HeLa TFIIF was added com- pared with the 30 ng of rTFIIF used in all the reactions. A2-fold excess of each of the other general factors was used. Only ex- cess TFIIF relieved squelching by SRF (Fig. 5). (A 2-fold excess of TFIIF gave similar results in a separate experiment (data not shown).) Beyond relieving inhibition, excess TFIIF actually increased activation by 40 ng of SRF. Higher amounts of SRF (60 ng) resulted in inhibition of this maximal level. This would be expected, since higher SRF should ultimately be able to bind up the excess TFIIF added. Excess TFIIH inhibited activation in this experiment, such that it did not give a clear result. In other experiments, however, excess TFIIH was not able to re- lieve SRF squelching (data not shown). The squelching of GALA-VP16 activation by SRF (Prywes and Zhu, 1992) could also be relieved by excess TFIIF (data not shown). These re- sults suggest that the “coactivator” with which SRF interacts is TFIIF. Since TFIIF is a general transcription factor, squelching of TFIIF would also be expected to affect basal transcription. One explanation for the lower effect on basal transcription is that SRF squelching is not effective in completely titrating out TFIIF. As described above, low levels of TFIIF are sufficient for basal transcription, such that small amounts of TFIIF, not bound by SRF, might be sufficient. Much higher amounts of SRF (200-400 ng) did in fact abolish basal transcription (data not shown), suggesting that very high amounts of SRF are sufficient to completely titrate out TFIIF.

Binding of RAP74 to an SRF.DNA Complex-The squelching experiments described above suggested that SRF may directly interact with TFIIF. We have detected such a direct interaction using a gel mobility shift assay. The probe, XF, used in these assays was a DNA fragment from pFC53X, a plasmid template used for the in vitro transcription assays, containing an SRF binding site 5’ to fos promoter sequence from -53 to +42. As

4.5 2 0.5-fold (S.E.).

3494 Role of TFIIF in SRF-activated Danscription

A Excess TFIIF - +

SRF(ng) - 5 40 60" - 5 40 60' Robe- -"."..

fosCAT Transcript - -- - . .) FIG. 5. TFIIF can relieve SRF squelching. A, increasing amounts of SRF were added, as indicated, to activate and then inhibit (squelch) transcription. The transcription factors used in these as- says were the same as described in Fig. 3 except that 1 p1 of a phosphocellulose 0.85 M KC1 fraction (Zhu et al., 1991) was used as the source of TFIID. In lams 5-8,3 pl of TFIIF purified from HeLa cytoplasmic extracts (30 nglpl) was added. B, as in A, except that a 2-fold excess of each of the other general factors was added as indi- cated. Transcripts detected in A and in similar experiments with excess levels of the other factors were quantitated using a PhosphorImager. The levels are ex- pressed as -fold activation compared with identical conditions without SRF.

B

shown in Fig. 6 A , the RAP74 subunit of TFIIF caused a slower mobility of the SRF complex (compare lanes 2 and 3). The speci- ficity of this complex was demonstrated by competition with a specific oligonucleotide containing an SRF binding site (lane 4 ) but not a mutant competitor (lane 3 1. RAP74 alone did not bind to DNA (lane 1 ). The free DNA probe was run off the gel to better visualize the change in mobility. When the free DNA was elec- trophoresed only to the bottom of the gel, no other specific com- plexes were observed (data not shown). Other transcription fac- tors, RAP30 and TFIIA, -B, -E, and -H did not change SRF's mobility (lanes 5-9). TFIID could bind to the probe, which con- tains a TATA box, but no cooperativity in binding of SRF and TFIID was observed (data not shown). The slower migrating band observed with addition of both SRF and TFIIF could be further shifted (supershifted) by both anti-SRF and anti-RAP74 sera, but not preimmune serum, indicating that the higher band contained both SRF and RAP74 (lanes 12-14). The RAP746RF-DNA complex could be shifted slightly higher by adding RAP30 (Fig. 6B, compare lanes 4 and 2) . We used the anti-RAP74 and anti-RAP30 sera to further demonstrate the presence of RAP30 and RAP74 in the complexes. Anti-RAP74 sera supershifted the RAP74.SRF-DNA (lane 6) and RAP30.RAP74.SRF.DNA (lane 7) complexes, but not the SRF.DNA complex (lane 5 and data not shown). Anti-RAP30 sera supershifted the RAP30.RAP74.SRF.DNA complex (lane IO), but not the RAP74.SRF-DNAcomplex (lane 8 ) or SRF.DNA complex (in the presence of RAP30, lane 9). These results are consistent with RAP74 binding to the SRF.DNA complex and RAP30 binding through its interaction with RAP74.

We tested whether the TFIIF interaction is specific to SRF or whether TFIIF can form complexes with other DNA binding factors. We used G U S DNA binding domain (amino acids 5-1471, Spl, and GAL4-VP16. The DNA probes were similar to that used for SRF, except for GAL4 or Spl binding sites in place of that for SRF. The gel mobility shift complex of neither

1 2 3 4 5 6 7 8

1

GU(5-147) nor Spl were affected by RAP74 (Fig. 7, lanes 2-7). Anti-RAP74 sera also did not affect the mobility of the complexes, further demonstrating that RAP74 did not bind with these factors (lane 4 and data not shown). GAL4-VP16 binding, however, was affected by RAP74 (lanes 8-11). The mobility of the RAP74.GAL4-VP16.DNA complex was super- shifted by anti-RAP74 sera, but not preimmune sera, demon- strating that RAP74 is in the complex. RAP74 caused a more diffuse GAL4-VP16 complex, perhaps because the RAP74- GAL4-VP16 complex dissociates during electrophoresis. As an additional control, DNAbinding by GAL4-Spl, containing Spl's transcriptional activation domain (Courey and Rian, 19881, was not affected by RAP74 (lanes 12 and 13).

In order to determine which domain of SRF interacts with RAP74, we first tested a chimeric protein, GM-SRF(245- 508), which consists of the DNA binding domain of GAL4 (ami- no acids 1-94) fused to the C-terminal part of SRF (amino acids 245-508) containing the transcriptional activation domain. This chimeric protein activates transcription to a similar ex- tent as SRF in As with GAL4(5-147), the mobility of a GAL4(1-94).DNAcomplex was not affected by RAP74 (data not shown). RAP74, however, did interact with GAIA-SRF(245- 5081, suggesting that SRF's transcriptional activation domain may mediate the interaction with RAP74 (Fig. 7, lanes 15-18).

We tested SRFs core DNA binding domain (SRF(141-244)) for RAP74 interaction and found that it also bound DNA with RAP74 (Fig. 7, lanes 20-23). We have found that SRF(141-244) activates transcription in vitro to about 50% the level with full-length SRF (about %fold; data not shown). Under similar conditions (but with a GAL4 site reporter template), GAL4's DNA binding domain (amino acids 5-147) activated 1.5-fold, suggesting that the activation is not simply due to occupation of a DNA binding site. These results suggest that SRF's DNA

F.-E. Johansen and R. F'rywes, unpublished data.

Role of TFIIF in SRF-activated Panscription 3495

A B SRF SRF

Ab TF 74 74 74 30 A B E H 74 74 74

I s P s 74 Pi Ab ' 74 74 74 30 30 30 RAP74 + + + + + + RAP30 + + + + + +

Cornp M M W

Y e, e

U

b 4 c

P 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 2 3 4 5 6 7 8 9 1 0

FIG. 6. Binding of RAP74 with SRF to DNA. Gel mobility shift assays were performed with 1 ng of probe ( X F ) containing an SRF binding site and fos promoter sequence from -53 to +42. The transcription factors (TF) used, as indicated, were: 1 ng of SRF, 20 ng of RAP74 (74) , 20 ng, of RAP30 (30). 1 pl of TFIIA (A) , 10 ng of ffFIIB ( B ) , 40 ng of rTFIIE ( E ) , and 0.2 pl of TFIIH ( H ) . Double-stranded oligonucleotides (25 ng) w t h wild type (W) or mutant (M) SRF binding sites were used as competitors. Antisera were added as indicated (Ab), including anti-SRF ( S ) , anti-RAP74 (74 ), or preimmune (P) sera (0.1 pl in A and 1 pl in B ) .

74 P"' 74 P" I 74 P ' ' 74 P" 74 PI R A P 7 4 + - + + + - + - + + + - + + - + + + + - + + + - + + +

"- I P " @' m

I

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 FIG. 7. Binding of RAP74 with other DNA-binding proteins. The indicated DNA-binding proteins were incubated with or without RAP74

in gel mobility shiff assays. Anti-RAP74 (74) or preimmune sera (P) (1 pl) were added, as indicated, to demonstrate whether RAP74 was in the shifted complexes. The "P-labeled DNA probes SF and GF were the same as XF (Fig. 6) except for Spl or GAL4 binding sites, respectively, in place of the SRF binding site. The following amounts of factors were used in the reactions: 20 ng of RAP74, 1 ng of SRF, 0.75 ng of Spl, 10 ng of GAL4(5-147), 5 ng of GACQ-VP16,O.Ol pl of GALA-Spl(A+B), 10 ng of GU-SRF(245-508), and 0.25 ng of SRF(141-244). Hemng sperm DNA (100 ng) was added for lanes 1 5 and 19-23. The reaction mixtures were loaded on native 4% polyacrylamide gels except for GALA(5-147) and SRF(141-244) where 8% gels were used.

binding domain contributes to activation by SRF. Thus, two domains in SRF can interact with RAP74 and contribute to activation in vitro, the core DNA binding domain (amino acids 141-244) and the C-terminal region (amino acids 245-508). However, since GAL4-SRF(245-508) can fully activate without SRF's DNA binding domain, the DNA binding domain is not required for activation.

DISCUSSION Three lines of evidence suggest that TFIIF is critical for

activation of transcription by SRF. First, higher levels of TFIIF were required for SRF-activated transcription than for basal-

or Spl-activated transcription. Second, squelching of transcrip- tional activation by SRF in vitro could be relieved by excess TFIIF, suggesting that SRF may bind TFIIF and make it inac- cessible to the initiation complex. Third, more direct evidence of an SRF-TFIIF interaction was found using a gel mobility shift assay. TFIIF or its RAP74 subunit bound in a complex with SRF, but not to DNA alone. We found that TFIIF did not affect SRF's DNase I protection pattern on its binding site, such that TFIIF does not appear to be binding to adjacent sequences (data not shown). These results suggest that SRF binds TFIIF directly and that this binding is involved in transcriptional activation.

3496 Role of TFIIF in SRF-activated !banscription

It is possible that SRF either recruits TFIIF to the transcrip- tional initiation complex or affects TFIIF's ability to function. The RAP30 subunit of TFIIF binds RNA polymerase I1 (Flores et al., 1991; Killeen and Greenblatt, 1992). Thus, binding of SRF to RAP74 could also recruit RNA polymerase I1 to the initiation complex or change the conformation of a larger com- plex. Gel mobility shift experiments have suggested that the order of addition of the general transcription factors to an ini- tiation complex is TFIID-TFIIA-TFIIB-(RNA polymerase II- TF1IF)-TFIIE-TFIIH (Buratowski et al., 1989; Flores et al., 1992). If TFIIF and RNA polymerase I1 addition is limiting, SRF could stimulate transcription by causing TFIIF and RNA polymerase I1 to enter the complex after TFIID, -A, and -B. Alternatively, SRF could change the conformation and activity of the initiation complex through its interaction with TFIIF without changing the rate of complex formation. Another pos- sibility involves TFIIF's effect on elongation (Flores et al., 1989; Price et al., 1989; Bengal et al., 1991). Recently, in fact, RAP74 was specifically found to affect an early stage in elongation (Chang et al., 1993). Although we found that TFIIF must be added during preinitiation complex formation, it is possible that SRF affects RAP74's subsequent ability to stimulate elon- gation.

The gel mobility shift and relief of squelching experiments suggest that SRF directly interacts with TFIIF. We have not, however, been able to detect this interaction by coimmunopre- cipitation with anti-RAP30 or anti-RAP74 sera. This may be due to the stability of the interaction under these conditions or a requirement for specific DNA sequences. We are currently in- vestigating other methods to detect the SRF-TFIIF interaction.

The reason why higher levels of TFIIF are required for SRF- activated transcription than basal transcription is unclear. It is possible that the stoichiometry of TFIIF in the activated tran- scription complex is higher than in a basal complex. Alterna- tively, SRF may only be able to associate with TFIIF in vitro when the TFIIF concentration is high. Thus, when higher TFIIF is present and activation occurs, more template becomes occupied by transcription complexes. There could also be a dif- ferent form of TFIIF required for activated versus basal tran- scription such that more of the mixture is required for activated transcription. This last possibility is somewhat unlikely since recombinant TFIIF functions in both types of transcription.

TFIIF formed complexes with SRF or GALA-VP16 but not with G U S DNA binding domain or with Spl. This correlates well with the requirement of these activators for higher levels of TFIIF. Spl did not interact with TFIIF and did not require higher levels of TFIIF to activate transcription. GALA-VP16 interacted with TFIIF, and higher levels of TFIIF increased GALA-VP16 activation. GAL4-VP16, however, could also acti- vate well with low levels of TFIIF. One possible explanation for the lack of an absolute requirement for high TFIIF levels is that GAL4-VP16 uses multiple mechanisms to activate tran- scription, These other mechanisms could involve its binding to TFIID or TFIIB, as has been reported (Stringer et al., 1990; Lin et al., 1991). Likewise, these results suggest that Spl uses a different mechanism than SRF to activate transcription. This could be its binding to the TBP-associated factor, TAFllO (Hoey et al., 1993).

SRF's transcriptional activation domain appears to lie in the C-terminal part of the protein, based on activation by GALA- SRF fusion proteins in HeLa cells (Johansen and Prywes, 1993). We have obtained slightly different results in vitro. De- letion of the C terminus of SRF reduced activation 50%, but did not abolish it (Prywes and Zhu, 1992), and we have found further that SRF's DNA binding domain (amino acids 141-244) can similarly activate to 50% of levels with full-length SRF (data not shown). It is possible that the in vitro results reflect

the effects of domains that contribute to activation but that are not required or sufficient for activation in vivo. Both the C- terminal and DNA binding domains of SRF were able to bind with RAP74 in the gel mobility shift assay, suggesting that there are two domains in SRF which can mediate an interac- tion with TFIIF. This is not surprising since a number of acti- vators have been found to have multiple activation domains (Courey and Tjian, 1988; Regier et al., 1993). Both of the SRF domains could activate transcription in vitro, albeit to different extents, such that there is a correlation with TFIIF interaction and activation. It will be worthwhile to examine more mutants of SRF to determine minimal regions of the activation domain required for TFIIF interaction and whether mutation of these regions affects activation.

Since the C-terminal activation domain of SRF activates more strongly than SRFs DNA binding domain, whereas both bind TFIIF in vitro, interaction with TFIIF may not be suffi- cient to fully account for activation of transcription by SRF. Interaction with other factors may also be required. This could be interaction with TFIID, since we found previously that the order of addition of SRF and TFIID was critical for activation (Zhu et al., 1991). In addition, TFIIF is not sufficient to account for activation of transcription when using recombinant TBP in place of HeLa TFIID. As with other activators, no activation by SRF was observed when using TBP (Zhu et al., 1991).

Different gene-specific transcription factors may target dif- ferent parts of the general transcription apparatus. In addition, a single factor may bind multiple components. This is sug- gested by the results that GALA-VP16 binds TFIID and TFIIB (Stringer et al., 1990; Lin et al., 19911, as well as binding DNA with TFIIF, as described here. Other factors have also been found to bind TBP, TFIIB, or TAFllO (Lee et al., 1991; Lieber- man and Berk, 1991; Seto et al., 1992; Ing et al., 1992; Hoey et al., 1993). The targeting of different general factors may also explain the synergy of activation observed with multiple factors in many systems (Herschlag and Johnson, 1993). The demon- stration here that TFIIF has a specific effect on SRF-activated transcription in vitro and that TFIIF can bind DNAin conjunc- tion with SRF suggests that TFIIF is an important target for activation of transcription.

Acknowledgments-We thank Kathy Wang, Philippe Pognonec, Rob- ert &der, and Zachary Burton for generous gifts of antibodies to TFIIF and Sherman Weisman and Zachary Burton for cDNA clones of RAP30 and RAp74. In addition, we thank Hui Ge for purified Spl, Timothy Hoey for purified G U - S p l , Violeta Tirnanic for purified G U - W l 6 , Finn-Eirik Johansen for purified GALA-SRF, Danny Reinberg for a TFIIB cDNA clone, and Robert Roeder for TFIIE cDNA clones.

REFERENCES

Bargonetti, J., Reynisdottir, I., Friedman, P., and Prives, C. (1992) Genes & Deu. 6,

Bengal, E., Flores, O., Krauskopf, A,, Reinberg, D., and Aloni, Y. (1991) Mol. Cell.

Buratowski. S., Hahn, S., Guarente, L., and Sharp, P. A. (1989) Cell 56,549-561 Cai, H., and Luse, D. S. (1987) J. Biol. Chern. 262, 298-304 Chang, C., Kostrub, C. F., and Burton, Z. F. (1993) J. Biol. Chern. 268,20482-20489 Chasman, D. I., Leatherwood, J., Carey, M., Ptashne, M.. and Komberg, R. D.

Courey, A., and Tjian, R. (1988) Cell 55,887-898 Dignam, J. D., Lebovitz, R. M., and Fbeder, R. G. (1983) Nucleic Acids Res. 11,

Dynlacht, B. D., Hoey, T., and Tjian, R. (1991) Cell 66,563676 Finkelstein, A,, Kostrub, C., Li, J., Chavez, D. P., Wang, B. Q., Fang, S. M.,

Greenblatt, J., and Burton, Z. F. (1992) Nature 366, 464-467 Flanagan, P. M., Kelleher, R. J., 111, Sayre, M. H., 'hchochner, H., and Kornberg, R. D. (1991) Nature 360,436-438

Flores, O., Maldonado, E,, and Reinberg, D. (1989) J. Biol. Chern. 264,891343921 Flores, O., Lu. H., Killeen, M., Greenblatt, J., Burton, Z. F., and Reinberg, D. (1991)

Flores, O., Lu, H., and Reinberg, D. (1992) J. Biol. Chem. 267,27862793 Gill, G., and Tjian, R. (1992) Cum Opin. &net Deu. 2, 236242 Ha, I., Lane, W. S., and Reinberg, D. (1991) Nature 552,689495 Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring

18861898

Biol. 11, 1195-1206

(1989) Mol. Cell. Biol. 9,4746-4749

1475-1489

Proc. Natl. Acad. Sci. U. S. A. 88,9999-10003

Harbor Laboratory, Cold Spring Harbor, NY

Role of TFIIF in SRF-activated D-anscription 3497 Herschlag, D., and Johnson, R. B. (1993) Genes & Deu. 7, 173-179 Hoey, T., Weinzierl, R. 0. J., Gill, G., Chen, J., Dynlacht, B. D., and Tjian, R. (1993)

Hoftinann, A,, and Roeder, R. (1991) Nucleic Acids Res. 19,63374338 Ing, N. H., Beekman, J. M., Tsai, S . Y., %ai, M.J., and O'Malley, B. W. (1992) J.

Jackson, S . , and Tjian, R. (1989) Proc. Natl. h a d . Sci. U. S. A. 86, 1781-1785 Johansen, F.-E., and Prywes, R. (1993) Mol. Cell. B i d . 13,464M647 Katagiri, F., Yamazaki, K., Horikoshi, M., Roeder, R. G., and Chua, N.-H. (1990)

Killeen, M. T., and Greenblatt, J. F. (1992) Mol. Cell. B i d . 12, 3 0 3 7 Lee, W. S. , Kao, C. C., Bryant, G. O., Liu, X., and Berk,A. J. (1991) Cell 67,365-376 Lieberman, P. M., and Berk, A. J . (1991) Genes & Deu. 5,2441-2454 Lin, Y. S . , and Green, M. (1991) Cell 64, 971-981 Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. (1991) Nature 353,

Manak, J. R., and Prywes, R. (1993) Oncogene 8,703-711 Manak, J. R., deBisschop, N., Kris, R. M., and Prywes, R. (1990) Genes & Deu. 4,

Meisterernst, M., Roy, A,, Lieu, H. M., and Roeder, R. (1991) Cell 66, 981-993 Mitchell, P., and Qian, R. (1989) Science 245,371478 Nakajima, N., Horikoshi, M., and Roeder, R. G. (1988) Mol. Cell. B i d . 8,4028-4040 Norman, C., Runswick, M., Pollock, R., and Treisman, R. (1988) Cell 55,989-1003 Ohkuma, Y., Sumimoto, H., Hoftinann, A., Shimasaki, S. , Horikoshi, M., and

Cell 72, 247-260

Biol. Chem. 267,17617-17623

Genes & Deu. 4, 1899-1909

569-571

955-967

Peterson, M. G., Tanses, N., Pugh, B. F., and Qian, R. (1990) Science 248, 1626-

Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1989) Mol. Cell. Biol. 9,14651475 Prywes, R., and Zhu, H. (1992) Nucleic Acids Res. 20, 513-520 Pugh, B. E, and Tjian, R. (1991) Genes & Deu. 5, 1935-1945 Pugh, B. F., and Tjian, R. (1992) J. Biol. Chem. 267,679-682 Regier, J. L., Shen, F., and Triezenberg, S . J. (1993) Proc. Natl. Acad. Sci. U. S. A.

90,883-887 Seto, E., Usheva, A., Zambetti, G. P., Momand, J., Horikoshi, N., Weinmann, R.,

Stringer, K. F., Ingles, C. J., and Greenblatt, J. (1990) Nature 345, 783-786 Levine, A., and Shenk, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,1202&12032

Suminoto, H., Ohkuma, Y., Sinn, E., Kato, H., Shimasaki, S. , Horikoshi, M., and

Tanese, N., Pugh, B. E, and Tjian, R. (1991) Genes & Deu. 5, 2212-2224 Treisman, R. (1992) Zkknds Biochem. Sci. 17,423-426 Wang, B. Q., Kostrub, C. F., Finkelstein, A., and Burton, Z. E (1993) Protein

White, J. H., Brou, C., Wu, J., Burton, N., Egly, J.-M., andchambon, P. (1991)Proc.

Zawel, L., and Reinberg, D. (1992) Cum Opin. Cell Biol. 4, 488-495 Zhu, H., and Prywes, R. (1992) Proc. Natl. Acad. Sci. U. S.A. 89, 5291-

Zhu, H., Roy, A. L., Roeder, R., and Prywes, R. (1991) New Biol. 3, 455464

Roeder, R. (1991) Nature 354,396401

1630

Roeder, R. (1991) Nature 354,401-404

Expression Purif. 4, 207-214

Natl. Acad. Sci. U. S. A. 88, 76767678

5295