/ . Biochem., 78, 845-858 (1975)
Purification and Some Properties of DNA-dependent RNA Polymerase from an Extreme Thermophile, Thermus thermophilus HB8 Takayasu DATE,1 Koichi SUZUKI, and Kazutomo IMAHORI Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, January 24, 1975
DNA-dependent RNA polymerase [EC 2.7.7.6] was purified from the thermophile Thermus thermophilus HB8 and some properties were investigated. 1. Two kinds of the enzyme (enzymes A and B) were found and separated from each other by phosphocellulose column chromatography. Enzyme A could utilize various DNA's as templates, while enzyme B was active only when alternating copolymer of deoxyadenylic and deoxythymidylic acids (poly d(A-T)) was used as a template. The specific activities of enzymes A and B at 65° were 30,000 and 16,000, respectively, which are markedly higher than the value obtained with E, coli enzyme. 2. The enzyme A was extremely thermostable. The activity did not decrease even after incubation for 3 hr at 70°. The maximal rate of RNA synthesis was found at 70°. 3. Both polymerases were resistant to rifampicin but were markedly sensitive to streptolydigin. 4. Four kinds of subunits were found in the enzymes by sodium dodecylsulfate (SDS)- gel electrophoresis; subunit I (M.W. 42,000), II (M.W. 58,000), III (M.W. 140,000), and IV (M.W. 180,000). The subunit compositions of enzymes A and B were 2I-II-III-IV and 2I-III-IV, respectively. A part of enzyme B contained 1 mole of extra polypeptide (named X) of molecular weight 100,000.
Thermus thermophilus HB3, an extreme thermophile, isolated from a hot spring at Mine, Shizuoka-ken, Japan, can grow at high temperatures up to 85° (1). To elucidate the thermophily and the adaptability which enables it
to grow at such high temperatures, where most ordinary biological macromolecules are denatured, we have undertaken studies on the chemical and physical nature of the cell constituents. All the cell constituents of the or-
1
Present address: Department of Chemistry, Faculty of Pharmaceutical Science, University of Kanazawa, Takara-machi, Kanazawa 920. Abbreviations: poly d(A-T), alternating copolymer of deoxyadenylic and deoxythymidylic acids; poly dGdC, mixture of homopolymers of deoxyguanylic acid and deoxycytidylic acid; EDTA, ethylenediaminetetraacetic acid; TCA, trichloroacetic acid; SDS, sodium dodecylsulfate; pCMB, ^-oxymercuribenzoate; DTT, dithiothreitol. Vol. 78, No. 4, 1975
845
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T. DATE, K. SUZUKI, and K. IMAHORI
ganism so far examined, enzymes, ribosomes, tRNA, and DNA, were highly thermostable compared with the counterparts from mesophilic sources (1—5). Studies on DNA from the thermophilic bacteria by means of melting profiles and cesium chloride equilibrium centrifugation suggested that the GC content was very high (about 68 mole %) (5). It is therefore interesting to investigate the nature of the DNA-dependent RNA polymerase [EC 2.7.7.6] from T. thermophilus and its interaction with DNA molecules. Thermophilic RNA polymerase has been isolated from B. stearothermophilus and some properties have been examined (6~). The enzymes from T. thermophilus are generally more thermostable than the corresponding enzymes from B. stearothermophilus (7). It is therefore expected that RNA polymerase of T. thermophilus will be highly thermostable. Thus, the thermostability of the enzyme would also allow us to investigate the effect of temperature on the various steps involved in RNA synthesis over a wide range. For these reasons we tried to isolate RNA polymerase from the thermophilic bacteria. Two kinds of enzymes (A and B) were isolated. The enzyme A seemed to be a holoenzyme and was active on various DNA's. The enzyme B, however, was active only when poly d(A-T) was used as a template and the subunit composition suggested that it was a core enzyme of enzyme A. In the present paper the isolation of the two enzymes and some properties of enzyme A are described. The characterization of enzyme B is described in an accompanying paper (8). MATERIALS AND METHODS Materials—Radioactive nucleoside triphosphates were obtained from New England and Schwartz Bioresearch. Other nucleoside triphosphates, catalase [EC 1.11.1.6] and j8galactosidase [EC 3.2.1.23] were purchased from Boehringer. Protamine sulfate was obtained from Yukigosei Co. Spermine tetrahydrochloride, spermidine trihydrochloride, putrescine dihydrochloride, 2,5-diphenyloxazole,
and 1, 4-bis[2-(5-phenyloxalyl)]benzene were obtained from Nakarai Chemicals. Poly d(A-T) and a mixture of homopolymers of deoxyguanylic acid and deoxycytidylic acid (poly dGdC) were the products of Miles. Calf thymus DNA of high molecular weight was purchased from Sigma. DNA preparations from T4 and T7 bacteriophage were prepared as described by Thomas and Abelson (9). Single-stranded fl DNA was kindly provided by Dr. K. Shishido, Institute of Physical and Chemical Research, Wako-shi, Saitama-ken. Millipore filters (HAWP 0.45 ft) were purchased from Millipore Corporation. Sephadex G-200 and DEAE-Sephadex A-50 were obtained from Pharmacia. Phosphocellulose Pll was a product of Whatmann. Hydroxyapatite gel was prepared according to Tiselius et al. (10). Streptolydigin and rifampicin were kindly supplied by Dr. A. Ishihama, Institute for Virus Research, Kyoto University, and Dr. J. Ikeda, Institute of Applied Microbiology, the University of Tokyo, respectively. DNA-dependent RNA polymerase from E. coli A19 was prepared by the procedure of Chamberlin and Berg (11) with slight modifications and was further purified by glycerol density gradient centrifugation. Other reagents were of the highest purity obtainable. Methods — Assay of E. coli RNA polymerase: The reaction mixture (0.1 ml) contained 0.05 M Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM disodium ethylenediaminetetraacetate dihydrate (EDTA), 10 mM 2-mercaptoethanol, •10—15 fig of calf thymus DNA, 0.4 mM each of ATP, GTP, CTP, and [3H]UTP (1-2 mCi/ mmole), 20 fig of bovine serum albumin and 5 fig of enzyme. The reaction was initiated by adding enzyme solution and was terminated by the addition of 1.5 ml of ice-cold 5% trichloroacetic acid (TCA) after incubation for 10 min at 37°. After 30 min, the resulting precipitates were collected on a Millipore filter and washed well with ice-cold 5% TCA. The filters were immersed in a scintillation fluid and the radioactivities were counted with a Hitachi-Horiba scintillation counter, type LS-500. The scintillation fluid contained 4 g of 2, 5-diphenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxalyl)]benzene in 1 liter of toluene. / . Biochem.
DNA-DEPENDENT RNA POLYMERASE FROM THERMOPHILIC BACTERIA
847
Assay of T. thermophilus RNA polymerase, enzyme A: The standard assay mixture (0.10 ml) contained 0.05 M glycine-NaOH (pH 8.2), 10 mM MgCl2) 2 mM MnCl2) 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 100 mM KC1, 0.4 mM each of ATP, GTP, and CTP, 0.4 mM [3H]UTP (1-2 mCi/mmole), and 4 fig of calf thymus DNA. After incubation for 5 min at 65° the reaction mixture was chilled in ice-cold 5% TCA. The precipitates were collected on a Millipore filter, washed and counted as described above. One unit of RNA polymerase activity is denned as the amount of protein which catalyzes the incorporation of 1 nmole of UMP into RNA in 60 min at 65° under the conditions described above.
layered on the gradient and the tubes were centrifuged in an RPS40 rotor with a Hitachi 55P centrifuge for 9 hr at 40,000 rpm at 4°. After centrifugation the tubes were punctured and 28 fractions were collected. Appropriate portions of the fractions were withdrawn and assayed. /3-Galactosidase and catalase were used as markers. The assays of catalase and j9-galactosidase were carried out essentially as described by Beers and Sizer (75) and Maruo et al. (16), respectively. Other Methods—The amount of protein was calculated from the absorbance at 280 and 260 nm according to Warburg and Christian (77). The concentration of DNA was determined using an extinction coefficient of
Enzyme B: The same procedure was used as described for enzyme A, except that 4 fig of poly d(A-T) was used in place of calf thymus DNA, and that GTP and CTP were omitted. Gel electrophoresis—Electrophoresis was carried out in 7.5% acrylamide gels at pH 8.5— 9.0 as described by Ornstein (72) and Davis (73). A sample solution (0.2 ml) containing about 50 fig of protein was applied to each gel (0.6x8.0 cm). After electrophoresis (3 ma per tube for 70 min) gels were stained for at least 2 hr in 0.2% Coomassie brilliant blue in methanol-acetic acid-water mixture ( 5 : 1 : 5 , v/v), and destained overnight in the same methanol-acetic acid-water mixture. SDS-polyacrylamide gel electrophoresis was performed in 7.5% gels according to the procedure of Weber and Osborn (14). The enzyme (5—30 fig) was preincubated in 0.1 M sodium phosphate, pH 7.2, containing 0.1% SDS, 2% 2-mercaptoethanol, and 5% glycerol for 3 hr at 37°. The mixtures were layered on 7.5% SDSpolyacrylamide gels (0.6x8 cm) and electrophoresis was performed for 4 hr at 8 ma per gel. Staining and destaining were carried out as described above. Destained gels were scanned on a Gilford 240 spectrophotometer fitted with a Hitachi QPD73 recorder and a Gilford gel-scanning attachment, model 2410. Glycerol Gradient Sedimentation—A 10— 30% glycerol gradient containing 0.04 M TrisHC1, pH 8.0, 10 mM MgCl2, 0.1 mM DTT, 0.1 mM EDTA, and 0.1 M KC1 was used. Enzyme solution (30—100 fig of protein in 100 fi\) was
Purification of DNA-dependent RNA Polymerase from T. thermophilus—Unless otherwise stated, all the operations were carried out at 4° and all centrifugation steps were-at 15,000 rpm for 15 min in a Tominaga UV90 centrifuge. Cells: Thermus thermophilus (strain HB8) was grown in a medium described previously (2). The cells were harvested in the late log phase and stored at —20°. Preparations of extracts: Frozen cells (200 g) were suspended in 210 ml of buffer A (10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT). The cells were disrupted in an Ohtake French Press at 200 kg/ cm2. The temperature was kept below 10° by cooling the container with ice. The homogenate was first centrifuged at 6,000xg for 15 min and the supernatant was then centrifuged in a Hitachi 65P centrifuge for 4 hr at 100,000 x g . The resulting supernatant was pooled (high-speed supernatant; Fraction 1). Protamine sulfate treatment: Fraction 1 was diluted with buffer A to bring its absorbance at 280 nm to 30. One percent protamine sulfate solution (24 ml) was added dropwise to the diluted solution (250 ml) with gentle stirring. The precipitate was collected by centrifugation after 15 min. The pellet was extracted with 50 ml of buffer A containing 0.05 M ammonium sulfate in a Potter-Elvehjem homogenizer. The milky solution was centrifuged and the supernatant was pooled (Fraction 2).
Vol. 78, No. 4, 1975
T. DATE, K. SUZUKI, and K. IMAHORI
848
DEAE-Sephadex column chromatography: Fraction 2 was applied to a DEAE-Sephadex A-50 column (2.4x25 cm) which had been equilibrated with buffer A containing 0.1 M KCl. The column was first washed with 100 ml of buffer A containing 0.16 M KCl and then eluted with a linear salt gradient of KCl from 0.16 to 0.50 M in buffer A (total volume: 500 ml). The flow rate was 50 ml per hr. The enzyme was eluted at KCl concentrations between 0.22 and 0.25 M. Fractions containing RNA polymerase activity were combined (Fraction 3). Hydroxyapatite column chromatography: Solid ammonium sulfate was added to fraction 3 to 40% saturation (20 g ammonium sulfate per 100 ml of solution). The pH of the solution was maintained at 7.5 by addition of 1 N NaOH. After 30 min the solution was centrifuged. The supernatant solution was adjusted to 55% saturation by further addition of solid ammonium sulfate (8 g per 100 ml). After 30 min the precipitate was collected by centrifugation at 25,000 rpm for 30 min. The pellet was extracted successively with 15 ml of 0.005 M phosphate buffer, pH 7.2, containing 0.1 mM DTT and was passed through a hydroxyapatite
a.
0.6
i
i
05
I
column (1.6x14 cm) at a flow rate of 5 ml/hr. The enzyme was then eluted from the column using a gradient from 0.005 to 0.4 M phosphate buffer containing 0.1 mM DTT (total volume: 140 ml) at a flow rate of 10 ml/hr. The enzyme began to elute at about 0.07 M phosphate (Fraction 4). Sephadex G-200 gel filtration: Solid ammonium sulfate was added to fraction 4 to 60% saturation. The precipitate was collected, dissolved in the smallest possible volume of 0.04 M Tris-HCl buffer, pH 7.4, containing 0.1 mM DTT and loaded on top of a column (2.5x70 cm), then the column was developed with buffer B (0.04 M Tris-HCl, pH 7.4, 0.1 mM DTT, 5% glycerol) containing 0.05 M KCl. The active fractions were pooled (Fraction 5). Phosphocellulose column chromatography: The pooled fraction (Fraction 5) was placed on a column of phosphocellulose (1.2x12 cm) which had been well-equilibrated with buffer B containing 0.05 M KCl. Addition of glycerol and a slow flow rate were used to minimize elution of the enzyme in the washing process. The column was then developed with 20 ml of buffer B containing 0.05 M KCl. The en-
B
0.4
A
0.3
^^
m
Purification and Some Properties of DNA-dependent RNA Polymerase from an Extreme Thermophile, Thermus thermophilus HB8 Takayasu DATE,1 Koichi SUZUKI, and Kazutomo IMAHORI Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, January 24, 1975
DNA-dependent RNA polymerase [EC 2.7.7.6] was purified from the thermophile Thermus thermophilus HB8 and some properties were investigated. 1. Two kinds of the enzyme (enzymes A and B) were found and separated from each other by phosphocellulose column chromatography. Enzyme A could utilize various DNA's as templates, while enzyme B was active only when alternating copolymer of deoxyadenylic and deoxythymidylic acids (poly d(A-T)) was used as a template. The specific activities of enzymes A and B at 65° were 30,000 and 16,000, respectively, which are markedly higher than the value obtained with E, coli enzyme. 2. The enzyme A was extremely thermostable. The activity did not decrease even after incubation for 3 hr at 70°. The maximal rate of RNA synthesis was found at 70°. 3. Both polymerases were resistant to rifampicin but were markedly sensitive to streptolydigin. 4. Four kinds of subunits were found in the enzymes by sodium dodecylsulfate (SDS)- gel electrophoresis; subunit I (M.W. 42,000), II (M.W. 58,000), III (M.W. 140,000), and IV (M.W. 180,000). The subunit compositions of enzymes A and B were 2I-II-III-IV and 2I-III-IV, respectively. A part of enzyme B contained 1 mole of extra polypeptide (named X) of molecular weight 100,000.
Thermus thermophilus HB3, an extreme thermophile, isolated from a hot spring at Mine, Shizuoka-ken, Japan, can grow at high temperatures up to 85° (1). To elucidate the thermophily and the adaptability which enables it
to grow at such high temperatures, where most ordinary biological macromolecules are denatured, we have undertaken studies on the chemical and physical nature of the cell constituents. All the cell constituents of the or-
1
Present address: Department of Chemistry, Faculty of Pharmaceutical Science, University of Kanazawa, Takara-machi, Kanazawa 920. Abbreviations: poly d(A-T), alternating copolymer of deoxyadenylic and deoxythymidylic acids; poly dGdC, mixture of homopolymers of deoxyguanylic acid and deoxycytidylic acid; EDTA, ethylenediaminetetraacetic acid; TCA, trichloroacetic acid; SDS, sodium dodecylsulfate; pCMB, ^-oxymercuribenzoate; DTT, dithiothreitol. Vol. 78, No. 4, 1975
845
846
T. DATE, K. SUZUKI, and K. IMAHORI
ganism so far examined, enzymes, ribosomes, tRNA, and DNA, were highly thermostable compared with the counterparts from mesophilic sources (1—5). Studies on DNA from the thermophilic bacteria by means of melting profiles and cesium chloride equilibrium centrifugation suggested that the GC content was very high (about 68 mole %) (5). It is therefore interesting to investigate the nature of the DNA-dependent RNA polymerase [EC 2.7.7.6] from T. thermophilus and its interaction with DNA molecules. Thermophilic RNA polymerase has been isolated from B. stearothermophilus and some properties have been examined (6~). The enzymes from T. thermophilus are generally more thermostable than the corresponding enzymes from B. stearothermophilus (7). It is therefore expected that RNA polymerase of T. thermophilus will be highly thermostable. Thus, the thermostability of the enzyme would also allow us to investigate the effect of temperature on the various steps involved in RNA synthesis over a wide range. For these reasons we tried to isolate RNA polymerase from the thermophilic bacteria. Two kinds of enzymes (A and B) were isolated. The enzyme A seemed to be a holoenzyme and was active on various DNA's. The enzyme B, however, was active only when poly d(A-T) was used as a template and the subunit composition suggested that it was a core enzyme of enzyme A. In the present paper the isolation of the two enzymes and some properties of enzyme A are described. The characterization of enzyme B is described in an accompanying paper (8). MATERIALS AND METHODS Materials—Radioactive nucleoside triphosphates were obtained from New England and Schwartz Bioresearch. Other nucleoside triphosphates, catalase [EC 1.11.1.6] and j8galactosidase [EC 3.2.1.23] were purchased from Boehringer. Protamine sulfate was obtained from Yukigosei Co. Spermine tetrahydrochloride, spermidine trihydrochloride, putrescine dihydrochloride, 2,5-diphenyloxazole,
and 1, 4-bis[2-(5-phenyloxalyl)]benzene were obtained from Nakarai Chemicals. Poly d(A-T) and a mixture of homopolymers of deoxyguanylic acid and deoxycytidylic acid (poly dGdC) were the products of Miles. Calf thymus DNA of high molecular weight was purchased from Sigma. DNA preparations from T4 and T7 bacteriophage were prepared as described by Thomas and Abelson (9). Single-stranded fl DNA was kindly provided by Dr. K. Shishido, Institute of Physical and Chemical Research, Wako-shi, Saitama-ken. Millipore filters (HAWP 0.45 ft) were purchased from Millipore Corporation. Sephadex G-200 and DEAE-Sephadex A-50 were obtained from Pharmacia. Phosphocellulose Pll was a product of Whatmann. Hydroxyapatite gel was prepared according to Tiselius et al. (10). Streptolydigin and rifampicin were kindly supplied by Dr. A. Ishihama, Institute for Virus Research, Kyoto University, and Dr. J. Ikeda, Institute of Applied Microbiology, the University of Tokyo, respectively. DNA-dependent RNA polymerase from E. coli A19 was prepared by the procedure of Chamberlin and Berg (11) with slight modifications and was further purified by glycerol density gradient centrifugation. Other reagents were of the highest purity obtainable. Methods — Assay of E. coli RNA polymerase: The reaction mixture (0.1 ml) contained 0.05 M Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM disodium ethylenediaminetetraacetate dihydrate (EDTA), 10 mM 2-mercaptoethanol, •10—15 fig of calf thymus DNA, 0.4 mM each of ATP, GTP, CTP, and [3H]UTP (1-2 mCi/ mmole), 20 fig of bovine serum albumin and 5 fig of enzyme. The reaction was initiated by adding enzyme solution and was terminated by the addition of 1.5 ml of ice-cold 5% trichloroacetic acid (TCA) after incubation for 10 min at 37°. After 30 min, the resulting precipitates were collected on a Millipore filter and washed well with ice-cold 5% TCA. The filters were immersed in a scintillation fluid and the radioactivities were counted with a Hitachi-Horiba scintillation counter, type LS-500. The scintillation fluid contained 4 g of 2, 5-diphenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxalyl)]benzene in 1 liter of toluene. / . Biochem.
DNA-DEPENDENT RNA POLYMERASE FROM THERMOPHILIC BACTERIA
847
Assay of T. thermophilus RNA polymerase, enzyme A: The standard assay mixture (0.10 ml) contained 0.05 M glycine-NaOH (pH 8.2), 10 mM MgCl2) 2 mM MnCl2) 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 100 mM KC1, 0.4 mM each of ATP, GTP, and CTP, 0.4 mM [3H]UTP (1-2 mCi/mmole), and 4 fig of calf thymus DNA. After incubation for 5 min at 65° the reaction mixture was chilled in ice-cold 5% TCA. The precipitates were collected on a Millipore filter, washed and counted as described above. One unit of RNA polymerase activity is denned as the amount of protein which catalyzes the incorporation of 1 nmole of UMP into RNA in 60 min at 65° under the conditions described above.
layered on the gradient and the tubes were centrifuged in an RPS40 rotor with a Hitachi 55P centrifuge for 9 hr at 40,000 rpm at 4°. After centrifugation the tubes were punctured and 28 fractions were collected. Appropriate portions of the fractions were withdrawn and assayed. /3-Galactosidase and catalase were used as markers. The assays of catalase and j9-galactosidase were carried out essentially as described by Beers and Sizer (75) and Maruo et al. (16), respectively. Other Methods—The amount of protein was calculated from the absorbance at 280 and 260 nm according to Warburg and Christian (77). The concentration of DNA was determined using an extinction coefficient of
Enzyme B: The same procedure was used as described for enzyme A, except that 4 fig of poly d(A-T) was used in place of calf thymus DNA, and that GTP and CTP were omitted. Gel electrophoresis—Electrophoresis was carried out in 7.5% acrylamide gels at pH 8.5— 9.0 as described by Ornstein (72) and Davis (73). A sample solution (0.2 ml) containing about 50 fig of protein was applied to each gel (0.6x8.0 cm). After electrophoresis (3 ma per tube for 70 min) gels were stained for at least 2 hr in 0.2% Coomassie brilliant blue in methanol-acetic acid-water mixture ( 5 : 1 : 5 , v/v), and destained overnight in the same methanol-acetic acid-water mixture. SDS-polyacrylamide gel electrophoresis was performed in 7.5% gels according to the procedure of Weber and Osborn (14). The enzyme (5—30 fig) was preincubated in 0.1 M sodium phosphate, pH 7.2, containing 0.1% SDS, 2% 2-mercaptoethanol, and 5% glycerol for 3 hr at 37°. The mixtures were layered on 7.5% SDSpolyacrylamide gels (0.6x8 cm) and electrophoresis was performed for 4 hr at 8 ma per gel. Staining and destaining were carried out as described above. Destained gels were scanned on a Gilford 240 spectrophotometer fitted with a Hitachi QPD73 recorder and a Gilford gel-scanning attachment, model 2410. Glycerol Gradient Sedimentation—A 10— 30% glycerol gradient containing 0.04 M TrisHC1, pH 8.0, 10 mM MgCl2, 0.1 mM DTT, 0.1 mM EDTA, and 0.1 M KC1 was used. Enzyme solution (30—100 fig of protein in 100 fi\) was
Purification of DNA-dependent RNA Polymerase from T. thermophilus—Unless otherwise stated, all the operations were carried out at 4° and all centrifugation steps were-at 15,000 rpm for 15 min in a Tominaga UV90 centrifuge. Cells: Thermus thermophilus (strain HB8) was grown in a medium described previously (2). The cells were harvested in the late log phase and stored at —20°. Preparations of extracts: Frozen cells (200 g) were suspended in 210 ml of buffer A (10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT). The cells were disrupted in an Ohtake French Press at 200 kg/ cm2. The temperature was kept below 10° by cooling the container with ice. The homogenate was first centrifuged at 6,000xg for 15 min and the supernatant was then centrifuged in a Hitachi 65P centrifuge for 4 hr at 100,000 x g . The resulting supernatant was pooled (high-speed supernatant; Fraction 1). Protamine sulfate treatment: Fraction 1 was diluted with buffer A to bring its absorbance at 280 nm to 30. One percent protamine sulfate solution (24 ml) was added dropwise to the diluted solution (250 ml) with gentle stirring. The precipitate was collected by centrifugation after 15 min. The pellet was extracted with 50 ml of buffer A containing 0.05 M ammonium sulfate in a Potter-Elvehjem homogenizer. The milky solution was centrifuged and the supernatant was pooled (Fraction 2).
Vol. 78, No. 4, 1975
T. DATE, K. SUZUKI, and K. IMAHORI
848
DEAE-Sephadex column chromatography: Fraction 2 was applied to a DEAE-Sephadex A-50 column (2.4x25 cm) which had been equilibrated with buffer A containing 0.1 M KCl. The column was first washed with 100 ml of buffer A containing 0.16 M KCl and then eluted with a linear salt gradient of KCl from 0.16 to 0.50 M in buffer A (total volume: 500 ml). The flow rate was 50 ml per hr. The enzyme was eluted at KCl concentrations between 0.22 and 0.25 M. Fractions containing RNA polymerase activity were combined (Fraction 3). Hydroxyapatite column chromatography: Solid ammonium sulfate was added to fraction 3 to 40% saturation (20 g ammonium sulfate per 100 ml of solution). The pH of the solution was maintained at 7.5 by addition of 1 N NaOH. After 30 min the solution was centrifuged. The supernatant solution was adjusted to 55% saturation by further addition of solid ammonium sulfate (8 g per 100 ml). After 30 min the precipitate was collected by centrifugation at 25,000 rpm for 30 min. The pellet was extracted successively with 15 ml of 0.005 M phosphate buffer, pH 7.2, containing 0.1 mM DTT and was passed through a hydroxyapatite
a.
0.6
i
i
05
I
column (1.6x14 cm) at a flow rate of 5 ml/hr. The enzyme was then eluted from the column using a gradient from 0.005 to 0.4 M phosphate buffer containing 0.1 mM DTT (total volume: 140 ml) at a flow rate of 10 ml/hr. The enzyme began to elute at about 0.07 M phosphate (Fraction 4). Sephadex G-200 gel filtration: Solid ammonium sulfate was added to fraction 4 to 60% saturation. The precipitate was collected, dissolved in the smallest possible volume of 0.04 M Tris-HCl buffer, pH 7.4, containing 0.1 mM DTT and loaded on top of a column (2.5x70 cm), then the column was developed with buffer B (0.04 M Tris-HCl, pH 7.4, 0.1 mM DTT, 5% glycerol) containing 0.05 M KCl. The active fractions were pooled (Fraction 5). Phosphocellulose column chromatography: The pooled fraction (Fraction 5) was placed on a column of phosphocellulose (1.2x12 cm) which had been well-equilibrated with buffer B containing 0.05 M KCl. Addition of glycerol and a slow flow rate were used to minimize elution of the enzyme in the washing process. The column was then developed with 20 ml of buffer B containing 0.05 M KCl. The en-
B
0.4
A
0.3
^^
m
