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Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês

10.1074/jbc.m311433200

ISSN

1083-351X

Autores

Matthew B. Renfrow, Nikolai A. Naryshkin, L. Michelle Lewis, Hung‐Ta Chen, Richard H. Ebright, Robert A. Scott,

Tópico(s)

Bacteriophages and microbial interactions

Resumo

Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions –40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection. Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions –40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection. Extensive structural and biochemical characterization of transcription initiation complexes has revealed similarity of transcription systems across the three domains of life (1.Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar, 2.Lee T.I. Young R.A. Annu. Rev. Genet. 2000; 34: 77-137Crossref PubMed Scopus (632) Google Scholar, 3.Ebright R.H. J. Mol. Biol. 2000; 304: 687-698Crossref PubMed Scopus (194) Google Scholar, 4.Woychik N.A. Hampsey M. Cell. 2002; 108: 453-463Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Transcription initiation in archaea closely resembles eukaryal class II transcription initiation and is mediated by a single RNA polymerase (RNAP) 1The abbreviations used are: RNAP, RNA polymerase; TBP, TATA-element-binding protein; TFB, transcription factor B; TFBc, C-terminal domain of TFB; TFBn, N-terminal domain of TFB; CSB, conserved sequence block. and two general transcription factors, TATA-element binding protein (TBP) and transcription factor B (TFB) (5.Soppa J. Mol. Microbiol. 1999; 31: 1295-1305Crossref PubMed Scopus (113) Google Scholar, 6.Bell S.D. Magill C.P. Jackson S.P. Biochem. Soc. Trans. 2001; 29: 392-395Crossref PubMed Scopus (56) Google Scholar). In vitro transcription initiation has been demonstrated with RNAP, TBP, and TFB in Pyrococcus (7.Hausner W. Wettach J. Hethke C. Thomm M. J. Biol. Chem. 1996; 271: 30144-30148Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8.Hethke C. Geerling A.C. Hausner W. de Vos W.M. Thomm M. 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Recent crystallographic structures of bacterial RNAP and eukaryal RNAP II reveal striking structural homology (3.Ebright R.H. J. Mol. Biol. 2000; 304: 687-698Crossref PubMed Scopus (194) Google Scholar, 14.Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 15.Cramer P. Bushnell D.A. Fu J. Gnatt A.L. Maier-Davis B. Thompson N.E. Burgess R.R. Edwards A.M. David P.R. Kornberg R.D. Science. 2000; 288: 640-649Crossref PubMed Scopus (472) Google Scholar, 16.Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (974) Google Scholar, 17.Armache K.J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (195) Google Scholar, 18.Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (449) Google Scholar, 19.Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. 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Nature. 1993; 365: 512-520Crossref PubMed Scopus (1016) Google Scholar, 30.Rowlands T. Baumann P. Jackson S.P. Science. 1994; 264: 1326-1329Crossref PubMed Scopus (147) Google Scholar). Archaeal TFB belongs to the TFIIB family, whose members bind promoter DNA, bind RNAP, and serve as bridges between the TBP-TATA-element complex and RNAP (26.Nikolov D.B. Chen H. Halay E.D. Usheva A.A. Hisatake K. Lee D.K. Roeder R.G. Burley S.K. Nature. 1995; 377: 119-128Crossref PubMed Scopus (485) Google Scholar, 27.Kosa P.F. Ghosh G. DeDecker B.S. Sigler P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6042-6047Crossref PubMed Scopus (145) Google Scholar, 28.Littlefield O. Korkhin Y. Sigler P.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13668-13673Crossref PubMed Scopus (135) Google Scholar, 29.Tsai F.T. Sigler P.B. EMBO J. 2000; 19: 25-36Crossref PubMed Scopus (142) Google Scholar). Archaeal TFB has the characteristic domain organization of other TFIIB family members (31.Buratowski S. Zhou H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5633-5637Crossref PubMed Scopus (96) Google Scholar, 32.Barberis A. Muller C.W. Harrison S.C. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5628-5632Crossref PubMed Scopus (74) Google Scholar), comprising a C-terminal domain (TFBc) that mediates interactions with the TBP-TATA-element complex (7.Hausner W. Wettach J. Hethke C. Thomm M. J. Biol. Chem. 1996; 271: 30144-30148Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 27.Kosa P.F. Ghosh G. DeDecker B.S. Sigler P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6042-6047Crossref PubMed Scopus (145) Google Scholar, 28.Littlefield O. Korkhin Y. Sigler P.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13668-13673Crossref PubMed Scopus (135) Google Scholar) and an N-terminal domain (TFBn) that mediates interactions with RNAP (33.Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 12934-12940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 34.Magill C.P. Jackson S.P. Bell S.D. J. Biol. Chem. 2001; 276: 46693-46696Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The TFB N-terminal domain consists of a conserved metal binding region (“zinc ribbon”; Refs. 35.Zhu W. Zeng Q. Colangelo C.M. Lewis M. Summers M.F. Scott R.A. Nat. Struct. Biol. 1996; 3: 122-124Crossref PubMed Scopus (124) Google Scholar and 36.Chen H.T. Legault P. Glushka J. Omichinski J.G. Scott R.A. Protein Sci. 2000; 9: 1743-1752Crossref PubMed Scopus (58) Google Scholar) followed by a short, highly conserved region (“conserved sequence block” (CSB); Ref. 33.Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 12934-12940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Substitutions in the conserved sequence block in archaeal TFB indicate a role in transcription-initiation NTP concentration dependence (33.Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 12934-12940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and substitutions in eukaryal TFIIB indicate roles in transcription-initiation efficiency and start-site selection (37.Pinto I. Wu W.H. Na J.G. Hampsey M. J. Biol. Chem. 1994; 269: 30569-30573Abstract Full Text PDF PubMed Google Scholar, 38.Bangur C.S. Pardee T.S. Ponticelli A.S. Mol. Cell. Biol. 1997; 17: 6784-6793Crossref PubMed Scopus (44) Google Scholar, 39.Pardee T.S. Bangur C.S. Ponticelli A.S. J. Biol. Chem. 1998; 273: 17859-17864Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 40.Cho E.J. Buratowski S. J. Biol. Chem. 1999; 274: 25807-25813Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 41.Hawkes N.A. Roberts S.G. J. Biol. Chem. 1999; 274: 14337-14343Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 42.Zhang D.Y. Carson D.J. Ma J. Nucleic Acids Res. 2002; 30: 3078-3085Crossref PubMed Scopus (23) Google Scholar). Although the roles of the TFB and TFIIB N-terminal domains in transcription initiation and start-site selection have been well characterized, the structural and mechanistic basis of these roles has remained undefined. In published work, systematic site-specific protein-DNA photocrosslinking has been used to characterize the structural organization of eukaryal transcription initiation complexes (43.Lagrange T. Kim T.K. Orphanides G. Ebright Y.W. Ebright R.H. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10620-10625Crossref PubMed Scopus (92) Google Scholar, 44.Kim T.K. Lagrange T. Wang Y.H. Griffith J.D. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12268-12273Crossref PubMed Scopus (91) Google Scholar, 45.Kim T.K. Ebright R.H. Reinberg D. Science. 2000; 288: 1418-1422Crossref PubMed Scopus (215) Google Scholar), bacterial transcription initiation complexes (46.Naryshkin N. Revyakin A. Kim Y. Mekler V. Ebright R.H. Cell. 2000; 101: 601-611Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 47.Naryshkin N. Kim Y. Dong Q. Ebright R.H. Methods Mol. Biol. 2001; 148: 337-361PubMed Google Scholar), and the part of an archaeal transcription initiation complex at and downstream of the transcription start site (positions –1 to +20 of the template strand (21.Bartlett M.S. Thomm M. Geiduschek E.P. Nat. Struct. Biol. 2000; 7: 782-785Crossref PubMed Scopus (39) Google Scholar)). Here, we report the use of systematic site-specific protein-DNA photocrosslinking to define the structural organization of the part of an archaeal transcription initiation complex upstream of the transcription start site (positions –40 to +1; nontemplate and template strands), the part that contains binding determinants for RNAP subunits and general transcription factors. Our results confirm the anticipated high degree of homology between the archaeal and eukaryal transcription systems and identify an unanticipated interaction between the TFB N-terminal domain and promoter DNA in the transcription-bubble region, upstream of and at the transcription start site. The results immediately suggest a mechanistic basis for the role of TFB and TFIIB N-terminal domains in transcription initiation and start-site selection. PfRNAP—Under anaerobic conditions, frozen Pyrococcus furiosus cell paste (15 g, prepared as in Bryant and Adams (48.Bryant F.O. Adams M.W. J. Biol. Chem. 1989; 264: 5070-5079Abstract Full Text PDF PubMed Google Scholar)) was resuspended in 82 ml of ice-cold 50 mm Tris (pH 7.5), 22 mm NH4Cl, 10 mm EDTA, and 10% glycerol, and cells were lysed in two passes through a French press operating at 1200 p.s.i. After the addition of Polymin P to 0.4% w/v and stirring for 30 min at room temperature, the sample was centrifuged at 100,000 × g for 4 h at 4 °C, the pellet was resuspended in 50 ml of the same buffer, and the sample again was centrifuged at 100,000 × g for 4 h at 4 °C. The pellet was extracted 3 times with 100 ml of 50 mm Tris (pH 7.5), 1.2 m NH4Cl, 10 mm EDTA, and 10% glycerol, extracts were pooled, ammonium sulfate was added to 70% saturation, the sample was stirred for 1 h on ice, and the sample was again centrifuged at 100,000 × g for 4 h at 4 °C. The resulting pellet was aerobically resuspended in 100 ml of buffer A (50 mm Tris (pH 7.5), 50 mm KCl, 10 mm EDTA, and 20% glycerol) and dialyzed against three changes of 1 liter of the same buffer for 12 h at 4 °C (12–14-kDa molecular weight cut-off dialysis tubing (SpectraPor)). The sample was centrifuged at 20,000 × g for 1 h at 4 °C. The sample was loaded onto a 220-ml DEAE-Sepharose column (Amersham Biosciences) pre-equilibrated in buffer A and eluted with 2 liters of a linear gradient of 100% buffer A to 100% buffer B (50 mm Tris (pH 7.5), 1 m KCl, 10 mm EDTA, and 20% glycerol). Fractions containing DNA-dependent RNA polymerase (RNAP) activity (49.Burgess R.R. Jendrisak J.J. Biochemistry. 1975; 14: 4634-4638Crossref PubMed Scopus (843) Google Scholar, 50.Thomm M. Robb F.T. Archaea: A Lab Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990: 297-301Google Scholar) were pooled, concentrated, and loaded onto a 30-ml heparin-Sepharose column (Amersham Biosciences) equilibrated with 15% buffer B. The column was eluted with a 300-ml linear gradient of 15 to 100% buffer B. Pooled fractions containing RNAP activity were concentrated and dialyzed against buffer A for 12 h at 4 °C. The sample was centrifuged at 20,000 × g for 30 min at 4 °C and loaded onto a Mono Q 10/10 column (Amersham Biosciences). The column was eluted with a 100-ml linear gradient of 10–100% buffer B. Pooled fractions containing polymerase activity were concentrated and stored as aliquots at –80 °C. Yield, ∼4.7 μg. PfTBP—Plasmid PfTBP/pT7–7, which encodes PfTBP under control of the bacteriophage T7 gene 10 promoter, was constructed by replacing the NdeI-BamHI segment of plasmid pT7–7 (51.Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2459) Google Scholar) with a NdeI-BamHI DNA fragment containing the PfTBP-coding sequence (prepared by add-on PCR using P. furiosus genomic DNA as template (a gift of F. Jenney, University of Georgia, Athens, GA)). Transformants of Escherichia coli strain BL-21 DE3 (Novagen) with plasmid PfTBP/pT7–7 were cultured at 37 °C in 4 liters of LB medium (Fisher) to an A600 of ∼0.9, induced by the addition of isopropyl β-d-thiogalactopyranoside to 0.1 mm, cultured an additional 4 h at 37 °C (A600 ∼ 2.0), and harvested by centrifugation at 300 × g for 20 min at 4 °C. Cell pellets (10–12 g) were frozen and stored at –80 °C until used. Cells were thawed by suspension in 60 ml of lysis buffer (50 mm potassium phosphate (pH 7.8), 10 mm 2-mercaptoethanol, 1 mm phenylmethanesulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml DNase I) and were lysed by sonication; the lysate was centrifuged at 30,000 × g for 50 min at 4 °C. After incubation of the supernatant for 30 min at 70 °C, the sample was centrifuged at 30,000 × g for 50 min at 4 °C, the supernatant was loaded onto a 2.5 × 10-cm Q-Sepharose benchtop column (Amersham Biosciences), the column was washed with 500 ml of 50 mm potassium phosphate buffer (pH 7.8), and the column was eluted with a 150-ml linear gradient of 0–600 mm KCl in the same buffer. Pooled fractions containing PfTBP (50 ml; ∼300 mm KCl) were desalted into 50 mm potassium phosphate (pH 7.8) and concentrated to 2 ml by ultrafiltration at 4 °C (200-ml ultrafiltration unit (Amicon) with YM3 membrane (Millipore)). The sample was loaded onto a 1 × 10-cm Mono Q column (Amersham Biosciences), and the column was eluted with a 55-ml linear gradient of 0–600 mm KCl in 50 mm potassium phosphate (pH 7.0). Pooled fractions containing PfTBP (5 ml; ∼400 mm KCl) were exchanged into 50 ml of 50 mm potassium phosphate (pH 7.8), 500 mm KCl, concentrated by ultrafiltration through a YM3 membrane (Millipore) at 4 °C, and stored in aliquots at –70 °C. SDS-PAGE indicated an ∼21-kDa protein, corresponding to the expected molecular mass of PfTBP. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed a single molecular ion series of 21,285 atomic mass units (predicted, 21,311 atomic mass units). The yield was ∼40 mg, and purity was ∼95%. PfTFB—Plasmid pMLtfb (52.Zeng Q. Lewis L.M. Colangelo C.M. Dong J. Scott R.A. J. Biol. Inorg. Chem. 1996; 1: 162-168Crossref Scopus (4) Google Scholar), which encodes PfTFB under control of the bacteriophage T7 gene 10 promoter, was introduced by transformation into E. coli strain BL-21 DE3 (Novagen). Growth, induction, harvesting, lysis, and clarification were performed as described above for PfTBP. The supernatant was loaded onto a 2.5 × 20-cm phosphocellulose column (Whatman), the column was washed extensively with 50 mm potassium phosphate buffer (pH 7.8), and the column was eluted with 150 ml of 50 mm potassium phosphate (pH 7.8) and 500 mm KCl. The sample was concentrated to ∼2 ml by ultrafiltration through a YM3 membrane (Millipore) at 4 °C and loaded onto a 2.6 × 60-cm Superdex 75 column (Amersham Biosciences), and the column was eluted in 5-ml fractions of the same buffer. Pooled fractions containing PfTFB (20 ml) were stored in aliquots of 500 μl at –70 °C. SDS-PAGE analysis indicated a ∼34-kDa protein, corresponding to the expected molecular mass of PfTFB. The yield was ∼25 mg, and the purity was ∼95%. Derivatized Promoter DNA Fragments—M13mp18-gdh and M13mp19-gdh were constructed by replacement of the EcoRI-SphI segments of, respectively, M13mp18 and M13mp19 (New England Biolabs) by EcoRI-SphI DNA fragments containing positions –75 to +45 of the P. furiosus gdh promoter (prepared by add-on PCR using as template plasmid pLU479 (Ref. 8.Hethke C. Geerling A.C. Hausner W. de Vos W.M. Thomm M. Nucleic Acids Res. 1996; 24: 2369-2376Crossref PubMed Scopus (68) Google Scholar; a generous gift from M. Thomm, University of Regensburg, Regensburg, Germany). Promoter DNA fragments containing a phenyl azide photoactivable cross-linking agent incorporated at a single, defined DNA phosphate and containing an adjacent 32P label were prepared using M13mp18-gdh (for analysis of the template strand) and M13mp19-gdh (for analysis of the nontemplate strand) as templates by the method of Naryshkin et al. (47.Naryshkin N. Kim Y. Dong Q. Ebright R.H. Methods Mol. Biol. 2001; 148: 337-361PubMed Google Scholar), except that after the primer extension and ligation steps products were digested with FcpI and AvaII, 5′ overhanging ends were filled in using DNA polymerase Klenow fragment (Promega) and dATP, dGTP, dCTP, and dTTP (Amersham Biosciences), and 5′ phosphates at ends and nicks were removed using calf intestinal alkaline phosphatase (Promega; 3 units for 45 min at 37 °C) (filling of 5′ overhanging ends and removal of 5′ phosphates from ends and nicks significantly reduced nonspecific crosslinking by end- and nick-bound PfRNAP 2M. B. Renfrow, unpublished data.). Site-specific Protein-DNA Photocrosslinking—Reaction mixtures (43 μl) contained 0 or 2 nm PfRNAP, 1 nm PfTBP, 1 nm PfTFB, 1 nm derivatized promoter DNA fragment (200 Bq/fmol), and 40 μg/ml heparin in transcription buffer (40 mm HEPES (pH 6.5), 250 mm KCl, 2.5 mm MgCl2, 0.1 mm EDTA, 10 mm β-mercaptoethanol, 6 μg/ml chymostatin, 1 μg/ml aprotinin, 0.4 μg/ml pepstatin, 0.4 μg/ml leupeptin, 0.2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, and 8% glycerol) at 70 °C. Reaction mixtures were prepared by the incubation of 2 nm PfTBP and 2 nm derivatized promoter DNA fragment (200 Bq/fmol) in 20 μl of transcription buffer for 10 min at 70 °C followed by the addition of0or2nm PfRNAP and 2 nm PfTFB in 20 μl of transcription buffer and incubation for 10 min at 70 °C. This was followed in experiments performed in the presence of PfRNAP by the addition of 3 μl of 583 μg/ml heparin and incubation for 2 min at 70 °C. Crosslinking, nuclease digestion, and product analysis were performed by a modification of the procedures of Naryshkin et al. (47.Naryshkin N. Kim Y. Dong Q. Ebright R.H. Methods Mol. Biol. 2001; 148: 337-361PubMed Google Scholar). Reaction mixtures were UV-irradiated for 60 s (11 mJ/mm2 at 365 nm) in a Spectrolinker XL-1000 UV crosslinker (Fisher). Reaction vessels were siliconized polypropylene microcentrifuge tubes contained within borosilicate glass culture tubes (16 × 100 mm) filled with water pre-equilibrated to 70 °C. Crosslinked polypeptides were identified by performing nuclease digestion (the addition of 2 μl of 100 mm CaCl2 and 10 units/μl DNase I (Sigma) and incubation for 10 min at 37 °C followed by the addition of 1 μl of 10% SDS and incubation for 5 min at 70 °C followed by the addition of 1 μl of 30 units/μl S1 nuclease (Roche Applied Science) and incubation for 10 min at 37 °C), SDS-PAGE, and autoradiography. Site-specific Protein-DNA Photocrosslinking—The procedure used consists of four steps (Fig. 1A; Refs. 43.Lagrange T. Kim T.K. Orphanides G. Ebright Y.W. Ebright R.H. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10620-10625Crossref PubMed Scopus (92) Google Scholar, 44.Kim T.K. Lagrange T. Wang Y.H. Griffith J.D. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12268-12273Crossref PubMed Scopus (91) Google Scholar, 45.Kim T.K. Ebright R.H. Reinberg D. Science. 2000; 288: 1418-1422Crossref PubMed Scopus (215) Google Scholar, 46.Naryshkin N. Revyakin A. Kim Y. Mekler V. Ebright R.H. Cell. 2000; 101: 601-611Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 47.Naryshkin N. Kim Y. Dong Q. Ebright R.H. Methods Mol. Biol. 2001; 148: 337-361PubMed Google Scholar) as follows: (i) Chemical and enzymatic reactions were used to prepare a DNA fragment containing a phenyl azide photoactivable crosslinking agent (“probe”) and an adjacent 32P radiolabel incorporated at a single, defined DNA phosphate (with a 9.7-Å linker between the reactive atom of the probe and the phosphorus atom of the phosphate and with an ∼11-Å maximum “reach” between potential crosslinking targets and the phosphorus atom of the phosphate). (ii) The multiprotein-DNA complex of interest was formed using the site-specifically derivatized DNA fragment, and the multiprotein-DNA complex was UV-irradiated, initiating covalent crosslinking with polypeptides in direct physical proximity to the probe. (iii) Extensive nuclease digestion was performed, eliminating uncrosslinked DNA and converting crosslinked DNA to a crosslinked, radiolabeled 3–5 nucleotide “tag.” (iv) The tagged polypeptides were identified using denaturing polyacrylamide gel electrophoresis and autoradiography. For analysis of the P. furiosus transcription initiation complex, we constructed 41 derivatized DNA fragments, each having a probe incorporated at a single, defined phosphate of the P. furiosus glutamate dehydrogenase promoter (gdh; each second phosphate on each strand from positions –40 to +1; Fig. 1B). For each DNA fragment, we analyzed crosslinking both in the PfTFB-PfTBP-promoter complex and in the PfRNAP-PfTFB-PfTBP-promoter complex. For each DNA fragment, we formed the complex, UV-irradiated the complex to initiate crosslinking, and identified crosslinked polypeptides. Experiments were performed at 70 °C. In vitro transcription assays indicate that the PfRNAP-PfTFB-PfTBP-promoter complex exhibits optimal activity at 70 °C (with high activity at 60–90 °C (8.Hethke C. Geerling A.C. Hausner W. de Vos W.M. Thomm M. Nucleic Acids Res. 1996; 24: 2369-2376Crossref PubMed Scopus (68) Google Scholar, 9.Hethke C. Bergerat A. Hausner W. Forterre P. Thomm M. Genetics. 1999; 152: 1325-1333Crossref PubMed Google Scholar, 10.Lewis, L. M. (2000) Structural and Functional Characterization of the Transcription Preinitiation Complex from Pyrococcus furiosus. Ph.D. dissertation, University of Georgia, AthensGoogle Scholar)). Potassium permanganate footprinting experiments indicate that the PfRNAP-PfTFB-PfTBP-promoter complex is an open complex at 70 °C (with a single-stranded “transcription bubble” extending from at least position –7toat least position +3at70 °C (53.Spitalny P. Thomm M. J. Biol. Chem. 2003; 278: 30497-30505Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar)). Heparin-challenge experiments further indicate that the PfRNAP-PfTFB-PfTBP-promoter complex is a promoter-specific and transcriptionally competent complex at 70 °C. 3N. Naryshkin and M. B. Renfrow, unpublished data. Complications due to crosslinking within nonspecific, non-productive complexes were avoided by inclusion of heparin (which disrupts nonspecific and non-productive complexes on double-stranded DNA (54.Cech C.L. McClure W.R. Biochemistry. 1980; 19: 2440-2447Crossref PubMed Scopus (60) Google Scholar)) and by inclusion of filling-in and dephosphorylation steps in DNA-fragment preparation (which prevents formation of heparin-resistant nonspecific and non-productive complexes at DNA ends and DNA nicks 3N. Naryshkin and M. B. Renfrow,

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