Promoter Activation via a Cyclic AMP Response Element in Vitro
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.32301
ISSN1083-351X
AutoresBranden S. Wolner, Jay D. Gralla,
Tópico(s)RNA and protein synthesis mechanisms
ResumoTranscription activation via activating transcription factor cyclic AMP response element binding (ATF/CREB) sites in vitro was explored using transcription and permanganate assay for open complex formation. These sites were used to drive transcription from an adenovirus major late core sequence. Under conditions where activation is strong, 20–50-fold, ATF/CREB is required for preinitiation complexes to reach the open complex stage. Complete opening requires activator, ATP, and initiating nucleotides. In exploration of postinitiation steps, no stimulation of promoter clearance was observed but a modest stimulation of the rate of continuous transcription occurred. High amounts of DNA template, commonly used in in vitro studies, allows some templates to open without activator, but leaves the nucleotide requirements intact. This leads to a drastic lowering of the dependence on ATF/CREB. Taken together, the data indicate that ATF/CREB activates this system primarily by stimulating the formation of functional preinitiation complexes. Transcription activation via activating transcription factor cyclic AMP response element binding (ATF/CREB) sites in vitro was explored using transcription and permanganate assay for open complex formation. These sites were used to drive transcription from an adenovirus major late core sequence. Under conditions where activation is strong, 20–50-fold, ATF/CREB is required for preinitiation complexes to reach the open complex stage. Complete opening requires activator, ATP, and initiating nucleotides. In exploration of postinitiation steps, no stimulation of promoter clearance was observed but a modest stimulation of the rate of continuous transcription occurred. High amounts of DNA template, commonly used in in vitro studies, allows some templates to open without activator, but leaves the nucleotide requirements intact. This leads to a drastic lowering of the dependence on ATF/CREB. Taken together, the data indicate that ATF/CREB activates this system primarily by stimulating the formation of functional preinitiation complexes. RNA polymerase II associates with promoters via multiprotein complexes and is subsequently released from these complexes to carry out transcript elongation. The multistep pathway involves both general transcription factors (GTFs) 1The abbreviations used are: GTF, general transcription factor; ATF, activating transcription factor; CREB, cyclic AMP response element binding; CRE, cyclic AMP response element; TF, transcription factor; PAGE, polyacrylamide gel electrophoresis. and promoter-specific activator proteins. The roles of many of the GTFs have been studied (reviewed in Refs. 1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (848) Google Scholar and 2Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar). These function primarily either as assembly factors for polymerase or to modify the properties of the polymerase after it has assembled at the promoter (3Zawel L. Reinberg D. Prog. Nucleic Acid Res. Mol. Biol. 1993; 44: 67-108Crossref PubMed Scopus (284) Google Scholar, 4Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 5Holstege F.C. van der Vliet P.C. Timmers H.T. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar, 6Krumm A. Hickey L.B. Groudine M. Genes Dev. 1995; 9: 559-572Crossref PubMed Scopus (192) Google Scholar, 7Tang H. Sun X. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (94) Google Scholar). Activators intervene to promote and facilitate various steps. Many studies suggest that this can occur by diverse mechanisms, including recruiting polymerase and GTFs to the promoter (reviewed in Refs.8Pugh B.F. Curr. Opin. Cell Biol. 1996; 8: 303-311Crossref PubMed Scopus (84) Google Scholar and 9Stargell L.A. Struhl K. Trends Genet. 1996; 12: 311-315Abstract Full Text PDF PubMed Scopus (102) Google Scholar) in a way that triggers open complex formation (10Jiang Y. Triezenberg S.J. Gralla J.D. J. Biol. Chem. 1994; 269: 5505-5508Abstract Full Text PDF PubMed Google Scholar), releasing polymerase from pause sites (6Krumm A. Hickey L.B. Groudine M. Genes Dev. 1995; 9: 559-572Crossref PubMed Scopus (192) Google Scholar, 11Blau J. Xiao H. McCracken S. O'Hare P. Greenblatt J. Bentley D. Mol. Cell. Biol. 1996; 16: 2044-2055Crossref PubMed Scopus (235) Google Scholar), or stimulating elongation rates (12Yankulov K. Blau J. Purton T. Roberts S. Bentley D.L. Cell. 1994; 77: 749-759Abstract Full Text PDF PubMed Scopus (208) Google Scholar). There is strong evidence supporting the recruitment model (see Refs. 8Pugh B.F. Curr. Opin. Cell Biol. 1996; 8: 303-311Crossref PubMed Scopus (84) Google Scholarand 9Stargell L.A. Struhl K. Trends Genet. 1996; 12: 311-315Abstract Full Text PDF PubMed Scopus (102) Google Scholar). There is less, but accumulating, evidence for postinitiation mechanisms. Postinitiation can be further subdivided into the steps of promoter clearance, elongation, and re-initiation. Several activators, including heat shock factor, VP16, E1a, and E2F (6Krumm A. Hickey L.B. Groudine M. Genes Dev. 1995; 9: 559-572Crossref PubMed Scopus (192) Google Scholar, 11Blau J. Xiao H. McCracken S. O'Hare P. Greenblatt J. Bentley D. Mol. Cell. Biol. 1996; 16: 2044-2055Crossref PubMed Scopus (235) Google Scholar, 12Yankulov K. Blau J. Purton T. Roberts S. Bentley D.L. Cell. 1994; 77: 749-759Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 13Shopland L.S. Lis J.T. Chromosoma (Berl.). 1996; 105: 158-171Crossref PubMed Scopus (39) Google Scholar), have been proposed to work at one or more of these steps. For other activators there is uncertainty concerning which steps are affected. One very important class of activators fits into this uncertain category, the activating transcription factor cyclic AMP response element binding (ATF/CREB) protein family. These proteins work via attachment to promoters containing upstream cAMP response elements (CREs) and mediate cAMP-dependent transcription responses (14Karin M. Smeal T. Trends Biochem. Sci. 1992; 17: 418-422Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 15Alberts A.S. Arias J. Hagiwara M. Montminy M.R. Feramisco J.R. J. Biol. Chem. 1994; 269: 7623-7630Abstract Full Text PDF PubMed Google Scholar, 16Rehfuss R.P. Walton K.M. Loriaux M.M. Goodman R.H. J. Biol. Chem. 1991; 266: 18431-18434Abstract Full Text PDF PubMed Google Scholar). There are many members of the protein family and their diversity is increased further by their ability to form heterodimers and to be phosphorylated (14Karin M. Smeal T. Trends Biochem. Sci. 1992; 17: 418-422Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 15Alberts A.S. Arias J. Hagiwara M. Montminy M.R. Feramisco J.R. J. Biol. Chem. 1994; 269: 7623-7630Abstract Full Text PDF PubMed Google Scholar, 17Kobayashi M. Shimomura A. Hagiwara M. Kawakami K. Nucleic Acids Res. 1997; 25: 877-882Crossref PubMed Scopus (20) Google Scholar). Members of the ATF/CREB family of activators have been suggested to work at both pre- and postinitiation steps (18Hai T.W. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 19Horikoshi M. Hai T. Lin Y.S. Green M.R. Roeder R.G. Cell. 1988; 54: 1033-1042Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar, 21Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (36) Google Scholar). Several members of the family have been shown to bind TFIID (19Horikoshi M. Hai T. Lin Y.S. Green M.R. Roeder R.G. Cell. 1988; 54: 1033-1042Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 22Ferreri K. Gill G. Montminy M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1210-1213Crossref PubMed Scopus (164) Google Scholar, 23Quinn P.G. J. Biol. Chem. 1993; 268: 16999-17009Abstract Full Text PDF PubMed Google Scholar, 24Xing L. Gopal V.K. Quinn P.G. J. Biol. Chem. 1995; 270: 17488-17493Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and in some cases TFIIB (24Xing L. Gopal V.K. Quinn P.G. J. Biol. Chem. 1995; 270: 17488-17493Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These interactions can occur directly or indirectly via coactivators (25Arias J. Alberts A.S. Brindle P. Claret F.X. Smeal T. Karin M. Feramisco J. Montminy M. Nature. 1994; 370: 226-229Crossref PubMed Scopus (681) Google Scholar, 26Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1282) Google Scholar). The contact between activator and TFIID apparently occurs whether or not the protein is phosphorylated (18Hai T.W. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 19Horikoshi M. Hai T. Lin Y.S. Green M.R. Roeder R.G. Cell. 1988; 54: 1033-1042Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 23Quinn P.G. J. Biol. Chem. 1993; 268: 16999-17009Abstract Full Text PDF PubMed Google Scholar, 24Xing L. Gopal V.K. Quinn P.G. J. Biol. Chem. 1995; 270: 17488-17493Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 27Xing L. Quinn P.G. J. Biol. Chem. 1994; 269: 28732-28736Abstract Full Text PDF PubMed Google Scholar). On this basis the ATF/CREB members have been suggested to be involved in recruitment of the GTFs and the polymerase. However, even individual proteins may contain multiple activation domains, which may function differently (23Quinn P.G. J. Biol. Chem. 1993; 268: 16999-17009Abstract Full Text PDF PubMed Google Scholar, 27Xing L. Quinn P.G. J. Biol. Chem. 1994; 269: 28732-28736Abstract Full Text PDF PubMed Google Scholar). Other studies, conducted in HeLa extracts containing a wide range of factors, have suggested that activation in such extracts occurs instead at the step of promoter clearance (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar, 21Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (36) Google Scholar). In these studies ATF/CREB was not required to form preinitiation complexes in which the DNA start site was melted. Instead the activator was proposed to allow the polymerase, prebound in an open complex, to clear the promoter in a way that led to the observed 3-fold activation of transcription. The protein isoform that bound the CRE and accomplished the activation in nuclear extract was reported to be unphosphorylated CREB-1 (28Narayan S. He F. Wilson S.H. J. Biol. Chem. 1996; 271: 18508-18513Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Recently, we found that the effect of a different activator, Sp1, on recruitment can to some extent be bypassed if high amounts (500–1000 ng) of template DNA are used (29Yean D. Gralla J. Nucleic Acids Res. 1996; 24: 2723-2729Crossref PubMed Scopus (7) Google Scholar). It is possible that some of the uncertainty concerning the step at which ATF/CREB functioned was due to variations in experimental conditions. An additional uncertainty arises from recent work with core promoter sequences analogous to those used in ATF/CREB studies. These suggested that promoter opening is more complex than had been appreciated previously in that initiating nucleotides are involved in addition to ATP (5Holstege F.C. van der Vliet P.C. Timmers H.T. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar). Because of these new uncertainties and the importance of activation via CRE sites, we re-evaluated the activation mechanisms. In contrast to earlier studies, we see little or no effect of ATF/CREB on the rate of promoter clearance. At high concentrations of DNA a strong effect of ATF/CREB on preinitiation complex formation is bypassed. When low amounts of DNA are used the stimulation by ATF/CREB is very great. Its main effect under these conditions is to nucleate formation of a preinitiation complex in which the DNA can be opened. Complete opening requires activator, ATP, and initiating nucleotides. In addition to this primary recruitment effect, there is a small secondary effect in which ATF/CREB stimulates the rate of transcription re-initiation events. Fig.1 summarizes the constructs used. The pBW1 promoter construct is identical to the pSH15 promoter (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar). To create this promoter, upstream from sequences used previously to facilitate transcript detection, two partially complementary oligonucleotides were synthesized. B1 has the sequence CCGGAATTCGTGACGTCACAACAGGCTATAAAAGGGGGTGGGGGCATGCCTCGTCCTC. B2 has the sequence CGCGGATCCCCCAGCTCCGGCGCGGCCGGGAAGAGAGTGAGGACGAGGCATGCCCCCA. The underlines represent the complementary sequences. Equimolar amounts were hybridized in 2 × Klenow buffer (2 × is: 20 mm Tris·Cl, pH 7.5, 10 mm MgCl2, 100 mm NaCl) in a total volume of 30 μl by heating to 95 °C and slowly cooling to room temperature. The overhanging ends were filled in with the Klenow fragment of DNA polymerase by mixing the 30-μl annealing reaction with 7.5 mm dithiothreitol, 33 μm each deoxyribonucleoside triphosphate (dNTP), and 3–5 units of Klenow enzyme (Life Technologies, Inc.) in a total volume of 60 μl and incubating for 1 h at room temperature. DNA was purified away from unincorporated nucleotides and buffer over a Qiaquick polymerase chain reaction column (Qiagen). The Klenow extension creates an EcoRI restriction site at the upstream end of the double-stranded oligonucleotide and a BamHI site at the downstream end (see Fig. 1). The product was cleaved with EcoRI and BamHI and was ligated into the EcoRI and BamHI sites of plasmid pSP72 (Promega). This construct was transformed into DH5α-competent cells (Life Technologies, Inc.), and EagI-sensitive clones were identified. Candidate clones were verified by direct sequencing. Promoter construct pBW2 is identical to promoter pAH1 (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar) and was created in the same manner as pBW1 except that oligonucleotide B3 (CCGGAATTCCGGCTATAAAAGGGGGTGGGGGCATGCCTCGTCCTC) replaced B1. Double-stranded competitor DNA was made by annealing two perfectly complementary 24-nucleotide CRE sequences (top strand: GGACGCGTGACGTCACAACACAGC, consensus CRE underlined). All primer extensions used the Inr primer (CCTTATGTATCATACACATACGATTTAGG), which hybridizes to the pSP72 vector up to position +93 relative to the transcription start site (indicated by an arrow in Fig. 1) except bottom strand probing in KMnO4 assays, which used primer BPM1 (CGCGTGACGTCACAACAG), which hybridizes to the region from −52 to −35 in promoter pBW1. Oligonucleotides were synthesized on a Gene Assembler Plus (Pharmacia Biotech Inc.) and purified on 20% urea-PAGE (19:1 acrylamide:bisacrylamide; 8 m urea). Plasmids were purified using Qiagen Maxiprep kits. This assay has been described (29Yean D. Gralla J. Nucleic Acids Res. 1996; 24: 2723-2729Crossref PubMed Scopus (7) Google Scholar,30Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9159) Google Scholar). HeLa nuclear extract was prepared as previously (29Yean D. Gralla J. Nucleic Acids Res. 1996; 24: 2723-2729Crossref PubMed Scopus (7) Google Scholar, 30Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9159) Google Scholar, 31Jiang Y. Gralla J.D. Mol. Cell. Biol. 1993; 13: 4572-4577Crossref PubMed Scopus (36) Google Scholar). The indicated amounts of supercoiled whole plasmid template DNA were mixed with 20 μl of HeLa nuclear extract (6 mg of protein/ml), 8 mm MgCl2, 500 ng of pGEM as carrier DNA and 500 mm each nucleoside triphosphate (NTP) in a total volume of 40 μl. Samples were then incubated at 30 °C for 30 min. 100 μl of stop buffer (10 mm EDTA, 0.3 m sodium acetate, pH 5.5, 0.2% SDS, 50 μg/ml yeast tRNA, 20 μg of proteinase K) was added, followed by incubation at room temperature for 30 min. RNA was isolated and copied using reverse transcriptase (Promega) extension of 5′-32P-labeled Inr primer (29Yean D. Gralla J. Nucleic Acids Res. 1996; 24: 2723-2729Crossref PubMed Scopus (7) Google Scholar). These labeled cDNA products were separated by 6% urea-PAGE (19:1 acrylamide:bisacrylamide, 8 m urea) and visualized and quantified using a PhosphorImager (Molecular Dynamics). For promoter clearance and re-initiation assays the above protocol was modified as follows: NTPs were omitted in the first step and preinitiation complexes were formed on the DNA template for 1 h at 30 °C. NTPs were then added to a final concentration of 500 mm each. Reactions were incubated at 30 °C for various times and the RNA quantified as described. In all transcription assays each data point was performed in duplicate except in the competitor titration assay. This assay has been described (31Jiang Y. Gralla J.D. Mol. Cell. Biol. 1993; 13: 4572-4577Crossref PubMed Scopus (36) Google Scholar, 32Jiang Y. Gralla J.D. J. Biol. Chem. 1995; 270: 1277-1281Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 33Jiang Y. Yan M. Gralla J.D. Mol. Cell. Biol. 1996; 16: 1614-1621Crossref PubMed Google Scholar, 34Wang W. Carey M. Gralla J.D. Science. 1992; 255: 450-453Crossref PubMed Scopus (164) Google Scholar). Briefly, preinitiation complexes were assembled as follows: the indicated amount of supercoiled template DNA was incubated with 20 μl of HeLa nuclear extract (6 mg of protein/ml), 8 mmMgCl2, and 200 ng of pBR322 (Promega) for 30 min at 30 °C. When present, deoxyadenosine triphosphate (dATP) was added to a final concentration of 500 μm, and NTPs were added to final concentrations of 100 μm each. Samples were further incubated at 30 °C for 2 min, after which potassium permanganate (KMnO4) was added to a final concentration of 6 mm. Two minutes later the KMnO4 was quenched with 3 μl of β-mercaptoethanol. DNA was purified by extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by extraction with chloroform:isoamyl alcohol (24:1) and ethanol precipitation. Pellets were dissolved in water and passed over 1-ml Sephadex G-50 (Sigma) columns. Eluate volumes were equalized and permanganate attack patterns analyzed by extension of the Inr or BPM1 primers in a polymerase chain reaction thermocycler (MJ Research). In the case where 10 ng of template DNA was used, the entire sample was analyzed. When 1 μg of DNA was used, only a 20-ng sample was analyzed. After polymerase chain reaction, samples were recovered and run on 6% urea-PAGE at 36 watts for 1.5 h. Gels were dried and exposed to PhosphorImager screens overnight. This assay was performed as described using dinucleotide primer and radioactive CTP (35Jiang Y. Yan M. Gralla J.D. J. Biol. Chem. 1995; 270: 27332-27338Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) except that CpA was the dinucleotide primer and a much higher concentration of DNA (1 μg/40 μl) was used. Labeled trinucleotide products were resolved on a 20% (19:1 acrylamide:bisacrylamide)/8 m urea gel and exposed to PhosphorImager screens (Molecular Dynamics) for 1–5 h. Prior experiments regarding activation via ATF/CREB sites in vitrohave primarily used plasmid DNA amounts in the range of 500–1000 ng (18Hai T.W. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 19Horikoshi M. Hai T. Lin Y.S. Green M.R. Roeder R.G. Cell. 1988; 54: 1033-1042Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar, 21Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (36) Google Scholar). In some cases the apparent mechanism of activation is known to depend on the amount of DNA with these amounts representing the highest used (29 and see below). We begin by exploring how these sites activate transcription at high concentrations of DNA. Fig. 2 a, lanes 1 and 8, compare in vitro transcription with and without a single consensus CRE site. Removal of the site (lane 8) leads to a 3-fold loss of transcription, consistent with prior studies done with the same promoter under the same conditions (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar, 21Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (36) Google Scholar). To confirm that this effect is mediated by binding to these sites, and to facilitate clearing ATF/CREB from the extract for experiments described below, a competition experiment was done. A double-stranded oligonucleotide encompassing a consensus CRE (see “Materials and Methods”) was added to in vitro transcription assays prior to addition of template DNA. Its purpose is to specifically sequester CRE-binding proteins (36Englander E.W. Widen S.G. Wilson S.H. Nucleic Acids Res. 1991; 19: 3369-3375Crossref PubMed Scopus (20) Google Scholar, 37Widen S.G. Wilson S.H. Biochemistry. 1991; 30: 6296-6305Crossref PubMed Scopus (33) Google Scholar). As more of this competitor is added the signal gradually decreases (lanes 3–7). At the highest concentration of competitor shown, the amount of transcription has been reduced to basal levels, that is, an amount equal to that formed from a template lacking a CRE site (compare lanes 7 and 8). The competitor has no effect on transcription from the basal template (data not shown). These data confirm that CRE binding proteins are responsible for the activation. The competitor titration experiment was repeated several times with varying amounts of template DNA. In all cases complete inhibition of activation occurred with approximately 250 ng of competitor oligonucleotide, as shown in Fig. 2 b. Thus we conclude that this amount of competitor is sufficient to sequester the endogenous ATF/CREB proteins in the nuclear extract. We use this amount in later studies of unactivated, basal conditions. The source of the 3-fold activation by ATF/CREB has not been settled and was originally suggested to occur after the opening of the DNA (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar). That is, it was reported that ATF/CREB is not required to create a permanganate-sensitive open complex at these promoters. There is also some confusion as to the nature of the opening reaction itself. Opening in general was reported to require ATP or dATP (31Jiang Y. Gralla J.D. Mol. Cell. Biol. 1993; 13: 4572-4577Crossref PubMed Scopus (36) Google Scholar, 32Jiang Y. Gralla J.D. J. Biol. Chem. 1995; 270: 1277-1281Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 34Wang W. Carey M. Gralla J.D. Science. 1992; 255: 450-453Crossref PubMed Scopus (164) Google Scholar). Subsequently, Holstege et al. (5Holstege F.C. van der Vliet P.C. Timmers H.T. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar) showed that at the adenovirus major late promoter, the sequence of which is largely preserved in the constructs used here (see Fig. 1), initiating nucleotides were needed in addition to ATP to obtain a substantial permanganate signal. Therefore we investigated the formation of open complexes at these promoters. We looked first at opening of the upper (nontemplate) strand of the DNA. The data show strong permanganate signals downstream of the start site, and weaker signals at upstream sites, when the initiating nucleotides ATP and CTP are added (Fig. 3 a, lane 3 versusthe lane 1 control). Addition of only dATP (lane 2) or only ATP (not shown) does not yield a significant permanganate signal. These nucleotide requirements are consistent with prior reports using the adenovirus major late promoter (5Holstege F.C. van der Vliet P.C. Timmers H.T. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar), the core sequence of which is retained in the promoter studied here. The open complexes formed in this manner appear to be functional in that the addition of nucleotides that allow elongation causes the disappearance of the signal (compare lanes 2 and 3 of Fig. 3 b). This is expected based on studies of open complexes at several promoters in which the melted bubble has been shown to be chased to downstream positions (31Jiang Y. Gralla J.D. Mol. Cell. Biol. 1993; 13: 4572-4577Crossref PubMed Scopus (36) Google Scholar, 33Jiang Y. Yan M. Gralla J.D. Mol. Cell. Biol. 1996; 16: 1614-1621Crossref PubMed Google Scholar). The formation of functional open complexes does not require ATF/CREB under these conditions. When an excess of competitor oligonucleotide is added to HeLa extract prior to the template, the permanganate signal still appears (compare Fig. 3 a, lane 6, to the negative control in lane 4). However, the permanganate signal (denoted by a bar) is weaker relative to background reactivity (region below the bar) when basal (Fig. 3 a, lane 6) and activated (Fig. 3 a, lane 3) conditions are compared. A similar result is seen when basal and activated templates are compared directly (Fig. 3 b); the permanganate signal persists on the basal template, but is somewhat weaker (compare lane 5 with lane 2). As expected from Fig.3 a the signal depends on addition of initiating nucleotides (Fig. 3 b, lane 5) and disappears when a full complement of elongation substrates is present (lane 6). We also explored the opening of the other DNA strand, since that strand had been reported previously to open in response to dATP alone (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar). In this experiment, dATP alone did not cause detectable opening (not shown, but see Fig. 3 a and below), but initiating nucleotides did (Fig.3 c). Overall, these data are in significant, but not full, agreement with prior studies. The promoter opening behavior is consistent with that observed at the analogous adenovirus ML promoter (5Holstege F.C. van der Vliet P.C. Timmers H.T. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar), but not the same as reported elsewhere (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar). As reported (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar), promoter opening did not require ATF/CREB. The stimulation of transcription under these conditions was 3-fold (Fig. 2), also as reported in prior studies (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar,21Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (36) Google Scholar). However, the permanganate results on the top strand showed some strengthening of the open complex signal by the activator. It appears that at least some of the 3-fold effect is due to the stimulation of open complex formation. In a prior study the 3-fold stimulation was attributed not to formation of open complexes but to postinitiation stimulation of promoter clearance (20Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar). To clarify this issue we re-evaluated the rate of promoter clearance. Preinitiation complexes were formed by a 1-h incubation of template with transcription extract in the absence of nucleoside triphosphates. This drives the DNA into closed transcription complexes that lack the nucleotides needed to complete the opening and elongation reactions. All four NTPs are then added to begin these reactions synchronously. At various subsequent times the appearance of the 93-nucleotide-long RNA is quantified. In the very short time course of this experiment each template that forms an open complex can only produce a single RNA (31Jiang Y. Gralla J.D. Mol. Cell. Biol. 1993; 13: 4572-4577Crossref PubMed Scopus (36) Google Scholar). Thus, this is a one-round transcription assay, and the amount of RNA is a direct measure of the number of preinitiation complexes that complete opening and clearance at the indicated times (31Jiang Y. Gralla J.D. Mol. Cell. Biol. 1993; 13: 4572-4577Crossref PubMed Scopus (36) Google Scholar). Fig. 4 shows the time required for the preinitiation complexes to complete these steps. The activated (pBW1) and basal (pBW2) templates are compared to assess the effect of activator. In both cases the addition of NTPs leads to an initial burst of RNA synthesis as the preinitiation complexes synchronously initiate and elongate the short RNA transcript (as seen in prior studies of other promoters (38Yean D. Gralla J. Mol. Cell. Biol. 1997; 17: 3809-3816Crossref PubMed Scopus (68) Google Scholar, 39White J. Brou C. Wu J. Lutz Y. Moncollin V. Chambon P. EMBO J. 1992; 11: 2229-2240Crossref PubMed Scopus (95) Google Scholar)). The activated templates appear to have assembled approximately 2.5 times the number of active preinitiation complexes, as indicated by the 2.5-fold greater amount of RNA produced. However, the results show no effect of ATF/CREB on the rate of promoter clearance. On both templates the reactions are mostly complete within the first minute. The approximate half-time (t½) for th
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