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In Vitro Transcription Assays Using Components from Methanothermobacter thermautotrophicus
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[6] In Vitro Transcription Assays Using Components from Methanothermobacter thermautotrophicus By Yunwei Xie and John N. Reeve Eukarya, Bacteria, and Archaea have diverged to form the three primary domains of life.1 Although Archaea are prokaryotes, archaeal RNA polymerases (RNAP) are most similar to eukaryotic RNA polymerase II,2,3 but transcribe both protein and stable RNA encoding genes. A functional archaeal RNA polymerase has been reconstituted from 12 individual recombinant subunits,3 and archaeal RNAP subunits were used as surrogates in determining the RNA polymerase II structure.4,5 However, the largest subunit of the archaeal RNAP does not contain a heptapeptide repeat-containing C-terminal domain (CTD), consistent with this regulatory domain having evolved later during eukaryotic divergence.6 In addition to RNAP, transcription initiation in Archaea requires at least two general transcription factors, archaeal homologs of the eukaryotic TATA box-binding protein (TBP) and transcription factor TFIIB, the latter known as TFB in Archaea.2 Archaea also contain a protein homologous to the N-terminal, zinc ribbon-containing domain of the subunit of eukaryotic TFIIE, and addition of this protein, designated TFE, stimulates transcription in vitro from some but not all archaeal promoters.7,8 Based on the available genome sequences, Archaea do not have homologs of the subunit of TFIIE, nor of TFIIA, TFIIF, or TFIIH and although some Archaea have histones,9 they do not appear to have their DNA packaged into regular chromatin. There is also no evidence for archaeal homologs of the multisubunit eukaryotic histone modification, chromatin remodeling, and transcription activation complexes. In contrast, the archaeal transcription regulators investigated so far appear to function in a manner analogous to bacterial repressors. They bind to the promoter region, 1
M. L. Wheelis, O. Kandler, and C. R. Woese, Proc. Natl. Acad. Sci. USA 89, 2930 (1992). J. Soppa, Adv. Appl. Microbiol. 50, 171 (2001). 3 F. Werner and R. O. J. Weinzierl, Mol. Cell 10, 635 (2002). 4 P. Cramer, D. A. Bushnell, J. Fu, A. L. Gnatt, B. Maier-Davis, N. E. Thompson, R. R. Burgess, A. M. Edwards, P. R. David, and R. D. Kornberg, Science 288, 640 (2000). 5 F. Todone, P. Brick, F. Werner, R. O. J. Weinzierl, and S. Onesti, Mol. Cell 8, 1137 (2001). 6 J. W. Stiller and B. D. Hall, Proc. Natl. Acad. Sci USA 99, 6091 (2002). 7 B. L. Hanzelka, T. J. Darcy, and J. N. Reeve, J. Bacteriol. 183, 1813 (2001). 8 S. D. Bell, A. B. Brinkman, J. van der Oost, and S. P. Jackson, EMBO Rep. 2, 133 (2001). 9 K. Sandman and J. N. Reeve, Adv. Appl. Microbiol. 50, 75 (2001). 2
METHODS IN ENZYMOLOGY, VOL. 370
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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preventing TBP and/or TFB binding to the TATA box/BRE region or they block RNAP access to the site of transcription initiation.10 This article describes the purification and assay of RNAP from the thermophilic, anaerobic Archaeon, Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum strain H11), and its use with recombinant M. thermautotrophicus TBP, TFB, and TFE to obtain promoter-dependent transcription in vitro.12,13 Growth of Methanothermobacter thermautotrophicus
Methanothermobacter thermautotrophicus is a thermophilic, obligately anaerobic autotroph.11 For growth it requires a highly reduced environment, H2 plus CO2 supplied at an 8:1 (v/v) ratio, and a buffered salt solution. Sufficient cell mass (20 g wet weight) for RNAP purification (see later) can be obtained from one 20-liter culture, inoculated with the cells from a 1- to 2-L culture, or the combined cell mass from several such smaller cultures (see Fig. 1). The M. thermautotrophicus salts solution contains (per L H2O), 4 g NaHCO3, 0.3 g KH2PO4, 1 g NH4Cl, 0.6 g NaCl, 100 mg MgCl26H2O, 60 mg CaCl22H2O, a 10-ml trace element (TES) solution, 0.5 g cysteine, 0.62 g Na-thiosulfate, and 1 ml resazurin (2 mg/ml). To make 1 liter of TES, 12.8 g nitrilotriacetic acid is dissolved in 200 ml H2O, the pH is adjusted to 6.5, and the following salts are added and dissolved: 50 mg AlCl36H2O, 100 mg CaCl22H2O, 100 mg CoCl26H2O, 25 mg CuCl22H2O, 1.35 g FeCl36H2O, 10 mg H3BO3, 100 mg MnCl24H2O, 1 g NaCl, 24 mg Na2 MoO42H2O, 26 mg Na2SeO 46H2O, 120 mg NiCl26H2O, and 100 mg ZnCl2. This solution can be sterilized by autoclaving at 121 for 20 min and it is then stable for an extended period if stored at 4 . The growth salts solution is sterilized in situ inside a sealed fermentor vessel (2 or 20 liter), allowed to cool to 65 , and then reduced by sparging with an 8:1 mixture of H2:CO2 (v/v). The resazurin redox indicator is initially pink but with reduction the medium becomes colorless and can then be inoculated using an 10% volume inoculum. Provided that strictly anaerobic conditions are maintained, aliquots of fully grown M. thermautotrophicus cultures can be stored for at least 3 months at room 10
S. D. Bell and S. P. Jackson, Curr. Opin. Microbiol. 4, 208 (2001). A. Wasserfallen, J. No¨ lling, P. Pfister, J. Reeve, and E. Conway de Macario, Int. J. System. Evol. Microbiol. 50, 43 (2000). 12 T. J. Darcy, in ‘‘In Vitro Analysis of Transcription from the Thermophilic Archaeon Methanobacterium thermautotrophicum Strain H,’’ Ph.D. dissertation, Ohio State University, 1999. 13 T. J. Darcy, W. Hausner, D. E. Awery, A. Edwards, M. Thomm, and J. N. Reeve, J. Bacteriol. 181, 4424 (1999). 11
68
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Fig. 1. Examples of fermentation equipment used to grow (A) small (1–2 liter) and (B) large ( 20 liter) cultures of M. thermautotrophicus at 65 under anaerobic conditions using a 8:1 gas mixture of H2:CO2. The rate of growth is dependent on the rate of gas dissolution16 with methanogenesis adding methane to the exhaust gas.
temperature for future use as inocula. The culture is incubated at 65 with continuous H2:CO2 sparging at a flow rate of 200 ml/min and impellor mixing at 600 rpm until the OD600 reaches 1. The cells should be harvested and maintained under anaerobic conditions. The resulting cell paste is transferred into an anaerobic work chamber for immediate use or is frozen rapidly by immersion in liquid N2 and stored at 70 in an airtight container. Purification of M. thermautotrophicus RNA Polymerase
Stock solutions A (1 M KCl, 10 mM MgCl2, 50 mM Tris–HCl), B (50 mM KCl, 10 mM MgCl2, 50 mM Tris–HCl), and C (10 mM MgCl2, 50 mM Tris–HCl) are adjusted to pH 8, and glycerol [20% (v/v) final concentration] and resazurin (100 l of 2 mg/ml) are added. After filtration, autoclaving at 121 for 20 min, and cooling, these solutions are transferred into the anaerobic chamber, 174 mg Na-thiosulfate is added per liter to each solution, and the solution colors should change with reduction from blue to pink to colorless. 16
J. N. Reeve, J. No¨ lling, R. M. Morgan, and D. R. Smith, J. Bacteriol. 179, 5975 (1997).
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Resuspend 20 g M. thermautotrophicus cell paste in 40 ml of solution B, rupture the cells by passage through a French pressure cell at 18,000 psi, and collect the resulting cell lysate into a centrifuge tube preflushed with N2 that contains 200 l of cysteine (150 mg/ml) and 200 l of Na-thiosulfate (186 mg/ml). Seal the tube and centrifuge at 10,000 g for 90 min at 4 . The supernatant is collected, and all subsequent chromatography steps are undertaken at room temperature inside an anaerobic chamber containing an atmosphere of 95% N2:5% H2. The supernatant is loaded at 2 ml/ min onto a 200-ml bed volume DEAE cellulose column (Whatman, Fairfield, NJ) preequilibrated with solution B, and after washing with 300 ml of solution B, bound protein is eluted using a 50 to 525 mM KCl gradient (800 ml) generated by mixing solutions A and B. The presence of RNAP activity in each fraction (9 ml) collected is determined by assaying [ -32P]UTP incorporation into trichloroacetic acid (TCA)-precipitable material using poly(dA-dT) as the DNA template. To do so, an aliquot (10 l) of each fraction is mixed with 90 l of 20 mM Tris–HCl (pH 8), 40 mM KCl, 10 mM MgCl2, 1 mM ATP, 0.1 mM UTP, 0.7 Ci [ -32P]UTP (3 kCi/mM), and 9 g poly(dA-dT) (ICN, Costa Mesa, CA) and the mixtures are incubated at 58 for 30 min. The reaction is stopped by the addition of 900 l ice-cold 5% TCA containing 165 mM NaCl and, after a 5-min incubation on ice, the resulting precipitates are collected by filtration onto glass microfiber filters (934-AH, Whatman). Each filter is washed three times using 10 ml of cold 5% TCA and once with 10 ml of cold 95% ethanol; after drying, the amount of radioactive material bound to the filters is determined by scintillation counting. Fractions containing RNAP activity are pooled and solution C is added to reduce the KCl concentration to 70 mM. Load this solution (1 ml/min) onto a 20-ml bed volume heparin–Sepharose (Amersham Pharmacia, Piscataway, NJ) column preequilibrated with solution B. Wash with 60 ml of solution B, and elute bound proteins using a 200-ml gradient of 50 mM to 1 M KCl made by mixing solutions A and B. Collect 4-ml fractions and identify the fractions containing RNAP activity as described earlier. Pool these fractions and add solution C to reduce the KCl concentration to 70 mM. Load this solution onto a 1-ml bed volume Mono Q column (Amersham Pharmacia) preequilibrated with solution B, wash with 3 ml of solution B, and elute bound proteins using a 15-ml gradient of 50 mM to 1 M KCl made by mixing solutions A and B. Collect 0.5-ml fractions and assay for RNAP activity. Equilibrate a Hi-Load 16/60 Superdex 200 gel filtration column (120-ml bed volume, 60 cm height; Amersham Pharmacia) for 20 h by pumping a 27:73 mixture of solutions A and B at 0.35 ml/min through the column. Then inject a 1-ml aliquot of the pooled RNAP-containing fractions from the Mono Q column and elute using the
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27:73 mixture of solutions A and B. Collect 50 fractions (2 ml) and identify the RNAP-containing fractions. Pool these fractions (usually fractions 30 and 31), add 2 l 1 M dithiothreitol (DTT) per fraction, aliquot (1 ml) into 1.5-ml microfuge tubes, freeze in liquid nitrogen, and store at 70 for future use. Such fractions retain full RNAP activity when stored at 70 for at least 1 year. Repeat the Superdex 200 gel filtration column chromatography, each time injecting a 1-ml aliquot of the RNAP-containing pooled material from the Mono Q column until all this material has fractionated. Preparation of Recombinant M. thermautotrophicus TBP, TFB, and TFE
Promoter-specific transcription initiation by M. thermautotrophicus RNAP requires the additional presence of at least two general transcription factors, TBP and TFB.12,13 TFE addition further stimulates transcription in vitro from some but not all M. thermautotrophicus promoters.7 Genes MTH1627, MTH0885, and MTH1669 which encode TBP, TFB, and TFE, respectively, have been amplified from the M. thermautotrophicus genome14 and cloned into expression vectors. They direct the synthesis of soluble (his)6-tagged recombinant versions of these transcription factors in Escherichia coli, which function in vitro. These proteins can be purified easily by standard Ni-NTA affinity-imidazole elution chromatography.7,12,13 Isopropyl- -d-thiogalactoside-inducible expression plasmids encoding TBP, TFB, and TFE, designated pTD105, pTD103, and pTrc1669, respectively, are available at O.S.U. on request. These transcription factor preparations should be dialyzed against 50 mM Tris–HCl, pH 8, 300 mM KCl, 10 mM MgCl2, 1 mM DTT, and 20% (v/v) glycerol to remove all traces of imidazole and to substitute KCl for NaCl and their concentrations determined by Bradford assays; they can be stored as aliquots frozen at 70 . Promoter-Specific in Vitro Transcription
Promoter-specific transcription initiation is obtained in vitro using native M. thermautotrophicus RNAP and recombinant M. thermautotrophicus TBP, TFB, and TFE, purified as described earlier, from some but not all M. thermautotrophicus promoters.7 The basis for the inactivity in vitro of some promoters known to be active in vivo is currently under investigation. Templates carrying the promoter for the archaeal histoneencoding gene hmtB15 can be used to establish the in vitro system as they consistently direct abundant transcription in vitro. DdeI digestion of 14
D. R. Smith et al., J. Bacteriol. 179, 7135 (1997).
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plasmid pRT741215 DNA (available on request) generates a convenient linear template from which the hmtB promoter directs the synthesis of a 193 nucleotide runoff transcript. In vitro transcription reaction mixtures (50 l) containing 20 mM Tris–HCl, pH 8, 120 mM KCl, 10 mM MgCl2, 2 mM DTT, 100 ng template DNA, 50 ng TBP, 300 ng TFB, 5 l RNAP, 30 M ATP, 30 M CTP, 30 M GTP, 2 M UTP, and 2 Ci of [ -32P]UTP (3 kCi/mM) are incubated at 58 . Although the concentrations of the individual reaction components can be varied slightly, this in vitro transcription system is particularly sensitive to the KCl concentration. Nonspecific (promoter-independent) transcription increases substantially at KCl concentrations below 100 mM, and all transcription is inhibited above 150 mM KCl.12 Based on runoff transcript accumulation, the rate of transcription remains linear for at least 30 min at 58 . Transcription is terminated by adding 30 l of 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol and placing the reaction mix ture at 95 for 3 min. The radioactively labeled transcripts synthesized can then be detected and quantitated by autoradiography or phosphorimaging after their separation by electrophoresis through a denaturing polyacryamide gel. Addition of TFE to the transcription reaction mixture does not stimulate hmtB promoter function in vitro, but has been shown to result in a one- to three-fold stimulation in transcription in vitro from several other M. thermautotrophicus promoters.7 As the M. thermautotrophicus genome sequence is available,14 template DNAs can be generated with any promoter of interest directly by polymerase chain reaction (PCR) amplification from M. thermautotrophicus genomic DNA. Such PCR-generated DNA molecules should be purified using a Qiaquick PCR cleanup kit (Qiagen, Valencia, CA) before being used as templates to direct in vitro transcription. Purification and Use of Stalled Transcription Ternary Complexes to Assay Transcription Elongation
Templates have been constructed on which the hmtB promoter directs transcription initiation at the start of a 24-bp sequence that can be transcribed in the absence of UTP. Such U-less cassette templates can be used to investigate the consequences of the addition of inhibitors or changes in reaction conditions on transcription elongation without concern for spurious concomitant effects on transcription initiation (Fig. 2A). After transcription of the first 24 nucleotides in the absence of UTP but in the presence of ATP, GTP, and [32P] CTP, a stable ternary complex is generated that contains the template DNA, a 32P-labeled stalled transcript, and 15
R. Tabassum, K. M. Sandman, and J. N. Reeve, J. Bacteriol. 174, 7890 (1992).
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Fig. 2. U-less template and stalled-transcript elongation. (A) A U-less cassette template DNA containing the TFB-responsive element (BRE) and TATA box from the hmtB promoter. Transcription initiated at the site indicated by the arrow results in a 24 nucleotide stalled transcript in the absence of UTP and in a 116 nucleotide runoff transcript in the presence of UTP. (B) Ternary complexes that contained the template DNA, M. thermautotrophicus RNAP, and 32P-labeled stalled 24 nucleotide transcript were purified by centrifugation through a Sephadex G-50 spin column. An aliquot of the 24 nucleotide transcript was isolated and subjected to electrophoresis in lane 1. The remaining complexes were incubated in a transcription reaction mixture that contained all four unlabeled rNTPs, and an aliquot of the resulting runoff transcripts was subjected to electrophoresis in lane 2. Radioactively labeled transcripts were then visualized by autoradiography.
RNAP. Such complexes can be separated from the transcription reaction mixture by centrifugation through a Sephadex G-50 spin column and then added back to a complete in vitro transcription reaction mixture that contains all four unlabeled ribonucleotide triphosphates to obtain stalled transcript elongation. The stalled, 32P-labeled transcripts are extended, resulting in full-length 32P-labeled runoff transcripts, but all newly initiated transcripts are unlabeled. The effects of a potential inhibitor or DNAbinding protein on transcription elongation, as opposed to initiation, are then determined easily by measuring the effects of their addition on the accumulation of 32P-labeled full-length transcripts. Acknowledgments Research in the authors’ laboratory is supported by grants from the Department of Energy (DE-FGO2-87ER13731) and the National Institutes of Health (GM53185).
RNA polymerase structure and properties
[6]
[6] In Vitro Transcription Assays Using Components from Methanothermobacter thermautotrophicus By Yunwei Xie and John N. Reeve Eukarya, Bacteria, and Archaea have diverged to form the three primary domains of life.1 Although Archaea are prokaryotes, archaeal RNA polymerases (RNAP) are most similar to eukaryotic RNA polymerase II,2,3 but transcribe both protein and stable RNA encoding genes. A functional archaeal RNA polymerase has been reconstituted from 12 individual recombinant subunits,3 and archaeal RNAP subunits were used as surrogates in determining the RNA polymerase II structure.4,5 However, the largest subunit of the archaeal RNAP does not contain a heptapeptide repeat-containing C-terminal domain (CTD), consistent with this regulatory domain having evolved later during eukaryotic divergence.6 In addition to RNAP, transcription initiation in Archaea requires at least two general transcription factors, archaeal homologs of the eukaryotic TATA box-binding protein (TBP) and transcription factor TFIIB, the latter known as TFB in Archaea.2 Archaea also contain a protein homologous to the N-terminal, zinc ribbon-containing domain of the subunit of eukaryotic TFIIE, and addition of this protein, designated TFE, stimulates transcription in vitro from some but not all archaeal promoters.7,8 Based on the available genome sequences, Archaea do not have homologs of the subunit of TFIIE, nor of TFIIA, TFIIF, or TFIIH and although some Archaea have histones,9 they do not appear to have their DNA packaged into regular chromatin. There is also no evidence for archaeal homologs of the multisubunit eukaryotic histone modification, chromatin remodeling, and transcription activation complexes. In contrast, the archaeal transcription regulators investigated so far appear to function in a manner analogous to bacterial repressors. They bind to the promoter region, 1
M. L. Wheelis, O. Kandler, and C. R. Woese, Proc. Natl. Acad. Sci. USA 89, 2930 (1992). J. Soppa, Adv. Appl. Microbiol. 50, 171 (2001). 3 F. Werner and R. O. J. Weinzierl, Mol. Cell 10, 635 (2002). 4 P. Cramer, D. A. Bushnell, J. Fu, A. L. Gnatt, B. Maier-Davis, N. E. Thompson, R. R. Burgess, A. M. Edwards, P. R. David, and R. D. Kornberg, Science 288, 640 (2000). 5 F. Todone, P. Brick, F. Werner, R. O. J. Weinzierl, and S. Onesti, Mol. Cell 8, 1137 (2001). 6 J. W. Stiller and B. D. Hall, Proc. Natl. Acad. Sci USA 99, 6091 (2002). 7 B. L. Hanzelka, T. J. Darcy, and J. N. Reeve, J. Bacteriol. 183, 1813 (2001). 8 S. D. Bell, A. B. Brinkman, J. van der Oost, and S. P. Jackson, EMBO Rep. 2, 133 (2001). 9 K. Sandman and J. N. Reeve, Adv. Appl. Microbiol. 50, 75 (2001). 2
METHODS IN ENZYMOLOGY, VOL. 370
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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preventing TBP and/or TFB binding to the TATA box/BRE region or they block RNAP access to the site of transcription initiation.10 This article describes the purification and assay of RNAP from the thermophilic, anaerobic Archaeon, Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum strain H11), and its use with recombinant M. thermautotrophicus TBP, TFB, and TFE to obtain promoter-dependent transcription in vitro.12,13 Growth of Methanothermobacter thermautotrophicus
Methanothermobacter thermautotrophicus is a thermophilic, obligately anaerobic autotroph.11 For growth it requires a highly reduced environment, H2 plus CO2 supplied at an 8:1 (v/v) ratio, and a buffered salt solution. Sufficient cell mass (20 g wet weight) for RNAP purification (see later) can be obtained from one 20-liter culture, inoculated with the cells from a 1- to 2-L culture, or the combined cell mass from several such smaller cultures (see Fig. 1). The M. thermautotrophicus salts solution contains (per L H2O), 4 g NaHCO3, 0.3 g KH2PO4, 1 g NH4Cl, 0.6 g NaCl, 100 mg MgCl26H2O, 60 mg CaCl22H2O, a 10-ml trace element (TES) solution, 0.5 g cysteine, 0.62 g Na-thiosulfate, and 1 ml resazurin (2 mg/ml). To make 1 liter of TES, 12.8 g nitrilotriacetic acid is dissolved in 200 ml H2O, the pH is adjusted to 6.5, and the following salts are added and dissolved: 50 mg AlCl36H2O, 100 mg CaCl22H2O, 100 mg CoCl26H2O, 25 mg CuCl22H2O, 1.35 g FeCl36H2O, 10 mg H3BO3, 100 mg MnCl24H2O, 1 g NaCl, 24 mg Na2 MoO42H2O, 26 mg Na2SeO 46H2O, 120 mg NiCl26H2O, and 100 mg ZnCl2. This solution can be sterilized by autoclaving at 121 for 20 min and it is then stable for an extended period if stored at 4 . The growth salts solution is sterilized in situ inside a sealed fermentor vessel (2 or 20 liter), allowed to cool to 65 , and then reduced by sparging with an 8:1 mixture of H2:CO2 (v/v). The resazurin redox indicator is initially pink but with reduction the medium becomes colorless and can then be inoculated using an 10% volume inoculum. Provided that strictly anaerobic conditions are maintained, aliquots of fully grown M. thermautotrophicus cultures can be stored for at least 3 months at room 10
S. D. Bell and S. P. Jackson, Curr. Opin. Microbiol. 4, 208 (2001). A. Wasserfallen, J. No¨ lling, P. Pfister, J. Reeve, and E. Conway de Macario, Int. J. System. Evol. Microbiol. 50, 43 (2000). 12 T. J. Darcy, in ‘‘In Vitro Analysis of Transcription from the Thermophilic Archaeon Methanobacterium thermautotrophicum Strain H,’’ Ph.D. dissertation, Ohio State University, 1999. 13 T. J. Darcy, W. Hausner, D. E. Awery, A. Edwards, M. Thomm, and J. N. Reeve, J. Bacteriol. 181, 4424 (1999). 11
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Fig. 1. Examples of fermentation equipment used to grow (A) small (1–2 liter) and (B) large ( 20 liter) cultures of M. thermautotrophicus at 65 under anaerobic conditions using a 8:1 gas mixture of H2:CO2. The rate of growth is dependent on the rate of gas dissolution16 with methanogenesis adding methane to the exhaust gas.
temperature for future use as inocula. The culture is incubated at 65 with continuous H2:CO2 sparging at a flow rate of 200 ml/min and impellor mixing at 600 rpm until the OD600 reaches 1. The cells should be harvested and maintained under anaerobic conditions. The resulting cell paste is transferred into an anaerobic work chamber for immediate use or is frozen rapidly by immersion in liquid N2 and stored at 70 in an airtight container. Purification of M. thermautotrophicus RNA Polymerase
Stock solutions A (1 M KCl, 10 mM MgCl2, 50 mM Tris–HCl), B (50 mM KCl, 10 mM MgCl2, 50 mM Tris–HCl), and C (10 mM MgCl2, 50 mM Tris–HCl) are adjusted to pH 8, and glycerol [20% (v/v) final concentration] and resazurin (100 l of 2 mg/ml) are added. After filtration, autoclaving at 121 for 20 min, and cooling, these solutions are transferred into the anaerobic chamber, 174 mg Na-thiosulfate is added per liter to each solution, and the solution colors should change with reduction from blue to pink to colorless. 16
J. N. Reeve, J. No¨ lling, R. M. Morgan, and D. R. Smith, J. Bacteriol. 179, 5975 (1997).
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Resuspend 20 g M. thermautotrophicus cell paste in 40 ml of solution B, rupture the cells by passage through a French pressure cell at 18,000 psi, and collect the resulting cell lysate into a centrifuge tube preflushed with N2 that contains 200 l of cysteine (150 mg/ml) and 200 l of Na-thiosulfate (186 mg/ml). Seal the tube and centrifuge at 10,000 g for 90 min at 4 . The supernatant is collected, and all subsequent chromatography steps are undertaken at room temperature inside an anaerobic chamber containing an atmosphere of 95% N2:5% H2. The supernatant is loaded at 2 ml/ min onto a 200-ml bed volume DEAE cellulose column (Whatman, Fairfield, NJ) preequilibrated with solution B, and after washing with 300 ml of solution B, bound protein is eluted using a 50 to 525 mM KCl gradient (800 ml) generated by mixing solutions A and B. The presence of RNAP activity in each fraction (9 ml) collected is determined by assaying [ -32P]UTP incorporation into trichloroacetic acid (TCA)-precipitable material using poly(dA-dT) as the DNA template. To do so, an aliquot (10 l) of each fraction is mixed with 90 l of 20 mM Tris–HCl (pH 8), 40 mM KCl, 10 mM MgCl2, 1 mM ATP, 0.1 mM UTP, 0.7 Ci [ -32P]UTP (3 kCi/mM), and 9 g poly(dA-dT) (ICN, Costa Mesa, CA) and the mixtures are incubated at 58 for 30 min. The reaction is stopped by the addition of 900 l ice-cold 5% TCA containing 165 mM NaCl and, after a 5-min incubation on ice, the resulting precipitates are collected by filtration onto glass microfiber filters (934-AH, Whatman). Each filter is washed three times using 10 ml of cold 5% TCA and once with 10 ml of cold 95% ethanol; after drying, the amount of radioactive material bound to the filters is determined by scintillation counting. Fractions containing RNAP activity are pooled and solution C is added to reduce the KCl concentration to 70 mM. Load this solution (1 ml/min) onto a 20-ml bed volume heparin–Sepharose (Amersham Pharmacia, Piscataway, NJ) column preequilibrated with solution B. Wash with 60 ml of solution B, and elute bound proteins using a 200-ml gradient of 50 mM to 1 M KCl made by mixing solutions A and B. Collect 4-ml fractions and identify the fractions containing RNAP activity as described earlier. Pool these fractions and add solution C to reduce the KCl concentration to 70 mM. Load this solution onto a 1-ml bed volume Mono Q column (Amersham Pharmacia) preequilibrated with solution B, wash with 3 ml of solution B, and elute bound proteins using a 15-ml gradient of 50 mM to 1 M KCl made by mixing solutions A and B. Collect 0.5-ml fractions and assay for RNAP activity. Equilibrate a Hi-Load 16/60 Superdex 200 gel filtration column (120-ml bed volume, 60 cm height; Amersham Pharmacia) for 20 h by pumping a 27:73 mixture of solutions A and B at 0.35 ml/min through the column. Then inject a 1-ml aliquot of the pooled RNAP-containing fractions from the Mono Q column and elute using the
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27:73 mixture of solutions A and B. Collect 50 fractions (2 ml) and identify the RNAP-containing fractions. Pool these fractions (usually fractions 30 and 31), add 2 l 1 M dithiothreitol (DTT) per fraction, aliquot (1 ml) into 1.5-ml microfuge tubes, freeze in liquid nitrogen, and store at 70 for future use. Such fractions retain full RNAP activity when stored at 70 for at least 1 year. Repeat the Superdex 200 gel filtration column chromatography, each time injecting a 1-ml aliquot of the RNAP-containing pooled material from the Mono Q column until all this material has fractionated. Preparation of Recombinant M. thermautotrophicus TBP, TFB, and TFE
Promoter-specific transcription initiation by M. thermautotrophicus RNAP requires the additional presence of at least two general transcription factors, TBP and TFB.12,13 TFE addition further stimulates transcription in vitro from some but not all M. thermautotrophicus promoters.7 Genes MTH1627, MTH0885, and MTH1669 which encode TBP, TFB, and TFE, respectively, have been amplified from the M. thermautotrophicus genome14 and cloned into expression vectors. They direct the synthesis of soluble (his)6-tagged recombinant versions of these transcription factors in Escherichia coli, which function in vitro. These proteins can be purified easily by standard Ni-NTA affinity-imidazole elution chromatography.7,12,13 Isopropyl- -d-thiogalactoside-inducible expression plasmids encoding TBP, TFB, and TFE, designated pTD105, pTD103, and pTrc1669, respectively, are available at O.S.U. on request. These transcription factor preparations should be dialyzed against 50 mM Tris–HCl, pH 8, 300 mM KCl, 10 mM MgCl2, 1 mM DTT, and 20% (v/v) glycerol to remove all traces of imidazole and to substitute KCl for NaCl and their concentrations determined by Bradford assays; they can be stored as aliquots frozen at 70 . Promoter-Specific in Vitro Transcription
Promoter-specific transcription initiation is obtained in vitro using native M. thermautotrophicus RNAP and recombinant M. thermautotrophicus TBP, TFB, and TFE, purified as described earlier, from some but not all M. thermautotrophicus promoters.7 The basis for the inactivity in vitro of some promoters known to be active in vivo is currently under investigation. Templates carrying the promoter for the archaeal histoneencoding gene hmtB15 can be used to establish the in vitro system as they consistently direct abundant transcription in vitro. DdeI digestion of 14
D. R. Smith et al., J. Bacteriol. 179, 7135 (1997).
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transcription assays
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plasmid pRT741215 DNA (available on request) generates a convenient linear template from which the hmtB promoter directs the synthesis of a 193 nucleotide runoff transcript. In vitro transcription reaction mixtures (50 l) containing 20 mM Tris–HCl, pH 8, 120 mM KCl, 10 mM MgCl2, 2 mM DTT, 100 ng template DNA, 50 ng TBP, 300 ng TFB, 5 l RNAP, 30 M ATP, 30 M CTP, 30 M GTP, 2 M UTP, and 2 Ci of [ -32P]UTP (3 kCi/mM) are incubated at 58 . Although the concentrations of the individual reaction components can be varied slightly, this in vitro transcription system is particularly sensitive to the KCl concentration. Nonspecific (promoter-independent) transcription increases substantially at KCl concentrations below 100 mM, and all transcription is inhibited above 150 mM KCl.12 Based on runoff transcript accumulation, the rate of transcription remains linear for at least 30 min at 58 . Transcription is terminated by adding 30 l of 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol and placing the reaction mix ture at 95 for 3 min. The radioactively labeled transcripts synthesized can then be detected and quantitated by autoradiography or phosphorimaging after their separation by electrophoresis through a denaturing polyacryamide gel. Addition of TFE to the transcription reaction mixture does not stimulate hmtB promoter function in vitro, but has been shown to result in a one- to three-fold stimulation in transcription in vitro from several other M. thermautotrophicus promoters.7 As the M. thermautotrophicus genome sequence is available,14 template DNAs can be generated with any promoter of interest directly by polymerase chain reaction (PCR) amplification from M. thermautotrophicus genomic DNA. Such PCR-generated DNA molecules should be purified using a Qiaquick PCR cleanup kit (Qiagen, Valencia, CA) before being used as templates to direct in vitro transcription. Purification and Use of Stalled Transcription Ternary Complexes to Assay Transcription Elongation
Templates have been constructed on which the hmtB promoter directs transcription initiation at the start of a 24-bp sequence that can be transcribed in the absence of UTP. Such U-less cassette templates can be used to investigate the consequences of the addition of inhibitors or changes in reaction conditions on transcription elongation without concern for spurious concomitant effects on transcription initiation (Fig. 2A). After transcription of the first 24 nucleotides in the absence of UTP but in the presence of ATP, GTP, and [32P] CTP, a stable ternary complex is generated that contains the template DNA, a 32P-labeled stalled transcript, and 15
R. Tabassum, K. M. Sandman, and J. N. Reeve, J. Bacteriol. 174, 7890 (1992).
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RNA polymerase structure and properties
[6]
Fig. 2. U-less template and stalled-transcript elongation. (A) A U-less cassette template DNA containing the TFB-responsive element (BRE) and TATA box from the hmtB promoter. Transcription initiated at the site indicated by the arrow results in a 24 nucleotide stalled transcript in the absence of UTP and in a 116 nucleotide runoff transcript in the presence of UTP. (B) Ternary complexes that contained the template DNA, M. thermautotrophicus RNAP, and 32P-labeled stalled 24 nucleotide transcript were purified by centrifugation through a Sephadex G-50 spin column. An aliquot of the 24 nucleotide transcript was isolated and subjected to electrophoresis in lane 1. The remaining complexes were incubated in a transcription reaction mixture that contained all four unlabeled rNTPs, and an aliquot of the resulting runoff transcripts was subjected to electrophoresis in lane 2. Radioactively labeled transcripts were then visualized by autoradiography.
RNAP. Such complexes can be separated from the transcription reaction mixture by centrifugation through a Sephadex G-50 spin column and then added back to a complete in vitro transcription reaction mixture that contains all four unlabeled ribonucleotide triphosphates to obtain stalled transcript elongation. The stalled, 32P-labeled transcripts are extended, resulting in full-length 32P-labeled runoff transcripts, but all newly initiated transcripts are unlabeled. The effects of a potential inhibitor or DNAbinding protein on transcription elongation, as opposed to initiation, are then determined easily by measuring the effects of their addition on the accumulation of 32P-labeled full-length transcripts. Acknowledgments Research in the authors’ laboratory is supported by grants from the Department of Energy (DE-FGO2-87ER13731) and the National Institutes of Health (GM53185).