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Properties of PC4 and an RNA Polymerase II Complex in Directing Activated and Basal Transcription in Vitro

1998; Elsevier BV; Volume: 273; Issue: 20 Linguagem: Inglês

10.1074/jbc.273.20.12492

ISSN

1083-351X

Autores

Shwu-Yuan Wu, Cheng-Ming Chiang,

Tópico(s)

RNA Research and Splicing

Resumo

A human RNA polymerase II (pol II) complex was isolated from a HeLa-derived cell line that conditionally expresses an epitope-tagged RPB9 subunit of human pol II. The isolated FLAG-tagged pol II complex (f:pol II) contains a subset of general transcription factors but is devoid of TFIID and TFIIA. In conjunction with TATA-binding protein (TBP) or TFIID, f:pol II is able to mediate both basal and activated transcription by Gal4-VP16 when a transcriptional coactivator PC4 is also provided. Interestingly, PC4, in the absence of a transcriptional activator, actually functions as a repressor to inhibit basal transcription. Remarkably, TBP is able to mediate activator function in this transcription system. The presence of TBP-associated factors, however, helps overcome PC4 repression and further enhance the level of activation mediated by TBP. Alleviation of PC4 repression can also be achieved by preincubation of the transcriptional components with the DNA template. Sarkosyl disruption of preinitiation complex formation further illustrates that PC4 can only inhibit transcription prior to the assembly of a functional preinitiation complex. These results suggest that PC4 represses basal transcription by preventing the assembly of a functional preinitiation complex, but it has no effect on the later steps of the transcriptional process. A human RNA polymerase II (pol II) complex was isolated from a HeLa-derived cell line that conditionally expresses an epitope-tagged RPB9 subunit of human pol II. The isolated FLAG-tagged pol II complex (f:pol II) contains a subset of general transcription factors but is devoid of TFIID and TFIIA. In conjunction with TATA-binding protein (TBP) or TFIID, f:pol II is able to mediate both basal and activated transcription by Gal4-VP16 when a transcriptional coactivator PC4 is also provided. Interestingly, PC4, in the absence of a transcriptional activator, actually functions as a repressor to inhibit basal transcription. Remarkably, TBP is able to mediate activator function in this transcription system. The presence of TBP-associated factors, however, helps overcome PC4 repression and further enhance the level of activation mediated by TBP. Alleviation of PC4 repression can also be achieved by preincubation of the transcriptional components with the DNA template. Sarkosyl disruption of preinitiation complex formation further illustrates that PC4 can only inhibit transcription prior to the assembly of a functional preinitiation complex. These results suggest that PC4 represses basal transcription by preventing the assembly of a functional preinitiation complex, but it has no effect on the later steps of the transcriptional process. In eukaryotes, transcription of protein-encoding genes requires general transcription factors (GTFs) 1The abbreviations used are: GTF, general transcription factor; pol, polymerase; PC4, positive cofactor 4; SRB, suppressor of RNA polymerase B; TBP, TATA-binding protein; TAF, TBP-associated factor; TFIID, transcription factor IID; f:pol II, a FLAG-tagged RNA polymerase II complex; USA, upstream stimulatory activity; PIC, preinitiation complex; ss, single-stranded; HSSB, human single-stranded DNA-binding protein.1The abbreviations used are: GTF, general transcription factor; pol, polymerase; PC4, positive cofactor 4; SRB, suppressor of RNA polymerase B; TBP, TATA-binding protein; TAF, TBP-associated factor; TFIID, transcription factor IID; f:pol II, a FLAG-tagged RNA polymerase II complex; USA, upstream stimulatory activity; PIC, preinitiation complex; ss, single-stranded; HSSB, human single-stranded DNA-binding protein. and RNA polymerase II (pol II), which are assembled on the promoter region to form a preinitiation complex (PIC) capable of producing RNAs when both ribonucleoside triphosphates and energy sources are provided (1Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 2Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (838) Google Scholar).In vivo, the PIC assembly is a rate-limiting step for the initiation of transcription and is often facilitated by gene-specific transcriptional activators (3Struhl K. Cell. 1996; 84: 179-182Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Ptashne M. Gann A. Nature. 1997; 386: 569-577Crossref PubMed Scopus (926) Google Scholar). Currently, there are two proposed pathways for the PIC assembly on TATA-containing promoters. In the sequential pathway, TFIID binding to the TATA box is followed, in a stepwise fashion, by the joining of TFIIB, pol II/TFIIF, TFIIE, and TFIIH, whereas in the two-component pathway, binding of TFIID is accompanied by a preassembled pol II complex that contains pol II, a subset of GTFs, SRBs (suppressors of RNA polymerase B mutations (5Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (529) Google Scholar, 6Kim Y.-J. Björklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (879) Google Scholar)), and other proteins involved in chromatin remodeling, DNA repair, or mRNA processing (5Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (529) Google Scholar, 6Kim Y.-J. Björklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (879) Google Scholar, 7Ossipow V. Tassan J.-P. Nigg E.A. Schibler U. 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Chem. 1997; 272: 24563-24571Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Transcriptional activators, in most cases, are able to increase the level of initiation by enhancing the recruitment of TFIID and/or other components of the basal transcription machinery to the promoter region (3Struhl K. Cell. 1996; 84: 179-182Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Ptashne M. Gann A. Nature. 1997; 386: 569-577Crossref PubMed Scopus (926) Google Scholar). This activation process often requires transcriptional coactivators. Thus far, two major classes of general coactivators required for activator function have been identified in mammalian cell-free transcription systems. One is TBP-associated factors (TAFs) in TFIID (14Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (620) Google Scholar, 15Verrijzer C.P. Tjian R. Trends Biochem. Sci. 1996; 21: 338-342Crossref PubMed Scopus (318) Google Scholar, 16Tansey W.P. Herr W. Cell. 1997; 88: 729-732Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and the other is protein cofactors derived from the upstream stimulatory activity (USA) found in the phosphocellulose P11 0.85 m KCl fraction of HeLa nuclear extracts (17Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Abstract Full Text PDF PubMed Scopus (225) Google Scholar, 18Kaiser K. Meisterernst M. Trends Biochem. Sci. 1996; 21: 342-345Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Positive cofactor 4 (PC4) was isolated from a crude USA fraction and was able to substitute for USA to mediate activator-dependent transcription in vitro(19Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 20Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 21Kaiser K. Stelzer G. Meisterernst M. EMBO J. 1995; 14: 3520-3527Crossref PubMed Scopus (90) Google Scholar). PC4 is a nonspecific DNA-binding protein, which shows a higher affinity toward single-stranded (ss) DNA molecule (20Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 21Kaiser K. Stelzer G. Meisterernst M. EMBO J. 1995; 14: 3520-3527Crossref PubMed Scopus (90) Google Scholar, 22Ballard D.W. Philbrick W.M. Bothwell A.L.M. J. Biol. Chem. 1988; 263: 8450-8457Abstract Full Text PDF PubMed Google Scholar). The ssDNA binding activity of PC4 can replace human ssDNA-binding protein (HSSB, also called replication protein A (RPA)) in supporting the T antigen-catalyzed unwinding of SV40 origin-containing duplex DNA (23Pan Z.-Q. Ge H. Amin A.A. Hurwitz J. J. Biol. Chem. 1996; 271: 22111-22116Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Nevertheless, PC4 cannot substitute for HSSB in other aspects of replication activities mediated by HSSB (23Pan Z.-Q. Ge H. Amin A.A. Hurwitz J. J. Biol. Chem. 1996; 271: 22111-22116Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Likewise, the transcriptional activity of PC4 cannot be replaced by other ssDNA-binding proteins (19Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (304) Google Scholar). The coactivator function of PC4 seems to correlate with its double-stranded DNA binding activity (21Kaiser K. Stelzer G. Meisterernst M. EMBO J. 1995; 14: 3520-3527Crossref PubMed Scopus (90) Google Scholar) and its interactions with transcriptional activators and with components of the general transcription machinery such as TFIIA (19Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (304) Google Scholar). Surprisingly, gene inactivation of a PC4 homologue in yeast does not lead to cell death, indicating that PC4 is nonessential in yeast (24Knaus R. Pollock R. Guarente L. EMBO J. 1996; 15: 1933-1940Crossref PubMed Scopus (94) Google Scholar, 25Henry N.L. Bushnell D.A. Kornberg R.D. J. Biol. Chem. 1996; 271: 21842-21847Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Since yeast PC4 also exhibits distinct properties from that of human PC4 (24Knaus R. Pollock R. Guarente L. EMBO J. 1996; 15: 1933-1940Crossref PubMed Scopus (94) Google Scholar, 25Henry N.L. Bushnell D.A. Kornberg R.D. J. Biol. Chem. 1996; 271: 21842-21847Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), it is likely that PC4 may function differentially in various organisms. This remains to be further investigated. Using an in vitro transcription system reconstituted with either TBP or TFIID and a preassembled pol II complex, we found that PC4 could function as a repressor to suppress basal transcription in the absence of an activator. Interestingly, TBP was able to mediate Gal4-VP16 activation in the absence of TAFs. This finding suggests that human TBP can indeed mediate activator function, as observed in the yeast system (26Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar, 27Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (250) Google Scholar, 28Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (129) Google Scholar). To understand the molecular mechanism of PC4 repression, we carried out template challenge and Sarkosyl disruption experiments using our two-component transcription system. The results indicate that PC4 represses transcription by preventing the assembly of a functional preinitiation complex when an activator is not present. A tetracycline-regulated human RPB9-expressing plasmid, pTetCMV-Fo:hRPB9, was first constructed by cloning the RPB9 cDNA (29Acker J. Wintzerith M. Vigneron M. Kedinger C. Nucleic Acids Res. 1993; 21: 5345-5350Crossref PubMed Scopus (25) Google Scholar), isolated from pBn-Fo:14.5 (provided by H. Ge) between NdeI andXbaI sites, to pTetCMV-Fo(AS) (30Wu S.-Y. Chiang C.-M. BioTechniques. 1996; 21: 718-725Crossref PubMed Scopus (18) Google Scholar) at the same enzyme-cutting sites. The expression plasmid, pF:TFIIA(55)-11d, was made by transferring the p55 insert from pET11aN1–376 (31DeJong J. Roeder R.G. Genes Dev. 1993; 7: 2220-2234Crossref PubMed Scopus (89) Google Scholar) into pF:TBP-11d (32Chiang C.-M. Roeder R.G. Peptide Res. 1993; 6: 62-64PubMed Google Scholar) after removing the TBP insert between NdeI and EcoRI sites. Similarly, plasmids pF:TFIIA(1–274)-11d, pF:TFIIA(275–376), and pF:TFIIAγ(hp12) were constructed by swapping individual TFIIA inserts from pJD1–274, pJDGEX2t(L)275–376, and pGEX2t(L)hp12 (33DeJong J. Bernstein R. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3313-3317Crossref PubMed Scopus (64) Google Scholar) with the TBP insert from pF:TBP-11d betweenNdeI and BamHI sites. Ten μg of PvuI-linearized pTetCMV-Fo:hRPB9 DNA was cotransfected with 50 μg of sheared calf thymus DNA and with 0.5 μg of SacI-linearized pREP4 (Invitrogen) into HtTA-1 (34Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4213) Google Scholar), a HeLa-derived cell line that constitutively expresses a tetracycline-controlled transactivator. Detailed procedures for establishing the tetracycline-regulated clonal cell lines had been described (30Wu S.-Y. Chiang C.-M. BioTechniques. 1996; 21: 718-725Crossref PubMed Scopus (18) Google Scholar). A cell line (hRPB9–3) that showed induced expression of FLAG-tagged hRPB9 in the absence of tetracycline was isolated after screening a dozen hygromycin-resistant cellular clones. To detect the presence of individual GTFs as shown in Fig. 1 A, 50 μl of HeLa nuclear extracts and 200 μl of f:pol II were mixed separately with an equal volume of the 2× protein sample buffer. The protein mixture was then separated by 10% SDS-polyacrylamide gel electrophoresis using a preparative mini-gel apparatus (Bio-Rad). After transfer to the nitrocellulose filters, samples were divided into multiple lanes using a Mini-Protean II multiscreen apparatus (Bio-Rad). Each sample lane was then incubated with a primary antibody, usually diluted 1000-fold unless otherwise specified, in a final volume of 600 μl. The rest of the procedures for Western blotting were performed as described (30Wu S.-Y. Chiang C.-M. BioTechniques. 1996; 21: 718-725Crossref PubMed Scopus (18) Google Scholar). The f:pol II complex was purified from hRPB9–3 as follows. The hRPB9–3 cell line was maintained in suspension culture with Joklik medium containing 5% calf serum in the presence of tetracycline (1 μg/ml) and selected with G418 (0.6 mg/ml, total weight) for 4 days before expansion for the preparation of nuclear extracts and S100. To induce protein expression, cells were pelleted and washed at least 3 times with 1× phosphate-buffered saline to remove tetracycline. Cells were then resuspended in fresh Joklik medium plus 5% calf serum. Nuclear extracts and S100 were prepared from hRPB9–3 cells, 4 days after removing tetracycline, as described (35Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9131) Google Scholar). To purify f:pol II, 10 ml of the hRPB9–3 nuclear extract or S100 was incubated with 250–500 μl of anti-FLAG M2-agarose beads (Kodak) at 4 °C for 6–12 h. Bound proteins were then washed and eluted with the synthetic FLAG peptide with 100 mm KCl-containing buffer as described previously for TFIID purification (36Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). Recombinant PC4 was purified from bacteria harboring pET11a/PC4, obtained from H. Ge, as described (37Ge H. Martinez E. Chiang C.-M. Roeder R.G. Methods Enzymol. 1996; 274: 57-71Crossref PubMed Scopus (58) Google Scholar). Purification of recombinant FLAG-tagged basal transcription factors including TFIIA (p55, p35, p19, and p12), TFIIB, TBP, TFIIEα, and TFIIEβ was performed as described previously (32Chiang C.-M. Roeder R.G. Peptide Res. 1993; 6: 62-64PubMed Google Scholar). TFIIA and TFIIF were then reconstituted from individually purified components following denaturation and renaturation. Typically, equal molar amounts of individual TFIIA (p55 and p12) or TFIIF (RAP30 and RAP74) subunits were mixed and adjusted to 6 m guanidine hydrochloride in BC100 (36Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). The mixture was incubated at room temperature for an hour and then sequentially dialyzed for 2 h each against 60 volumes of 2, 0.5, and 0.1m guanidine hydrochloride in BC100 and eventually against BC100. After centrifugation, the reconstituted proteins were dispensed into small aliquots and stored at −80 °C. Purification of FLAG-tagged TFIID (36Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar), core-pol II (38Reinberg D. Roeder R.G. J. Biol. Chem. 1987; 262: 3310-3321Abstract Full Text PDF PubMed Google Scholar), Gal4-VP16 (39Chasman D.I. Leatherwood J. Carey M. Ptashne M. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 4746-4749Crossref PubMed Scopus (140) Google Scholar), and FLAG-tagged Gal4 fusion proteins (40Chiang C.-M. Roeder R.G. Science. 1995; 267: 531-536Crossref PubMed Scopus (351) Google Scholar) was performed as described. FLAG-tagged TFIIH was purified from S100 derived from a tetracycline-regulated cell line that conditionally expresses the FLAG-tagged p62 subunit of human TFIIH (30Wu S.-Y. Chiang C.-M. BioTechniques. 1996; 21: 718-725Crossref PubMed Scopus (18) Google Scholar) following the procedure described for f:pol II purification. Recombinant histidine-tagged RAP30 and histidine-tagged RAP74 were purified from bacteria containing pET23d/RAP30 (41Fang S.M. Burton Z.F. J. Biol. Chem. 1996; 271: 11703-11709Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and pET23d/RAP74NspV (42Wang Q. Lei L. Burton Z.F. Protein Expression Purif. 1994; 5: 476-485Crossref PubMed Scopus (39) Google Scholar), respectively, following the published protocols (43Wang Q. Kostrub C.F. Finkelstein A. Burton Z.F. Protein Expression Purif. 1993; 4: 207-214Crossref PubMed Scopus (44) Google Scholar). In vitro transcription was typically carried out in a 25-μl reaction mixture containing 50 ng of pG5HMC2AT, 20 ng of pMLΔ53, 42 ng of renatured TFIIA (35 ng of p55 and 7 ng of p12), 10 ng of TFIIB, 10 ng of TBP, or 1 μl of FLAG-tagged TFIID (which contains approximately 1 ng of TBP as judged by Western blotting), 20 ng each of TFIIEα and TFIIEβ, 28 ng of renatured TFIIF (20 ng of RAP74 and 8 ng of RAP30), 1 μl (∼7.5 ng) of FLAG-tagged TFIIH, and 0.6 μl (∼18 ng) of core-pol II or f:pol II using the conditions as described previously (17Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Abstract Full Text PDF PubMed Scopus (225) Google Scholar). For activator-dependent transcription, 100 ng of PC4 and 50 ng of Gal4-VP16 were also included as specified. In the minimal transcription system, 3 μl (∼90 ng) of f:pol II was used in conjunction with either 2 ng of TBP or 2 μl of FLAG-tagged TFIID. The amount of FLAG-tagged Gal4 fusion proteins used in the minimal transcription reaction was: 3 ng of Gal4 (1–94), 6 ng of Gal4-Pro, 6 ng of Gal4-Gln, and 30 ng of Gal4 (1–147). Reactions were then performed and analyzed as described (36Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). A two-step incubation procedure described by Kaiser et al. (21Kaiser K. Stelzer G. Meisterernst M. EMBO J. 1995; 14: 3520-3527Crossref PubMed Scopus (90) Google Scholar) was used with a minor modification. Briefly, 50 ng of pG5HMC2AT and 20 ng of pMLΔ53 were preincubated with either 2 μl of FLAG-tagged TFIID (or 2 ng of TBP) or 3 μl of f:pol II at 30 °C for 50 min. Ribonucleoside triphosphates (NTPs) and the other component required for transcription (f:pol II or TFIID or TBP) were added after the preincubation period to initiate transcription. Reactions were continued at 30 °C for 60 min and then analyzed for RNA formation (36Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). Gal4-VP16 and PC4, when added, were used at 50 and 100 ng, respectively. The challenge template (500 ng of pG5HMC2AT or 500 ng of pMLΔ53) was included either at the beginning or at the end of the preincubation period as depicted in Fig. 3 A. In this experiment, 0.015% Sarkosyl was added either at the beginning or at the end of the preincubation period, which was performed in the presence of TBP, f:pol II, and both pG5HMC2AT and pMLΔ53 DNA templates as described in the template challenge experiments. Ribonucleoside triphosphates were then added to initiate transcription. One hundred nanograms of PC4 were added at various time points as outlined at the bottom of Fig. 4. A human pol II complex was purified from a clonal HeLa-derived cell line (hRPB9–3) that conditionally expresses the FLAG-tagged RPB9 subunit of human pol II (see "Experimental Procedures"). The purified FLAG-tagged pol II complex (f:pol II) contains not only pol II subunits as detected by Western blotting with antibodies against RPB1, RPB2, RPB6, RPB8, and RPB9 2S.-Y. Wu and C.-M. Chiang, unpublished data. but also a subset of GTFs including TFIIB, TFIIE, TFIIF, and TFIIH (Fig.1 A). The f:pol II complex contains stoichiometric amounts of TFIIB and TFIIF but substoichiometric quantities of TFIIE and TFIIH (Fig. 1 A, compare relative signals detected in nuclear extracts and f:pol II), as also evidenced by quantitative Western blotting using purified recombinant proteins as standards. 3The f:pol II complex contains approximately 50 fmol μl−1 of pol II subunits, 70 fmol μl−1 of TFIIB, 135 fmol μl−1 of TFIIF, 1.2 fmol μl−1 of TFIIE, and 3.2 fmol μl−1of TFIIH. TFIID and TFIIA were not detected in f:pol II at a sensitivity of 1 ng with anti-TBP and anti-TAFII55 antibodies and at a sensitivity of 0.1 ng with anti-TFIIA p35 antibodies (Fig. 1 A).2 Our f:pol II, enriched approximately 200-fold after immunoaffinity purification, did not seem to contain other transcriptional cofactors such as PC4 and Dr1 and transcriptional activators including Sp1, YY1, USF, p53, pRB, and the p50 subunit of NFkB.2 Recombinant human TFIIA, TFIIB, TFIIE, TFIIF, and FLAG-tagged TFIID and FLAG-tagged TFIIH were used in conjunction with either core-pol II (i.e. traditionally defined pol II) or immunoaffinity-purified f:pol II for transcriptional analysis. Both core-pol II and f:pol II, normalized by the content of RPB2 in the purified complexes, showed comparable levels of transcriptional activities irrespective of whether TBP or TFIID was used as the TATA-binding factor (Fig. 1 B, top andbottom panels, lanes 1 and9). The pG5HMC2AT template contains 5 Gal4-binding sites preceding the HIV-1 TATA box and the adenovirus major late (ML) initiator element in front of a G-less cassette of approximately 380 nucleotides, whereas pMLΔ53, which lacks the activator-binding sites, has a shorter G-less cassette (∼280 nucleotides) driven only by the major late promoter TATA and initiator elements. In our transcription system, TFIIB, TFIIF, and a TATA binding activity (either TBP or TFIID) were essential for transcription by core-pol II (Fig. 1 B, lanes 3, 4, and6), whereas TFIIE and TFIIH, although not necessary for transcription from supercoiled DNA templates (44Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 45Parvin J.D. Shykind B.M. Meyers R.E. Kim J. Sharp P.A. J. Biol. Chem. 1994; 269: 18414-18421Abstract Full Text PDF PubMed Google Scholar, 46Timmers H.T.M. EMBO J. 1994; 13: 391-399Crossref PubMed Scopus (90) Google Scholar) (Fig.1 B, lanes 5 and 7), are required for transcription from linearized DNA molecules.2Interestingly, transcription from different promoter elements seem to require differential amounts of TFIIE and TFIIH, as leaving out TFIIE and TFIIH affected transcription from pG5HMC2AT more dramatically than from pMLΔ53 (Fig. 1 B, lanes5 and 7). In contrast, transcription by f:pol II required only a TATA-binding factor (Fig. 1 B, lanes 9–16), although leaving out TFIIE significantly reduced basal transcription from both DNA templates. The transcription data not only functionally confirm the identities of GTFs detected in our purified f:pol II (Fig. 1, A and B) but further suggest that a two-component pathway comprised of preassembled f:pol II and a TATA-binding factor is probably sufficient for the assembly of a functional preinitiation complex (see below). Obviously, TFIIA and TAFs were not needed for basal transcription from either DNA template (Fig.1 B, compare top and bottom panels, lanes 1 versus 2 and lanes 9 versus 10). We also examined the factor requirement for activator-dependent transcription in our highly purifiedin vitro transcription system. All GTFs, except TFIIA, were necessary, in conjunction with a transcriptional activator (Gal4-VP16) and a coactivator (PC4), for activated transcription by core-pol II (Fig. 1 C, lanes 1–10). In contrast, f:pol II only requires TFIID, PC4, and Gal4-VP16 for activated transcription from pG5HMC2AT (Fig. 1 C, lanes 11–20). Again, leaving out TFIIE and TFIIH showed some effect on activated transcription by f:pol II, reflecting their substoichiometric amounts in purified f:pol II (Fig. 1 A). Apparently, TFIIA was not required for activated transcription by either core-pol II or f:pol II in this highly purified in vitro transcription system (Fig. 1 C, compare lanes 1 and2 with lanes 11 and 12). Presumably TFIIA is only needed to antagonize the repressive effect from some negative factors that may be present in cruder systems (2Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (838) Google Scholar). Indeed, we have found that TFIIA becomes essential whenever a partially purified E/F/H or USA fraction is used in the transcription assay. 4S.-Y. Wu, E. Kershnar, and C.-M. Chiang, manuscript in preparation. In addition, the requirement for TFIIA can be affected by the amount of TFIID and the promoter structure of the DNA templates used in the reaction (47Lieberman P.M. Ozer J. Gürsel D.B. Mol. Cell. Biol. 1997; 17: 6624-6632Crossref PubMed Scopus (45) Google Scholar). Nevertheless, this analysis indicates that, in addition to f:pol II and TFIID, a transcriptional activator (Gal4-VP16) and a coactivator such as PC4 were minimally required for activated transcription in vitro. To investigate the role of TBP and TAFs in activator-dependent transcription, we compared in parallel the transcriptional activities of TBP and TFIID in a transcription system comprised of f:pol II, TBP or TFIID, PC4, and Gal4-VP16. In this assay, TFIID was the only source of TAFs, which were not found in f:pol II as judged by Western blotting (Fig. 1 A) and transcriptional assays (Fig. 1 B, lanes 12 inupper and lower panels, and Fig. 1 C,lane 14). As expected, no transcription could be detected in the absence of a TATA binding activity provided by either TBP or TFIID (Fig. 2 A, lanes 1and 6). When TBP or TFIID, containing an equivalent amount of TBP, was also included, basal transcription could be detected from both pG5HMC2AT and pMLΔ53 DNA templates (Fig.2 A, lanes 2 and 7). If Gal4-VP16 was added to the system without PC4, only minor if any enhancement of transcription was observed (Fig. 2 A, compare lanes 2 and 3, and 7 and 8), confirming the importance of additional cofactors other than TAFs in mediating activator function (17Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Abstract Full Text PDF PubMed Scopus (225) Google Scholar, 36Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). Surprisingly, the coactivator PC4 in the absence of an activator acts as a repressor to suppress basal transcription mediated by TBP (Fig. 2 A, compare lanes 2 and 4). PC4 repression was not obvious in the case of TFIID (Fig. 2 A, compare lanes 7 and9), indicating that TAFs can overcome PC4 repression. Surprisingly, when Gal4-VP16 was also provided, we observed transcriptional activation mediated by both TBP and TFIID (Fig.2 A, compare lanes 2 and 5, and7 and 10). These data suggest that human TBP, in the absence of TFIID TAFs, can also mediate transcriptional activation in a mammalian cell-free transcription system, as previously shown in yeast (5Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (529) Google Scholar). The presence of TAFs, however, help overcome PC4 repression and further enhance the level of activation mediated by TBP (Fig.2 A, compare lanes 7 and 9, and7 and 10). To see if TBP- and TFIID-mediated activation was unique to the acidic type of activation domains as exemplified by Gal4-VP16, we also tested the ability of other Gal4 fusion proteins in activating transcription in our minimal transcription system. Both Gal4-Pro and Gal4-Gln, which contain proline-rich and glutamine-rich activation domains linked, respectively, to the Gal4 DNA-binding domain, were able to activate transcription mediated by TBP, mainly at the level of antirepression (Fig. 2 B, lanes 1–4). In contrast, Gal4-(1–94), which was the portion used

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