Characterization of the Rhodobacter capsulatus Housekeeping RNA Polymerase
1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês
10.1074/jbc.272.43.27266
ISSN1083-351X
AutoresPaul J. Cullen, Charles K. Kaufman, W. C. Bowman, Robert G. Kranz,
Tópico(s)Microbial Community Ecology and Physiology
ResumoTo begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, theRhodobacter capsulatus RNA polymerase (RNAP) that contains the ς70 factor (R. capsulatusRNAP/ς70) was purified and characterized using two classical ς70 type promoters, the bacteriophage T7A1 and the RNA I promoters. Transcription from these promoters was sensitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed against the Escherichia coli ς70 subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c 2(cycA) and fructose-inducible (fruB) promoters and genes induced in photosynthesis (puf andpuc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated byR. capsulatus RNAP/ς70 indicated a preference for the sequence TTGAC at the −35 region for strong in vitro transcription. To test the −35 recognition pattern, theR. capsulatus nifA1 promoter, which exhibits only three of the five consensus nucleotides at the −35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1wild type promoter showed no transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/ς70 enzyme. Similarities and differences between the RNAPs and the promoters ofR. capsulatus and E. coli are discussed. To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, theRhodobacter capsulatus RNA polymerase (RNAP) that contains the ς70 factor (R. capsulatusRNAP/ς70) was purified and characterized using two classical ς70 type promoters, the bacteriophage T7A1 and the RNA I promoters. Transcription from these promoters was sensitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed against the Escherichia coli ς70 subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c 2(cycA) and fructose-inducible (fruB) promoters and genes induced in photosynthesis (puf andpuc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated byR. capsulatus RNAP/ς70 indicated a preference for the sequence TTGAC at the −35 region for strong in vitro transcription. To test the −35 recognition pattern, theR. capsulatus nifA1 promoter, which exhibits only three of the five consensus nucleotides at the −35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1wild type promoter showed no transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/ς70 enzyme. Similarities and differences between the RNAPs and the promoters ofR. capsulatus and E. coli are discussed. As elucidated by many in vitro studies during the last 30 years, bacterial transcription requires a core RNA polymerase (RNAP) 1The abbreviations used are: RNAP, RNA polymerase; ς70, sigma 70 factor; Rifr and Rifs, rifampicin-resistant and -sensitive, respectively; PCR, polymerase chain reaction; bp, base pair; PEG, polyethylene glycol; DTT, dl-dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography. 1The abbreviations used are: RNAP, RNA polymerase; ς70, sigma 70 factor; Rifr and Rifs, rifampicin-resistant and -sensitive, respectively; PCR, polymerase chain reaction; bp, base pair; PEG, polyethylene glycol; DTT, dl-dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography. enzyme and ς factors that recognize specific promoter elements. RNAP has been purified from a variety of bacterial species but has been characterized most thoroughly in Escherichia coli (for review, see Ref.1Record M.T. Reznikoff W.S. Craig M.L. McQuade K.L. Schlax P.J. Neidhardt F.C. Escherichia coli and Salmonella. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1996: 792-821Google Scholar). RNAP from the photosynthetic bacterium Rhodobacter sphaeroides has been purified and shown to have a subunit composition similar to that of other Gram-negative bacteria (2Kansy J.W. Kaplan S. J. Biol. Chem. 1989; 264: 13751-13759Abstract Full Text PDF PubMed Google Scholar). A report of a partially purified preparation of Rhodobacter capsulatus RNAP was published a decade ago (3Forrest J.E. Beatty J.T. FEBS Lett. 1987; 212: 24-28Crossref Scopus (6) Google Scholar), but from that study it was not possible to determine definitively which, if any, was the housekeeping ς70 subunit nor to evaluate promoter recognition determinants. In vitro transcription of R. sphaeroides RNAP has been demonstrated from templates that containE. coli ς32 and ς70 type promoters and from an R. sphaeroides rrn promoter (4Karls R.K. Jin D.J. Donohue T. J. Bacteriol. 1993; 175: 7629-7638Crossref PubMed Google Scholar). In the present report we describe in vitro studies on the transcription apparatus of R. capsulatus and promoters that are recognized by this system. Most of the studies to date on gene regulation in R. capsulatus have concerned genetic characterization of signal transduction pathways or the in vivo analysis of mRNAs. From these investigations unique activators and repressors are theorized to regulate ς70-dependent transcription at a variety of promoters in photosynthetic bacteria. InR. capsulatus, operons involved in photosynthesis are regulated by light and oxygen (for review, see Ref. 5Bauer C.E. Bird T.H. Cell. 1996; 85: 5-8Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). For example,puc, puf, and puh encode the structural polypeptides for the photosynthetic complexes, andbch encodes bacteriochlorophyll biosynthetic enzymes. The promoters of some of these genes have been studied by deletion andin vivo primer extension analysis (e.g. 6Bauer C.E. Young D.A. Marrs B.L. J. Biol. Chem. 1988; 263: 4820-4827Abstract Full Text PDF PubMed Google Scholar, 7Young D.A. Bauer C.E. Williams J.C. Marrs B.L. Mol. Gen. Genet. 1989; 218: 1-12Crossref PubMed Scopus (98) Google Scholar, 8Bauer C. Buggy J. Yang Z. Marrs B. Mol. Gen. Genet. 1991; 228: 433-444Crossref PubMed Scopus (49) Google Scholar). Although bch and puc are thought to be transcribed by the R. capsulatus RNA polymerase/ς70 holoenzyme, based on promoter sequences, it is unclear what ς factor(s) recognize the puf andpuh genes. Site-directed mutational analysis of thebchC promoter demonstrated that nucleotides typical of ς70 promoters in the −10 and −35 hexamers upstream of the transcriptional start site are important for in vivotranscription (9Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar). Light-dependent stimulation of transcription from the puf and puh operons requires the hvrA gene (10Buggy J.J. Sganga M.W. Bauer C.E. J. Bacteriol. 1994; 176: 6936-6943Crossref PubMed Google Scholar). The oxygen-regulatedpuf, puh, and puc operons require theregA/regB-encoded two-component system, although it is unknown whether these promoters are directly activated by such proteins (11Sganga M. Bauer C. Cell. 1992; 68: 945-954Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 12Mosley C.S. Suzuki J.Y. Bauer C.E. J. Bacteriol. 1994; 176: 7566-7573Crossref PubMed Google Scholar). The recently discovered CrtJ is required for aerobic repression of the puf, puc, puh, andbch operons by an unknown mechanism (13Ponnampalam S. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar). In some casescis-DNA elements upstream of these promoters have been proposed to mediate light and oxygen regulation by binding of the regulatory proteins (e.g. 9, 11). In the present study it is shown that the cytochrome c 2 gene (cycA) and the fructose-inducible gene fruB (14Duport C. Meyer C. Naud I. Jouanneau Y. Gene (Amst.). 1994; 145: 103-108Crossref PubMed Scopus (17) Google Scholar) may also be activated by the R. capsulatusRNAP/ς70. Other signal transduction pathways in R. capsulatus elucidated mainly by genetic studies include nitrogen sensing (for review, see Refs. 15Kranz R.G. Cullen P.J. Blankenship R.E. Madigan M. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishing, Norwell, MA1995: 1191-1208Google Scholar and 16Masepohl B. Klipp W. Arch. Microbiol. 1996; 165: 80-90Crossref Scopus (65) Google Scholar) and the FnrL regulon. 2Zeilstra-Ryalls, J. H., Gabbert, K. K., Mouncey, N. J., Kaplan, S., and Kranz, R. G. (1997) J. Bacteriol., in press. 2Zeilstra-Ryalls, J. H., Gabbert, K. K., Mouncey, N. J., Kaplan, S., and Kranz, R. G. (1997) J. Bacteriol., in press. As an important step toward in vitro reconstitution of these signal transduction pathways, the R. capsulatus housekeeping RNAP holoenzyme was characterized. ς70-Dependent transcription from a variety of promoters using linear or supercoiled templates was demonstrated. A consensus for optimal ς70type promoters in this bacterium was tested further by mutating the nitrogen-regulated nifA1 promoter (18Foster-Hartnett D. Cullen P.J. Monika E.M. Kranz R.G. J. Bacteriol. 1994; 176: 6175-6187Crossref PubMed Google Scholar) toward the consensus and engineering each mutant promoter upstream of a transcriptional terminator on a supercoiled plasmid. Results of in vitrotranscription studies on these mutant promoters confirmed the importance of specific −35 recognition elements for the holoenzyme. The bacterial strains and plasmids used in this study are shown in TableI. pUC:SBrif and pUC:B10rif contain the sequences that encode the rifampicin binding domain of rpoBstrains SB1003 (Rifr) and B10 (Rifs), respectively. The plasmids were constructed by polymerase chain reaction (PCR) of chromosomal DNA of the two strains using primers described in Table II. The R. capsulatus rpoB sequence for design of primers was provided by Dr. Robert Haselkorn (University of Chicago). The PCR products were digested with BamHI and PstI and cloned into pUC118. The rpoB RNAP ॆ subunit fragments were sequenced using the Sequenase enzyme according to company protocols (Amersham Corp.).Table IBacterial strains and plasmidsStrain/plasmidDescriptionRef.E. coli TB1F− araΔ(lac-proAB)rpsL(Strr)43Baldwin T. Focus. 1984; 6: 7Google Scholar[φ80dlacΔ(lacZ)M15]hsdR(rk−mk−)R. capsulatus B10Wild type44Yen H.C. Marrs B. Arch. Biochem. Biophys. 1976; 181: 411-418Crossref Scopus (90) Google Scholar SB1003Rifr mutant of B1044Yen H.C. Marrs B. Arch. Biochem. Biophys. 1976; 181: 411-418Crossref Scopus (90) Google ScholarPlasmid pUC118, 119Ampr, M13 intergenic region45Vieira J. Messing J. Gene (Amst.). 1982; 19: 259-268Crossref PubMed Scopus (3781) Google Scholar pHP45Ampr, Specr, cassette with flanking transcriptional/translational terminators20Prentki P. Krisch H.M. Gene (Amst.). 1984; 29: 303-313Crossref PubMed Scopus (1342) Google Scholar pC42Ampr, 500-nt1-ant, nucleotide. PCR insert ofcycA in pUC11946Beckman D.L. Trawick D.R. Kranz R.G. Genes Dev. 1992; 6: 268-283Crossref PubMed Scopus (147) Google Scholar pCL185Ampr, 2-kb1-bkb, kilobase. T7A1 promoter in pBR32219Heisler L.M. Suzuki H. Landick R. Gross C.A. J. Biol. Chem. 1993; 268: 25369-25375Abstract Full Text PDF PubMed Google Scholar pRPSLH2Ampr, pucCBA genes EcoRI fragment in pBR32217Youvan D.A. Ismail S. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 58-62Crossref PubMed Scopus (65) Google Scholar pCB701ΩAmpr,puhA-lacZ fusion8Bauer C. Buggy J. Yang Z. Marrs B. Mol. Gen. Genet. 1991; 228: 433-444Crossref PubMed Scopus (49) Google Scholar p1255exAmpr,pufQ EcoRI fragment in pBR3226 pDAY23Ampr, specr bchC gene7Young D.A. Bauer C.E. Williams J.C. Marrs B.L. Mol. Gen. Genet. 1989; 218: 1-12Crossref PubMed Scopus (98) Google Scholar pA1-P16Ampr, 300-nt R. capsulatus nifA1promoter18Foster-Hartnett D. Cullen P.J. Monika E.M. Kranz R.G. J. Bacteriol. 1994; 176: 6175-6187Crossref PubMed Google Scholar pUCTAmpr, PCR terminatorPstI-HindIII in pUC118This study pUC:SBrifAmpr, PCR Rifr ॆ-subunit domain of SB1003This study pUC:B10rifAmpr, Rifs ॆ-subunit domain of B10This study pUC:T7Ampr, T7A1 promoter from pCL185 in pUC118This study pUC:bchCAmpr, bchCpromoter from pDAY23 in pUC118This study pUC:pucCAmpr, pucC promoter from pRPSLH2 in pUC118This study pUC:pufQAmpr, pufQpromoter from p1255ex in pUC118This study pUC:fruBAmpr, fruB promoter from SB1003 in pUC118This study pUCT: pucC, pufQ, bchC, fruB, nifA1Ampr, terminator PstI-HindIII cloned downstream of pUC: pucC, pufQ, bchC, fruB, nifA1 constructsThis study1-a nt, nucleotide.1-b kb, kilobase. Open table in a new tab Table IIPrimers for PCRTemplatePlasmid madeUpstream oligonucleotide (5′ to 3′) for PCRDownstream oligonucleotide (5′ to 3′) for PCRpA1-P16pA1M1CGGACGCGTCGGAAGACTTGCCTTTTTTCGCCCATAACAGCTATGACCATGpA1-P16pA1M2CGGACGCGTCGGAAGACGTGACTTTTTTCGCCCATAACAGCTATGACCATGpA1-P16pA1M3CGGACGCGTCGGAAGACTTGACTTTTTTCGCCCATAACAGCTATGACCATGpCBADEpUCT:pucCCGGGATCCGGGGGTGGCCGAATTTGCAACTGCAGCTGGGATCATTGGGAACGTTpCB701pUCT:puhACGGGATCCGTGGCGATGATGGTGGTCAACTGCAGTCCGATTTCGGTCACApDAY23pUCT:bchCCGGGATCCGCGGACCCTGCGCCCCTTAACTGCAGACTTGCGTTTCCATTTCTTp1255expUCT:pufQCGGGATCCTTATCTGGCCGAAACCAAGGAACTGCAGCGACTTGGCCGCCGAAChromosomepUCT:fruBGCCGGTGACGAATTCAAGCTTCACCGCCCCTGGTCAGGGGTACCATATGTCCTCCTCGGCpHP45pUCTGGGGTACCTGCAGGATCCGGTGGATGACCTTTTTGATTGAGCAAGCTTTATGCTTTCTAGACCGTTChromosomepUC:SBrif and pUCB10rifCGGGATCCTCGGTCGAGATCGACACGGTGAAACTGCAGCCGTATTTGTTGACGCGCGCGA Open table in a new tab Linear templates for transcription reactions were created by PCR using the primers indicated in Table II. For supercoiled templates, all DNA fragments that contained putative promoters were cloned into pUC118 directly upstream of a strong transcriptional terminator. The fruB promoter was cloned by PCR of SB1003 chromosomal DNA using primers described in Table II, based on Ref. 14Duport C. Meyer C. Naud I. Jouanneau Y. Gene (Amst.). 1994; 145: 103-108Crossref PubMed Scopus (17) Google Scholar. The 300-bp fruB promoter fragment was digested with KpnI and EcoRI and cloned into pUC118 to create pUC:fruB. Plasmid pUC:T7 that contains 150 bp of the T7A1 promoter was made by excision of the T7A1 promoter fragment with BamHI and EcoRI from plasmid pCL185 (19Heisler L.M. Suzuki H. Landick R. Gross C.A. J. Biol. Chem. 1993; 268: 25369-25375Abstract Full Text PDF PubMed Google Scholar) and ligation into pUC118. pUCT that contains 150 bps of the T4 bacteriophage gene 32 ρ-independent transcriptional terminator was made by PCR of plasmid pHP45 that contained the terminator (20Prentki P. Krisch H.M. Gene (Amst.). 1984; 29: 303-313Crossref PubMed Scopus (1342) Google Scholar) using the primers described in Table II. The PCR product was digested withPstI and HindIII and cloned into pUC118. Templates that contained ς70-dependent promoters (pucC, pufQ, puhA,bchC) were created by PCR of each promoter fragment using a 5′-(upstream) primer that contained a PstI site and a 3′-(downstream) primer that contained a BamHI restriction site (see Table II). The PCR products were digested withBamHI and PstI and cloned into pUC118 to create pUC:pucC, pufQ, puhA, bchC, nifA1. The transcriptional terminator was cloned downstream of each of the promoters by excision of the 125-bp terminator from pUCT withPstI and HindIII and into pUC:pucC, pufQ, bchC, to create the supercoiled templates pUCT:pucC, pufA, bchC. The pUCT:fruB was made by excision of the fruB promoter region from pUC:fruB with EcoRI and PstI and ligation into pUCT. The pUCT:nifA1 template was created by excision of thenifA1 promoter from pA1-P16 with SalI andPstI followed by ligation into pUCT. Templates were confirmed by restriction and sequence analysis and purified in CsCl gradients for all reactions. The nifA1 promoter mutants A1Mut1, A1Mut2, and A1Mut3 were generated by PCR of plasmid pPA1-P16 using the primers described in Table II. The PCR products were digested with MluI andPstI and cloned into pUCT:nifA1 to create pA1M1, pA1M2, pA1M3. The mutations were confirmed by sequence analysis, and the plasmids were purified in CsCl gradients for in vitrotranscription reactions. R. capsulatus RNAP was purified from 12 liters of R. capsulatus cells (strain SB1003) grown aerobically to mid exponential phase (∼17 h,A 600 2.0) in RCV medium (21Avtges P. Kranz R.G. Haselkorn R. Mol. Gen. Genet. 1985; 201: 353-369Crossref Scopus (38) Google Scholar) at 34 °C. These conditions, although aerobic, still allowed the synthesis of some photosynthetic pigments, albeit at lower levels than fully anaerobic, light-grown cells. The rifampicin-sensitive strain R. capsulatus B10 was used for some experiments, where noted. Otherwise, R. capsulatus RNAP refers to the enzyme from SB1003. Cells were harvested by centrifugation and stored as a cell pellet at −80 °C. Purification was based on procedures described previously (22Gross C. Engbaek F. Flammang T. Burgess R. J. Bacteriol. 1976; 128: 382-389Crossref PubMed Google Scholar, 23Chamberlin M. Kingston R. Gilman M. Wiggs J. DeVera A. Methods Enzymol. 1983; 101: 540-568Crossref PubMed Scopus (80) Google Scholar) with modifications. Cell lysis was performed on ice, and the purification was at 4 °C unless otherwise noted. Cells (∼200 g, wet weight) were lysed by resuspension in 108 ml of sucrose solution (10 mm Tris-HCl, pH 8, 257 sucrose, 100 mm NaCl) for 15 min, followed by the addition of 24 ml of lysozyme solution (300 mm Tris-HCl, pH 8, 100 mm EDTA, and 4 mg/ml lysozyme) for 5 min and addition of 135 ml of lysis solution (1 m NaCl, 20 mm EDTA, 110 mg of sodium deoxycholic acid). Lysis was allowed to proceed for 10 min at 10 °C. Following cell lysis, the RNA polymerase-nucleic acid complexes were precipitated by the addition of 380 ml of PEG solution (177 polyethylene glycol 8000 (PEG, Sigma), 157 mm NaCl, 1 mm DTT), followed by centrifugation at 7,000 rpm for 10 min in a Sorvall centrifuge. The supernatant was removed, and proteins were eluted from the PEG pellet by the addition of 60 ml of high salt solution (10 mm Tris-HCl, pH 8, 57 PEG, 2 mNaCl, 1 mm DTT). The PEG supernatant containing the R. capsulatus RNAP was diluted in 430 ml of column buffer (10 mm Tris-HCl, pH 8, 10 mm MgCl2, 1 mm EDTA, 1 mm DTT, and 7.57 glycerol) to 300 mm NaCl and loaded onto 5 × 4-ml heparin-agarose (Sigma) columns that were run at ∼0.5 ml/min using a peristaltic pump. The columns were washed with 20 column volumes (320 ml total) of column buffer that contained 300 mm NaCl, and RNA polymerase was eluted with 48 ml of column buffer in 450 mm NaCl. Fractions that contained peak RNA polymerase activity (∼5 ml; see below) were diluted to 135 ml in column buffer to 150 mm NaCl and loaded onto 3 × 4-ml DEAE-Sepharose (Sigma) columns run at 0.6 ml/min. The columns were washed with 20 column volumes (320 ml total) of column buffer in 150 mm NaCl, and the RNA polymerase was eluted off of the column in 80 ml of column buffer in 300 mm NaCl. Fractions that contained the peak RNAP activity (∼9 mls) were ammonium sulfate precipitated and stored at 2 mg/ml in 307 glycerol at −80 °C. TheE. coli RNAP was purified using the above procedure from 2 liters of cells (∼10 g wet weight; strain TB1) which were grown aerobically for 17 h in Luria broth (24Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) at 37 °C. RNA polymerase from the Rifs R. capsulatus strain B10 (2 liters grown to mid exponential phase at 34 °C) was purified by PEG precipitation and heparin-agarose chromatography as described above. Proteins were quantitated by BCA assays (Pierce) and SDS-PAGE analysis using serial dilutions of 2 mg/ml bovine serum albumin as a control. The E. coli RNAP HPLC-purified RNAP/ς70 holoenzyme was provided by Dr. Robert Landick (University of Wisconsin, Madison). In vitro transcription assays using the T7A1 promoter were used to determine the fractions that contained RNAP/ς70 holoenzyme of the highest specific activity, which were used for subsequent experiments. Under these conditions, R. capsulatus RNAP was stable forin vitro transcription assays for at least 1 year with less than a 3-fold loss of activity. Dialysis was not performed as it resulted in a dramatic loss of holoenzyme activity and caused precipitation of R. capsulatus RNAP when at high concentrations (2 mg/ml). Separation of the core R. capsulatus RNAP from the R. capsulatusRNAP/ς70 was attempted with a Bio-Rex 70 column (Bio-Rad), but this caused dissociation of the R. capsulatusRNAP subunits, whereas the E. coli RNAP core was stable under these same conditions. We do not understand the basis for this difference. For R. capsulatus RNAP/ς70, initial elution fractions from the heparin-agarose column were enriched for ς70 activity and later fractions for core RNAP, as assayed by in vitro transcription reactions and by SDS-PAGE. Sonication of R. capsulatus cells followed by the above PEG and heparin-agarose purification also resulted in purification of core-enriched R. capsulatus RNAP and a loss of the ς70 subunit, as assayed by SDS-PAGE, Western analysis, and in vitro transcription assays. The heparin-agarose-purified E. coli RNAP contained a contaminating nuclease that was not present in R. capsulatusRNAP preparations, and therefore the DEAE-purified E. coliRNAP was always used, whereas the R. capsulatus RNAP heparin-agarose-purified fraction could be used for specific experiments, when noted. Samples that contained RNA polymerase were assayed for activity in reactions (20 ॖl) which contained 25 mm Tris-HCl, pH 8; 10 mmMgCl2; 1 mm EDTA; 0.25 mm ATP, CTP, GTP, and UTP, (fast protein liquid chromatography grade, Pharmacia Biotech Inc.); 0.8 ॖm [3H]UTP (NEN Life Science Products); 1 mm DTT; 150 mm NaCl; 250 ॖg/ml salmon sperm DNA; 250 ॖg/ml bovine serum albumin; 37 glycerol; and 1 mm K2HPO4 (to inhibit polynucleotide phosphorylase). For nonspecific templates, salmon sperm DNA was transcribed approximately 2-fold more efficiently than poly(dA-dT) DNA and so was used for these assays. Samples that contained RNAP (2.5 ॖl) were added to initiate the reactions, which were incubated at 30 °C for 15 min and terminated by spotting onto DE81 DEAE-cellulose filters (Whatman). The filters were washed five times in 57 sodium phosphate buffer, washed twice in water, and once in 957 ethanol to remove unincorporated label. Filters were counted in a Beckman liquid scintillation counter. One unit of RNA polymerase activity corresponds to 1 nmol of UTP incorporation in 15 min at 30 °C. The cell lysis solution, PEG precipitation fractions, column flow-through, wash, and elution fractions for the heparin-agarose and DEAE-Sepharose columns were assayed for RNAP activity to determine the most active fractions. In vitrotranscription reactions in 16 ॖl total (e.g. Ref. 25Popham D.L. Szeto D. Keener J. Kustu S. Science. 1989; 243: 629-635Crossref PubMed Scopus (315) Google Scholar) were performed in transcription buffer (50 mm Tris-HCl, pH 8, 100 mm potassium acetate, 8 mmMgCl2, 1 mm DTT, 3.57 PEG, and 1 ॖl of RNasin (Promega). The RNAP (40 nm) and DNA (approximately 40 nm for supercoiled templates and either 600 nm for linear templates or 3 nm for the T7A1 template) were incubated for 10 min at 24 °C in transcription buffer before the simultaneous addition of heparin (50 ॖg/ml) and nucleotide triphosphates (0.4 mm ATP, GTP, and UTP; 0.01 mm CTP; 0.5 ॖl of 25 ॖCi [α-32P]CTP, NEN Life Science Products). The reactions were incubated for 30 min at 24 °C, which was determined to be the optimal temperature forR. capsulatus RNAP. The reactions were terminated by the addition of 7 ॖl of stop solution (807 urea, 270 mmTris-HCl, pH 8, 270 mm boric acid, 6 mm EDTA, and 0.17 bromphenol blue), heated to 70 °C for 5 min, and loaded onto an 87 acrylamide sequencing gel. Radiolabeled DNA size markers were prepared as described (18Foster-Hartnett D. Cullen P.J. Monika E.M. Kranz R.G. J. Bacteriol. 1994; 176: 6175-6187Crossref PubMed Google Scholar). Western analysis was performed using peroxidase detection reagents from Pierce. Monoclonal antibody 2G10 was kindly provided by Dr. Richard Burgess and Nancy Thompson (University of Wisconsin, Madison). Antibodies to the R. capsulatus RNAP were generated by immunization of New Zealand White rabbits with the holoenzyme of greater than 957 purity. In vivo primer extension analysis was performed as described (26Foster-Hartnett D. Kranz R.G. Mol. Microbiol. 1992; 6: 1049-1060Crossref PubMed Scopus (42) Google Scholar). The primer 5′-TGTTGAATTCTTTTTCGCCCTTCGCGGCGT-3′ was used for the reverse transcription reaction for the cycA gene. The R. capsulatus RNAP was purified to characterize ς70 type promoters and to study activator- and repressor-regulated transcription in R. capsulatus. TheR. capsulatus RNAP protein was purified by PEG precipitation followed by heparin-agarose and DEAE-Sepharose chromatography to at least 957 homogeneity by SDS-PAGE analysis (Fig.1). The purification was assayed by nonspecific transcription assays (see below) and SDS-PAGE. Analysis of the R. capsulatus RNAP by SDS-PAGE showed subunits that by molecular weight correspond to an α, ॆ, ॆ′, and ω, as well as the 90-kDa major (housekeeping) ς70 (Fig. 1, lane 6). The E. coli RNA polymerase (E. coliRNAP, strain TB1) was purified using the same procedure (Fig. 1, seelanes 1–3), which was compared with the HPLC-purifiedE. coli RNAP/ς70 holoenzyme (Fig. 1,lane 7). A doublet at approximately 150 kDa was confirmed by SDS-PAGE analysis of underloaded DEAE-Sepharose pure R. capsulatus RNAP fraction; moreover, a Bio-Rex 70 column was also able to separate these two 150-kDa bands (data not shown). The ratio of intensity of the R. capsulatus RNAP α polypeptides to the ॆ and ॆ′ bands correspond to 2:1:1 stoichiometry, similar to theE. coli RNAP (Fig. 1, compare lane 3 withlane 6). An ω subunit occasionally purifies with the coreE. coli RNAP (27Gentry D.R. Burgess R.R. Gene (Amst.). 1986; 48: 33-40Crossref PubMed Scopus (29) Google Scholar) but has no known function in vivo (28Gentry D.R. Xiao H. Burgess R. Cashel M. J. Bacteriol. 1991; 173: 3901-3903Crossref PubMed Scopus (62) Google Scholar). The polypeptide observed by SDS-PAGE at approximately 15 kDa may be the R. capsulatus ω subunit homolog, but this has not been investigated further. The approximately 90-kDa polypeptide observed by SDS-PAGE in theR. capsulatus RNAP DEAE-Sepharose fraction (Fig. 1,lane 6) was shown to be the major (housekeeping) R. capsulatus ς70 factor. Monoclonal antibody 2G10 is specific for a 15-amino acid epitope in region 3.1 of E. coli ς70 (29Breyer M.J. Thompson N.E. Burgess R.R. J. Bacteriol. 1997; 179: 1404-1408Crossref PubMed Scopus (18) Google Scholar). 2G10 also cross-reacts with the major ς factor from a variety of bacterial species, including R. sphaeroides (4Karls R.K. Jin D.J. Donohue T. J. Bacteriol. 1993; 175: 7629-7638Crossref PubMed Google Scholar). Western analysis demonstrated that 2G10 cross-reacts with the 90-kDa subunit observed by SDS-PAGE analysis of the R. capsulatus RNAP purification fractions (not shown). Initially, transcriptional activities of R. capsulatus RNAP and E. coli RNAP enzymes at sequential purification steps were measured by a nonspecific transcription assay that determines the accumulation of RNA product using salmon sperm DNA as template and radiolabeled UTP (Table III). TheR. capsulatus RNAP purification resulted in a 140-fold enrichment (407 yield) of R. capsulatus RNAP and a total of 1.8 mg of greater than 957 pure protein from 12 liters of cells. TheE. coli RNAP purification resulted in a similar enrichment (150-fold) and a higher yield (757), which was comparable to results published previously (e.g. 26). Nonspecific transcription activity of R. capsulatus RNAP (as well as the E. coli RNAP) was dependent upon MgCl2, DNA template, and all four nucleotide triphosphates (not shown).Table IIISummary of RNA polymerase purification from R. capsulatus and E. coliStrainFractionVolumeTotal proteinTotal activity3-aFor nonspecific assays, 1 unit represents incorporation of 1 nmol of [3H]UTP in 15 min at 30 °C.Specific activityYieldFold purificationmlmg/mlunitsunits/mg7E. coli TB1PEG supernatant96.567911.6E. coliTB1Heparin-agarose31.42,31455110036E. coliTB1DEAE-Sepharose1.250.267252,23175150R. capsulatus SB1003PEG supernatant604.64,88517.7R. capsulatusSB1003Heparin-agarose151.18,64652410035R. capsulatusSB1003DEAE-Sepharose90.23,4691,92740140The starting material was 200 g of cells (wet weight) for R. capsulatus SB1003 and 10 g of cells (wet weight) for E. coli TB1. Each assay was repeated at least twice.3-a For nonspecific assays, 1 unit represents incorporation of 1 nmol of [3H]UTP in 15 min at 30 °C. Open table in a new tab The starting material was 200 g of cells (wet weight) for R. capsulatus SB1003 and 10 g of cells (wet weight) for E. coli TB1. Each assay was repeated at least twice. The antibiotic rifampicin specifically inhibits transcription of bacterial RNA polymerase by binding to the ॆ subunit (30Jin D.J. Gross C.A. J. Mol. Biol. 1988; 202: 45-58Crossref PubMed Scopus (542) Google Scholar, 31Severinov K. Soushko M. Goldfarb A. Nikiforov V. Mol. Gen. Genet. 1994; 244: 120-126Crossref PubMed Scopus (58) Google Scholar) of RNAP and preventing elongation of the nascent RNA chain past a few nucleotides. The traditional R. capsulatus strain that is often used for genetic studies, called SB1003, is a rifampicin-resistant (Rifr) derivative of strain B10 (32Marrs B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 971-973Crossref PubMed Scopus (186) Google Scholar). To confirm that the nonspecific transcription activity was caused by transcription by R. capsulatus RNAP, we characterized the RNAP from the Rifs R. capsulatus strain B10 and Rifr SB1003. The RNAP from R. capsulatusstrain B10 (B10RNAP) w
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