PURIE‘ECATECN ANS C§*%'ARACTEREATEON 0F DN‘A‘fifl-‘EHBEi‘éT Rh‘fi‘ POLYMERASE F530 M figfiUfiOé‘sfi 4A3 PUTIDA A. 3. 12 Thesis far the Degree of M. S. MECHEGI =3? STATE UNIVERSiTY JAM-ES CARL 10HP€30N 19.87 ‘4‘ 4 173543 -‘ A‘anmn- Q. J L IF R A R Y Michigan State University ABSTRACT wsxncuion AND CHARACTERIZATION or DNA-DEPENDENT RNA POLYMEBASE mom 3 ouou 3 mpg; A.3.12 by James Carl Johnson The purification of DNA-dependent RNA polymerase has been described. The enzyme has been purified to a specific activity of 5, 590 up moles of CTP converted to a 'i‘CA insoluble fora per hour per m3 of protein. The increase in specific activity was 160 fold over that of the Initial Extract and the recovery of activity was 20$. The enzyme could be stored at -196°c for several months without detectable losses of activity. The synthesis of RNA by RNA polynerase from 2. punti‘dg was absolutely DNA dependent. The reaction exhibited a broad pH Optimum between 8 and 9. RNA synthesis did not occur below pH 7. No RNA synthesis was observed in the absence of divalent cations. as” and/or Hn'H' stimulate the synthesis of RNA and maximum stimulation was observed when both cations were present. when assayed at either high substrate concentrations or high DNA concentrations, an inhibition of RNA synthesis was observed. When assayed at temperatures below 30°C, the synthesis of RNA was observed to have a lag phase which was dininidied by preincubating the enzyme with DNA. James Carl Johnson A.new and unique assay for the pyrophosphate formed by the catalytic action of RNA.polynerase during RNA synthesis was develOped. The pyrophosphate linked formation of NADPE was observed in a coupled reaction which involved the enzymes ‘uridinediphosphoglucose perphosphorylase, phosphoglucomutase and glucose-l-phosphate dehydrogenase. It was established that the formation of perphosphate as observed by this assay was catalyzed by DNApdependent RNA polymerase. PURIFICATION AND CHARACTERIZATION OF DNA-DEPENDENCE RNA POLIMERASE FROM ESEUDOfiOEAS 1111'ng A.3.12 By James Carl Johnson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1967 DEDICATION To Phyl ACKNOWLEDGMENTS The author wishes to express his words of gratitude and recognition of Dr. John A. Boezi whose guidance and counsel have been invaluable during the course of this study. I wish to thank Dr. R. G. Hansen and 8am Bass for the gift of the UDPG-perphosphorylase and the advice on its use. words of thanks are also given to Dr. Lucy F. Lee, Dr. R. L. Amstrong, Ken Payne and Gary Gerard for helpful discussions and to Mrs. M. DeBacker for technical assistance. I also wish to recognize the Department of Biochemistry of Michigan State University and the National Institutes of Health from which financial support was received. 11 TABLE OF CONTENTS INTRODUCTION................... NATERIALSANDNETRODS............... Growth of Pseudomonas 222125 A.3.12 . . . . . Growth and Purification of Bacteriophage gh-l Growth and Purification of BacteriOphage T4 . Growth and Purification of [we] Thymidine “bald ButOrlOphase T7 0 e e e e e e e 0 Purification of Bacteriophage DNA . . . . . . mriflcatlon of as Butida Aeaelz DNA. e e e e Preparation of PR] Uracil labeled RNA . . . Determination of DNA, RNA and Protein Concentrations.............. Procedure for the Measurement of Radioactivity. TCA Insoluble Radioactivity. . . . . . . TCA Soluble Radioactivity . . . . . . . Radioactive Assay of RNA Polymerase . . . . . Spectrophotometric Assay of RNA Polymerase. . Method for the Assay of Deoxyribonuclease . . Method for the Assay of Ribonuclease. . . . . Characterization of the RNA Synthesized by RNA Polymerase................ General Methods and Materials . . . . . . . . Chromatography “[38] CTP. . . . . . . . . . Purification of DNA-Dependent RNA Polymerase. Initilextract ............ High 3p,ed Supernatant Fraction. . . . . iii Page \Immml" (D 10 fit: 12 12 13 15 15 16 18 19 20 20 20 TABLE OF CONTENTS (continued) Streptomycin Precipitate. . . . . . 30-60% Ammonium.Sulfate Fraction I. Peak DEAR-Cellulose Fraction. . . . 60% Ammonium Sulfate Fraction II. . uwms................... Summary of the Purification Procedure. . Preperties of 60% Ammonium.Su1fate Fraction II Comments Concerning the Purification Procedure Kinetics of the Synthesis of RNA by DNAP Depmdent “A Palmerase. e e e e e e e e 0 Effect of RNA Polymerase Concentration on Rate Reaction.................... of Effect of DNA Concentration on Rate of Reaction. . EffOOtOprOnBNAsynthelifleeeeeeeeeee Effect Of Tmporatur. on RNA mthe818 e e e e e 0 Effect of Mg++ and Nn++'Concentration on RNA SMthflaiseeeeeeeeeeeeeeeeeee Effect of Substrate Concentration on RNA. synthesiBeeeeeeeeeeeeeeeeeee Effect of various DNA Templates on RNA Synthesis . Kinetics of Pyrophosphate Formation by RNA Polymerase................... Effect of RNA Polymerase Concentration on the Rate of Pyrophcsphate Formation. . . . . . . . . . . Effect of Inhibitors, RNase or DNase on Perphos- phatel'ormation................ Comparison of the Radioactive Assay and the Spectrophotometric Assay. e e e e e e e e 0 Characterization of the am Synthesized by mu Palmerase................. mmon O O O O O O O O O O O O O O O O O O O O 0 mass. 0 O O O O O O O O O O O O O O C O O O 0 iv Page 20 21 22 22 23 23 26 27 30 30 30 37 37 #2 #2 42 42 51 51 51 6O 62 69 Table I. II. III. V. VI. VII. LIST OF TABLES Page Summary of the Purification Procedure . . . . . 25 Effect of pH on the Rate of GNP Incorporation . 39 Effect of Mg“ and Mn” Concentration on the BateofCHPInoorporation........... 44 Effect of Sibstrate Concentration on the Rate ofCHPIneorporation.............. “6 Effect of Various DNA Templates on the Rate of ' synthOflBOfBNAeeeeeeseeeeeeeee “8 Effect of Ribonuclease Concentration on the Rate or Perphosphate POMtion e e e e e e e e e e 5? Characterization of the RNA Synthesized by DNA- Dependent RNA Polymerase. . . . . . . . . . . . 61 L181| OF FIGURES Figure Page 1. 2. 3. ll. 5. 6. 7. 8. 9. Chromatography of 30- 60% Anonium Silfate Mtionleeeeeeeeeeeeeeeeeee 29 Kinetics of the Synthesis of RNA by DNA- 9013011491113 RNA POI-”01333.0 e e e e e e e e e e e 32 ma Polymerase Concentration Verms Rate of CHPInoorporatlon..........e.... 3" Effect of gh-l DNA Concentration on the Rate of CMPInoorporation............... 36 Effect of Temperature on Rate of GNP Incorporation 41 Kinetics of Pyrophosphate Fanation by RNA P01n9r3”eeeeeeeeeeeeeeeeee 50 Effect of RNA Polymerase Concentration on the Bit. of Perphosphate ”mtion e e e e e e e e 53 Effect of Inhibitors, RNase or DNase on PINPhOBPH‘DCPOthOneeeeeeeeeeee 55 Comparison of the Formation of Pyrophc sphate as Measured by the Spectrophotometric Assay mth0m1030t170A8meeeeeeeeeee 59 vi m INTRODUCTION Bacterial RNA synthesis has been one of the general areas of interest in this laboratory. An avenue of approach to the investigation of RNA metabolize is the study of the enzymes which are responsible for the synthesis and modifica- tion of RNA. An inpcrtant enzyme which catalyzes the incorporation of nucleoside monophosphates into RNA is DNA-dependent RNA polymerase. ATP or? 143'” and/or an“ A mm + H, GTP DNA Template ’ 1 UT? me present study presents the results of the purifica- tion and characterization of the partially purified DNA- dependent RNA polymerase from g. LEE-.22 A.3.12. The results presented here do not represent a complete study of the most highly purified enzyme possible, but are only a partial characterization of the enzyme at the level of purification achieved to this time. A more complete study of the physical and chemical characteristics of the enzyme awaits further purification. DNA-dependent RNA polymerase has been purified from many sources (see Bloom, 1965). The most extensive purifica- tions have been*perfcned with extracts of gicrococgus ufleiktus and Escherichia ccii (Chamberlin and Berg, 1962; 1 2 Furth, Rurwitz and Anders, 1962; Nakuoto, Fox and Noise, 1961!; Stevens and Henry, 1961+; Zillig, Fuchs and Nillette, 1966). The specific activity of the most highly purified fraction from bacterial sources has reached 30 )1 moles of nucleotide polymerized per hour per mg of protein. RNA synthesis occurs in five steps}. (1) attachment of the enzyme to specific sites on the DNA template (2) initiation: (3) polymerization (it) termination of RNA synthesis at specific sites (5) release of necent RNA and enzyme from the template The results obtained from studies performed with the bacterial enzymes have brought about some understanding of the first three steps. Nith regard to steps 1, 2 and 3, the following sequences of reactions can be written: (1) attachment DNA + enzyme g 1‘ [DNk-onznél The binding of RNA polymerase to DNA is reversible. Binding occurs at specific sites on the DNA duplex. For coliphage T7 DNA there are approximately 50 binding sites for the g. 22}; :RNA polymerase. The binding of the enzyme to DNA which oceurs at low ionic strengths is inhibited at high ionic strengths (Berg, Kornberg, Fanoher, and Dieckmann, 1965; Crawford, tCrawford, Richardson and Slayter, 1965; Jones and Berg, 1966; Richardson, 1966; Stetnberger and Stevens, 1966; Stead and Jones, 1967). (2) initiation [DNA-enzyme] + purine nucleo side tripho sphate ——-) [DNA- enzyme-pppPu] me DNA-enzyme complex reacts preferentially with purine nucleoside triphosphates to form a complex which is stable in solutions of high ionic strength. The exact nature of the complex has not been establimed. (Anthony, Zeszotek and Goldthwait, 1966). ( 3 ) polymerization [DNA-enzyme-pppPu] + NTP -_-——-) DNA-enzyme-pppPu(pN )n + PP, where NTP stands for ATP, GTP, CTP or UTP. Polymerization occurs by the sequential addition of one ' nucleoside monophosphate to the 3/ end of the growing RNA chain (Bremer, Konrad, Gaines and Stent, 1965; Naitra and Rurwitz, 1965). The sequence of ribonucleotides incorporated into RNA is determined by the DNA strand which is transcribed. Transcription of DNA in antic with several DNA species has been town to be asymetric. with bacteriophage oi DNA and with the duplex derivative of 33X 17“ DNA, asymetric transcrip- tion involves transcription of one strand exclusively. The strand that is copied in £115.29. is the same strand that is copied .15 _v_i_zg (Tocchint-Valentini, Stodolsky, Aurisicchio, ”at, Graziosi, Noise and Geiduschek, 1963; Rayamii, . Rayadii and Spiegelmsn, 1963). with bacteriOPheso >\DNA, transcription is also asymetric; however, portions of each A complementary strand are copied in 21:32 and in zi_v_g (Cohen and Rurwitz, 196?; Taylor, Rradecna, and Szybalski, 1967). Nith bacteriOphage A DNA as a template for the g. 293;; enzyme, transcription is further limited to the AT-rich half of the DNA duplex. In LEV—0.9 the AT-rich half programs the synthesis of early messenger RNA (Nano and Gros, 1966; Cohen, Maitra and Rurwitz, 1967). Transcription of Ta DNA is also limited to the portion of the molecule which in 3113 programs the synthesis of early messenger RNA (Geiduschek, Snyder, Conill and Sarnat, 1966). In fig studies with the highly purified bacterial enzyme have not led to an understanding of the mechanim involved in steps I) and 5. RNA, synthesized in _v_i_t__r9_ on T1; or T7 DNA templates by the _E_. as}; RNA polymerase, remains attached to the template and enzyme (Bremer and Konrad, 1964; Richardson, 1966). Few, if any, RNA molecules are released from the complex during the course of the reaction. The relatively rapid decrease in the rate of RNA synthesis has been attributed to an interaction between the necent RNA and the enzyme. Stent (196“ has suggested that ribosomes are involved in an active process by which ascent RNA is released from the DNA-enzyme-RNA complex. The most highly purified preparations of RNA polymerase have been characterized with respect to the physical properties of the enzyme. The E. 321;; polymerase has a sedimentation coefficient of 21 when measured in buffers of low ionic 5 strength and 13 when measured at high ionic strengths (Richardson, 1966). The molecular weight of the 21 8 species was estimated to be 8.8 x 105 and that of the 13 3 species to be 4.1!» x 105. However, in buffers of intermediate ionic strength, a variety of forms exist with sedimentation coefficients of lb, 16 and 19.5 S (Stevens, hery, and Sternberger, 1966). Electron micrographs of the g. 99;; RNA polymerase have remlted in a model showing the enzyme to consist of six cylindrical subunits surrounding a hollow core in a hexagonal array forming a short hollow cylinder with dimensions of 125 I in diameter and 95 I in length (Zillig, Fuchs and Millette, 1966). N MATERIALS AND METHODS Growth of Pseudomoggs putida A4012 DNA-dependent RNA polymerase was extracted from Pseudomonas 222-2 A.3.12 which was grown in a Fermacell, model F-130, 130 liter batch fermentor. The growth media contained the following in grams per liter: yeast extract, 5; glucose, 8; NaCl, 8; (NRb)2RP0u, 6; mayo“, 3; NgSOu’7R20, l; and FeClB, 0.005. A 6 to 8 liter innoculum of an over- night culture was added to 100 liters of sterile media. The temperature of growth was 33°C. The sreation rate was 6 to 8 cubic feet per minute, and stirring was maintained at 300 rev/min. Cell density was measured at 660 uni. An optical density of 1.5 units/ml was equivalent to a cell density of 5 x 108 cells/ml. The doubling time for 2. 233.33! A. 3.12 grown under these conditions was 1&5 minutes. when an optical density of the culture reached 3.0 units/m1, it was centrifuged in a marples continuous flow centrifuge, type A. 3-12. The yield was M0 to #50 g wet weight of packed cells. After harvesting, the cells were stored at -20°C. For periods of 10 months no decrease in RNA polymerase activity was observed. growth and Enrication of ggcteriOBhgge Q-l Bacteriophage gh-l was grown on a 6 to 8 liter culture of 2- m A. 3.12 in a Nicroferm Laboratory Fermentor. Growth and purification of the bacteriophage were performed according to the procedure of Lee and Boezi (1966). when the 6 7 cell density of the exponentially growing culture reached 5 x lo8 cells/ml, bacteriophage gh-l, at a multiplicity of 5, was added. After 2 hours, the lysed culture was centrifuged at 4,000 x g for 10 minutes to remove cell debris followed by centrifugation at 16,000 x g for 2 hours to collect the bacteriophage. Following suspension of the viral pellet in tris (hydroxymethyl) aminomethane (Tris) adjusted to pH 8.0 with R01, containing 0.2 M NaCl, the suspension was passed through a diethylaminoethyl (DEAE) cellulose column (3 by 20 cm) equilibrated with the same buffer. The fractions contain- ing gh-l were concentrated by centrifugation at 16,000 x g for 2 hours, and the resulting pellets were mispended in the buffer described above. Purified gh-l was stored at u°c. Growth and Purification of Bacteri'Ophage Ty], Bacteriophage T1} was grown on g. 92;; B in Fernback _ flasks containing basal C medium (Roberts, Abelson, Cowie, Bolton and Britten, 1957) with 0.1“ glucose. when the cell density of an exponentially growing culture reached 5 x 108 cells/ml, (an optical density of 1.0 unit/m1 was equivalent to a cell density of 5 x 108 cells/ml) bacteriophage T1} was added at a multiplicity of 3 to 6. Simultaneously, L- tryptophan was added to 20 )ig/ml. Following lysis of the culture, the bacteriophage were purified by a series of differential centrifugations at 10,000 x g for 10 minutes and 16,000 x g for 2 hours. After each high speed centrifugation 8 the viral pellets were suspended in 0.01 N Tris-RC1, pH 7.16 containing 0.2 N NaCle Purified Th were stored at WC. 1“ owth and ification of ' 'idine Labeled c or op age 7 BacteriOphage T7 labeled with PACE] thymidine was a gift of Dr. Lucy F. Lee. It was grown on a thymidine requiring mutant of 5% 2211.3, purified by differential centrifugation and stored at 4°C. Purification of gggteriophgge pug Bacteriophage deoxyribonucleic acid (DNA) was purified according to the method of Abelson and Thomas (1966). ‘me purified phage preparation was adjusted to a concentration having an absorbency at 260 m}: of 8 to 20 units/ml. An equal volume of fredily distilled, water saturated phenol was added, and the mixture was rolled at 60 rev/min for 30 minutes at room temperature. Following centrifugation at 3,000 x g for 10 minutes, the phenol layer and material from the interface were rueoved from the aqueous layer with a Pasteur pipet. An equal volume of phenol was added and the aqueous layer was extracted an additional 30 minutes. Following centrifugation at 3,000 x g for 10 minutes, the aqueous layer was transferred to a dialysis sec for dialysis overnight against 5 liters of 0.01 M Tris, pH 8.0, containing 0.1 M NaCl. In the morning the dialysis sac was transferred to 5 liters of mm buffer solution for an additional 2% 9 hours of dialysis. The DNA obtained by this method was stored at 4°C. The specific activity of EAC] thymidine labeled T7 DNA was 6,500 counts/min per pg. Purification of 2. guide 5.3.12 DNA The DNA from g. pgtida A. 3.12 was purified according to the procedure of Thomas, Berns and Kelley (1966). Frozen cells (1 g) were suspended in 50 ml of standard saline- citrate (sec: 0.15 ll NaCl, 0.015 N trisodium citrate, pH 7.0). morose was dissolved in the suspension to 27% (w/v). A solution of pronase (10 ng/nl) was prepared by dissolving the lylophilized powder in 0.01 M sodium acetate buffer which had been adjusted to a pH of 5.0 with acetic acid. Both the pronase solution and cell suspension were incubated at 75°C for 10 minutes, followed by cooling to room temperature. The pronase solution was added to the cell suspension to a concentration of l ng/nl, followed by sodium dodecyl nlfate to 1% (w/v). The aispension was incubated at 37°C for 3 hours after which an additional 0.5 ng/nl of pronase solution was added and the suspension further incubated at 37°C for 3 hours. An equal volume of distilled, water saturated phenol was added, and the mixture was rolled at 60 rev/min for 30 minutes at room temperature. Following centrifugation at 3,000 x g for 10 minutes, the phenol layer and material from the interface were removed from the aqueous layer with a Pasteur pipet. The aqueous layer was transferred to a . dialysis sac and dialyzed overnight against 0.01 M Tris, 10 pH 8.0, containing 0.1 :4 NaCl. Pancreatic ribonuclease (RNase) which was prepared by boiling a solution (1 mg/ml) for 10 minutes was added to the dialyzate to a concentration of 50 fg/ml. The mixture was incubated at 37°C for 2 hours. Following two additional phenol extractions of the aqueous phase using the techniques described above, the DNA was precipitated with 2 volumes of cold ethanol and collected on a glass rod. The precipitate was dissolved in 0.01 N Tris- sc1, pH 8.0 to which we been added 0.3 14 sodium acetate. Isopropanol was added to this solution to 0.5:. volume with continuous stirring. The DNA was collected on a glass rod and dissolved in 0.01 H Tris-HCl, pH 8.0, containing 0.1 1! NaCl. Fibers of DNA which were not collected on the glass rod were collected by centrifugation at 3,000 x g for 10 ainutes and dissolved in the buffer solution containing the precipitate from the glass rod. The solution was dialyzed overnight against 5 liters of the same buffer. The yield of DNA from 1 g of frozen cells was 1.1! mg. Its ratio of absorbance at 260 m}: to that at 280 up was 1.55. The solution of DNA was stored at 4°C. Egparation of lfil green Labeled m [ uracil labsled ribonucleic acid (RNA) with a specific activity of #78 counts/min per pg was a gift of Dr. Robert L. Anstrong. It had been extracted and purified froa ;. 22;; B using the IB-phenol method (Armstrong, 1966) and was stored at -2o°c. ll Qetemination of DNA, RNA and Protein Concentrations DNA and RNA concentrations were calculated from the absorbency at 260 mp by use of an extinction coefficient of 20 cmz/mg. Protein concentrations were determined by the method of Lowry (1951) using bovine serum albumen as a standard or was calculated from the absorbency values at 280 and 260 :91 using the formula of Layne (1957): Protein concentration (mg/m1) = 1.55 A280- 0.76 A260 Procedure for the geamrement of Radioactivity. 2c; Insoluble mioaotivitz' . The sample was mixed with 5 ml of cold 10$ (w/v) trichloroacetic acid (TCA.) and 250 pg of carrier salmon sperm DNA. The precipitate that formed in 15 minutes at 0 to hoe was collected by filtering the sample, using gentle suction applied by a water aspirator, through a nitrocellulose membrane filter. Each sample tube and filter was wadied with three, 5 ml portions of cold 10$ TCA. The filter was blotted free of excess liquid, placed in a liquid scintillation vial, and dried in a 95°C oven. After the filter had cooled to room temperature, 5 ml of a fluor containing 0.1 g of l,l+-bis- 2-(5-phenyloxazolyl) - benzene (POPOP) and ”.0 of 2,-5 diphenyloxazole (PPO) per liter of tolune was added. The sample was counted in a Packard Tri Carb, model 3003, liquid scintillation spectrom- eter with gain and window discriminator settings for the radioisotopes as follows: [33] gain 58}, window discriminator 50-10008 [lac] gain 16%, window discriminator 50-1000. 12 Duplicate counts each of 10 minutes were taken for the sample and the numerical average calculated. :0; Soluble Radioactivity. The sample was mixed with 0.5 ml of cold 10% (w/v) TCA and 250 pg carrier salmon spem mu. The precipitate that formed in 15 minutes at o to u°c was removed by filtration through a nitrocellulose membrane filter. The filtrate was collected and duplicate 0.1 ml samples were transferred to liquid scintillation vials containing 5 ml of a fluor composed of 60 g naphtalene, 0.0 g PPO, 0.2 g POPOP, 100 ml MeOH and 20 ml ethylene glycol per liter of p-dioxane. The sample was counted as described in A above. Mioactive gag; of RNA. Polymerase The radioactive assay measured the conversion of I: 33] CTP into a TCA insoluble form. The reaction mixture contained 20 )1 moles Tris-3C1, pH 8.0, 2.0 )1 moles MgClZ; 0.5 )1 mole NnCIZ; 1.5 )1 mole Z-mercaptoethanol; 0.005 )1 mole MA: 0-195 pg DNA from various sources; 0.215 )1 mole each of ATP, GTP, UTP and [3H] CTP; and 0-36 units of RNA polymerase in 0.5 ml total volume. [33] CTP had a specific activity of 11% counts/min per mu mole. During the incubation of the reaction mixture, 0.1 ml samples were withdrawn and 5 ml of .cold 10% TCA were added to stop the synthesis of RNA. One unit of enzyme activity corresponds to the amount of enzyme necessary to incorporate l y: mole of GNP per hour under the conditions described above using gh-l DNA as the template. 13 The specific activity of the RNA polymerase is the number of units per mg of protein. aectrophotonetric Assay of an; p.12...“ The assay employed for determining the formation of pyrophosphate by RNA polymerase measured the formation of NADPR. NADPR synthesis was coupled to pyropho sphate forma- tion through the enzymatic reactions illustrated below. (Reactions l-lt). RNA polymerase catalyzes the synthesis of RNA and pyrophosphate (Reaction 1). For each mole of ribonucleoside ATP + GTP DNA 4- ) RNA 4- PP (1) CTP RNA 4' Polymerase UTP tripho sphate incorporated into RNA as the ribonucleoside monopho sphate, a mole of pyropho sphate is toned. Pyrophos- phate in the presence of uridine diphosphcglucose (UDPG) and uridinedipho sphogluccse pyropho sphorylase under proper conditions is converted to glucose-l-phosphate and UTP (Reaction 2). The available glucose-l-phosphate is changed to glucose-é-phosphate by phosphogluccmutase (Reaction 3). PP1 + UDPG r > gluco se-l-phosphate + UTP UDPG pyropho sphorylase . ( 2) glucose-l-phosphate é ‘ ) glucose-6-pho sphate pho sphoglucomutase ( 3) As glucose-é-phosphate is formed, it is acted upon by gluco”- 6-phosphate dehydrogenase in the presence of NADP" to give 115 6-phosphogluconate, 3+ and NADPH (Reaction it). g1ucose-6-phosphate + NADP" 7‘ 5-Ph03Ph0- gluco se- 6-phc sphate gluconate + R" + NADPR dehydrogenase (it) From the reactions above it is observed that one mole .of NADPR is synthesized for each mole of perphosphate formed. Calculation of the number of moles of NADPR synthesized was made using the molar extinction coefficient of 6.22 x 106 one/mole at 340 mu (Kornberg, 1955). The change in absorbency at 3&0 mu was followed using a Beck-an DU spectrophotometer equipped with a Gilford automatic sample changer and a Sargent recorder. The temperature of incubation was controlled using Rechan thenal plates and a Raake circulating water bath. The reaction mixture contained 20 )1 moles Tris-R01, pH 8.0; 20 )1 moles NgClzg 0.5): mole NnClZ; 1.5 )1 mole 2- mercaptoethanol; 0.005 )1 mole ETA; 0-100 )1g gh-l DNA; 0.215 )1 mole each of ATP, GTP, UTP and CTP; 0.22 )1 mole NADP; 0.20 )1 mole UDPG; 10 )1g glucose-6-phosphate dehydrogenase; 1 )1g uridine diphosphcglucose pyropho sphorylase and 0-29 units of RNA polymerase in a total volume of 0.5 ml. The reaction mixture minus RNA polymerase was incubated at the reaction temperature (30°C) for #0 minutes. During this incubation some reduction of NADP+ which was not the result of the RNA polymerase reaction was observed. After the completion of this reaction, RNA synthesis and perphosphate fonation was initiated by the addition of RNA polymerase. 15 nethod fo; the Asa: of Qeomibonuclease The time-dependent decrease in [1&8] thymidine labeled T 7 measure of DNase activity. The reaction mixture used for DNA which was insoluble in 10% TCA was employed as a the detection of DNase activity in the 60$ Amonium axlfate Fraction 11 contained 6 )1 moles NgClzg 3 pg [lac] thymidine labeled T7DNA; and 10 pg 605 Ammonium Sulfate Fraction II in a total volume of 0.6 ml. The reaction mixture was incubated at 37°C. Samples of 0.1 ml were periodically moved from the reaction mixture and mixed with 5 m1 of cold 10$ (w/v) TCA and 250 pg of carrier salmon sperm DNA. The precipitate which formed was collected according to the procedure described in part A of Procedure for Radioactivity Measurement. Control experiments of two types were perfolmed. The first followed the time dependent decrease in radioactivity of the TCA insoluble [lite] labeled T7DNA when no 60$ Ammonium Sulfate Fraction II was added to the reaction mixture. The second experiment followed the pameter measured above when DNase at a final concentration of 0.017 and 0.17 pg/ml were added to reaction mixtures not containing 60$ Ammonium Sulfate Fraction II. eth for the sa of bonuclease The time dependent increase in TCA soluble [3R] uracil labeled 5. gal; RNA radioactivity was employed as a measure of RNase activity. The reaction mixture used for the detection 16 of RNase activity in the 60% Ammoniun.8ulfate Fraction II contained 20 )1 moles Tris-RC1, pH 8.0; 0.005 )1 mole RDTA; 2 p moles NgClZ; 0.5 )1 mole NnClzx 1.5 p moles 2- mercaptoethanol; 1‘! )1g [-33] uracil labeled RNA and 10 pg of 605 Ammonium Sulfate Fraction II in a total volume 0.5 ml. The reaction mixture was incubated at 37°C. Samples of 0.1 ml were periodically removed over an hour of incubation and mixed with 0.5 ml of cold 10% TCA and 250 pg of carrier salmon spelm DNA. Radioactivity was assured in the filtrate according to part B of Procedure for Radioactivity Measurement. Control experiments of two types were performed. The first followed the time dependent increase in TCA soluble [BR] uracil labeled g. 92;; RNA radioactivity when no 601 Ammonium Sulfate Fraction II was added to the reaction mix- ture. The second experiment followed the time dependent increase in TCA soluble radioactivity when RNase at a final concentration of 0.11 )1g/ml was added to the reaction mixture in the absence of 60% Ammonium Sulfate Fraction II. Characterization of the an m " thesized big; page... The HA product of RNA polymerase was characterized by its susceptibility to degradation by RNase and 0.3 N NOR and by its resistance to degradation by DNase. A reaction mixture containing 80 p moles Tris-R01, pH 8.0; 8.0 )1 moles NgClzg 2.0 )1 moles NnClz; 6.0 p moles 2-mercaptoethanol; 0.02 p mole MA; 380 pg gh-l DNA; 0.86 )1 mole each of ATP, CTP, UTP and [331 CTP; and 0.2 units of 1? RNA polymerase (60$ Ammonium Sulfate Fraction II) in a total volume of 2.0 ml was incubated at 30°C for one hour. Part of the reaction mixture (0.5 ml) was heated at 100°C for 10 minutes, then cooled in an ice bath to 0°C. The remaining 1.5 ml of reaction mixture was made 0.2x (w/v) with respect to abs. The mixture was incubated at 37°C for an additional 10 minutes, then cooled to 0°C in an ice bath. The precip- itate which famed overnight was removed by centrifugation at 5,000 x g for 10 minutes. The pellet was discarded and the supernatant fluid was extensively dialyzed against 0.01 M sodium acetate buffer adjusted to pH 5.2 with acetic acid. Aliquots of the reaction mixture which had been heated at 100°C for 10 minutes were subjected to the following treamentss (l) Incubation for 1 hour at 37°C in the presence of 1 pg/ml RNase. (2) Incubation for 3 hours at 37°C in the presence of 10 pg/ml RNase. (3) Incubation for 30 minutes at 37°C in the presence of 1 pg/ml DNase. Aliquots of the reaction mixture which had been incubated with SDS were subjected to ,the following treatment as (1) Incubation for 30 minutes at 37°C in the presence of 25 pg/ml RNase. The pH of the reaction was brought to 8.0 with Tris-RC1 buffer. (2) Incubation for 200 minutes at 37°C in o. 3 n XOR. 18 Following treatment as described above, the samples were mixed with cold 10% TCA. TCA insoluble radioactivity was determined. General gethods and Eaterials Glass beads, purchased from R. R. Sargent & Co., were prepared by washing successively in 0. 5 H NaOR, water and 0.5 1! R01. They were rinsed free of traces of acid with water then dried. Streptomycinmlfate was purchased from ' Calbiochem. It was made to 10% (w/v) with water. Cellex D, a product of Bio Rad Laboratories, having an exchange capacity of 0.75 meq/g was prepared by washing extensively in 0.1 M NaOH followed by 0.1 H 301. It was rinsed with water until traces of acid were removed. The fine particles were removed by decantation. Dialysis tubing was prepared by boiling in 5} sodium bicarbonate. when used for DNA preparations it was further treated by boiling in 0.001 I! ETA. mass and electrophoretically purified DNase were purchased from worthington Biochemicals Corporation. Pronase, B grade, was purchased from Calbiochu. Nogaluycin and Actinomycin D were gifts of the Upjohn Company and Herck and Company respectively. Salmon sperm DNA, type III. was purchased from Sigma Chemical Company. Bact-T-flex nitro- cellulose membrane filters were obtained from Carl Schleicher and achuell Co. Unlabeled nucleo side triphosphates were purchased from the r. L. Laboratories Inc. [311] err with a specific activity 19 of 1.2 c/m mole was obtained from Schwarz Bioresearch, Inc. NADP+. UDPG and the enzymes used in the assay of pyrophosphate were the generous gifts of Dr. R. G. Hansen. Pho sphoglucomutase was prepared by di ssolving an ammonium milfate suspension in water to a final concentration of 1.0 mg/ml. One mg converted 50 p moles glucose-l-phosphate to g1ucose-6-phosphate per minute at pH 7.10 and at 30°C. Glucose-6-phosphate dehydrogenase was prepared by diluting the crystalline suspension to 0.2 mg/ml in water. One mg converted 130 p moles of NADP+ to mom per ninnte at pH 7.» at 25°C. UDPG-perphosphorylase was prepared by making a l to 100 dilution of the crystalline mspension (10 mg/ml) with 0.1 n Tris-acetate buffer, pH 8.0. One mg would convert 2‘60 p moles of pyropho sphate to glucose-l-phosphate and UTP per minute at pH 8.0 at 25°C. hromato of whatman #1, acid washed paper, was spotted 11 cm from the edge with [321] err. CTP, GDP, and our. The unlabeled nucleoside triphosphates were applied at 90 to 100 pg per spot. [33] CTP was spotted with approximately 3 x 105 counts/ min. The chromatogrsm was developed with isobutyric acid, concentrated muoa and water (66/1/33) using decending chromatography at 23°C. During in hours of development, the front moved 3? cm. The chroaatogram was air dried and read by vimal inspection of ultraviolet light quenching upon illumination with a hineralight model 81., 2537 lamp. The 20 lanes containing [3H1 CTP were cut into 3/b inch squares and placed in liquid scintillation vials to which was added 5 ml of the fluor described in part A of Procedure for Radioactivity Measurement. The radioactive content of the squares was determined. Betwaen 80 and 85$ of the radio- activity co-chromatographed with the CTP‘Iarker. Purification of DEA-Dependent RHA Palmerase Initial Extract. All of the following purification procedures were performed at 0 to 10°C unless otherwise stated. To prevent extensive loss of enzyme activity, it was necessary to avoid any delay in the completion of the purification. Frozen cells (25 g) were mixed with glass beads (62.5 g) and disrupted by grinding using a mortar and pestle. Follow- ing rupture of the cells in 15 to 20 minutes, 62.5 ml of cold buffer A (0.01 H Tris-R01, pH 8.0; 0.01 H HgClzs and 0.001 x mm) was added. After centrifugation at 25,000 x g for 15 minutes, the aipernatant fluid was carefully decanted and saved. The pellet was suspended in 31 m1 of cold buffer A and centrifuged as above. The combined 96 ml of mipernatant fluid is referred to as the Initial Extract. gig Speed Supernatant Mtion. The Initial Extract was centrifuged at 150,000 x g for 90 minutes. The High Speed Supernatant Fraction (83 ml) was collected by deconta- tion. fireptmcin Precipitate. Streptomycin ailfate was added to the High Speed Supernatant Fraction at a final 21 concentration of 0. 5} (w/v). The streptomycin sulfate solution was added slowly with continuous stirring. A fibrous precipitate which formed was collected on a glass rod, squeezed to remove excess liquid and added to 25 ml of buffer A containing 0.2 H (NHu)ZSOu. After 30 minutes the precipitate which was not collected with the glass rod was collected by centrifugation at 25,000 x g for 15 minutes. The pellet was added to the solution containing the fibrous precipitate. The combined precipitates were stirred slowly for it to 5 hours to dissolve the material. 30-605! monium Sulfate gm h ' tion I. DHase was added to the Streptomycin Precipitate at a final concentration of 1 pg/ml. The solution was dialyzed in the cold room (h°c) for 12 hours against 6 liters of a buffer solution prepared at 22 to 24°C containing 0.005 K 2-mercaptoethanol in buffer A. Approximately #0 to 501 of the initial absorbency at 260 71 was recovered following dialysis. Anonium allfate (saturated at room temperature and adjusted to pH 7.0 with ammonium hydroxide) was added to a final concentration of 30$ of saturation. The mixture was stirred 15 minutes and a precipitate was removed by centrifuga- tion at 25,000 x g for 10 minutes. Saturated ammonium sulfate was added to the mpernatsnt fluid at 60$ of saturation. The mixture was stirred 30 minutes, and the precipitate was collected by centrifugation at 25,000 r g for 15 minutes. The precipitate was dissolved in MO ml of buffer H (0.029 8 Tris-RC1, pH 8.0; 0.000,09 1! MA; 0.013 K HgClzg 0.001 K 22 nnClZ; and 0.01% n 2-mercaptoethanol). The protein concentra- tion was h to 6 mg/il. Peak 2§A§:Cellulose Fraction. The 30-60% Ammonium allfate Precipitate was diluted to a protein concentration of about 2 mg/hl with buffer A.containing 0.005 K 2-mercapto- ethanol. A.DHAD-cellulose column.(1 x 12 cm) was equilibrated ‘with the same buffer. The diluted solution.was passed on to the column at a rate of l mllhin. The column was washed with buffer A.containing 0.005 n 2~mercaptoethanol until the absorbency of the effluant was less than.0.05 unitswml at 260 mp. (A second wash of #0 ml of buffer A containing 0.005 K 2-mercaptoethanol and 0.1 H Neel was passed through the column. The enzyme was eluted from the column with buffer A containing 0.005 M deercaptoethanol and 0.2 H Ha01. Enzyme activity appeared within.10 to 12 m1 of the latter eluant. No appreciable activity was eluted with buffers containing higher salt concentrations. The flow rate was maintained at 1 m1/min throughout the chromatography. ' 602 Aggonium.8ulfate Fraction 1;. Immediately after elation, the enzyme was concentrated. A saturated ammonium sulfate solution.was added to the fractions containing RNA polymerase activity at 60$ of saturation. Following 15 minutes of precipitation during which the solution was occasionly stirred, the precipitate was collected by centrifugation at 37,000 x g for 30 minutes and dissolved in 0.5 m1 of buffer B. The protein concentration of the resultant solution.was 5 Isl-1- BESJUI‘S m of the Erigication Procedure The mammary of the purification procedure is presented in Table I. Reallts of the purification through the Streptomycin Precipitate fraction (step 3) are presented for one preparation of the enzyme. This Streptomycin Precipitate fraction was not purified further. A second purification was performed which was carried through the 60% Ammonium ailfate Fraction II to obtain the results of steps 10, 5 and 6. The remilts of the first three steps of the second purification were not typical and are not presented in Table I. The assays of the first three steps of the second purification were not performed immediately following the isolation of the fraction. Since activity of RNA polymerase is lost upon storage of fractions 1, 2 and 3, the rate of CNP incorporation into RNA was less than that from fremly prepared fractions. The best estimate of the total purification was obtained by compiling the remilts of the two separate purifications. The assays used for the compilation of Table I were of two types. The enzyme fraction was either assayed in the presence of 98 pg/ml gh-l DNA(+) or was assayed in the absence of any exogenous DNA(-). CTP was converted to an acid insoluble form when DNA was omitted from reaction mixtures containing RNA polymerase from fractions 1 through It. Evidently, there is mifficient DNA in the enzyme fraction to function as a template for RNA polymerase. Invariably, addition of DNA to 23 24 .eflmnfle no and? esp epeasoaso on can: ass degassed." no seasons n no oepenoauoosa man .790» ea. .edshdsde huabdpoeoaoca you mesa» 36ng as grandsons one: :a .73 moaned»... .noosvez use madness: a." confluence end—puns soapoeeh d: n6 gouache esp a." Down as eons-as new moapoenu cabana seem .9333: open as: house en» s." can: 420 Hun» no soapenpseosoo we .Tv couscous an." a." no T; «an .753 no nun—\ml no no eoseeeha on» an Henge oe-ficuuea one: ensued a .0563qu sodasoauansa on» no anal-8n .H sands 25 na.n can an c.c an.“ ccc.aa ccr.ea + no nonsense cpeuanm c.c an.“ o c . seahorse «on ea.e men nu c.cmn.n H~.c ccm.m~ caa.a + concerns c.c H~.c c c . ccoaeaac01u4nn seem .n m.aa n c.~rH ca.n ccm.n one + a nonsense enceanm o.~ cm.m n: «H u nuancana accacn .r c.mna mm.a one.» new caopaaaccnm n.a~ an c.e~a nn.a com.mn cam.a . cheesecacaan .m soaaoeam w.nm ~.HH oouamm 3mm +_ unsaesuemsn aH.~ and c.na «.Ha cc~.~a can . ocean near .u c.aa «.ma con.a~ mam + u can n.rn «.ma cca.ae ran a cocoons acaaanu .H unchanged utcccm assesses sexes, beads as can each a enhancer oneness Hopes \waaap_ muse concerns nodvdodhahsm on» no humminn .H Hands 26 any fraction before purification step It resulted in a decreased rate of RNA synthesis. However, the extent of RNA synthesis was greater in the presence of exogenous DNA than in its absence over long periods of incubation. The highest specific activity reported for this purifica- tion was 5,590 mp moles of CTP converted into a TCA insoluble form per hour per mg protein. Homogeneous RNA polymerase from g. 93;; has a specific activity of 7,000 mp moles of CTP converted into an acid insoluble form per hour per mg (Richardson, 1966b). Following DEAE-cellulose chromatography, it was necessary to concentrate the enzyme, since dilute enzyme solutions lost activity rapidly. Concentration by anemium sulfate remilted in a relatively stable enzyme. The specific activity of the 605 Ammonium ailfate Fraction II apparently decreases. This enzyme fraction was inadvertently assayed under conditions in which total RNA polymerase activity was not detected. The concentration of gh-l DNA in this assay limited the rate of RNA synthesis by 1&0 to 501 (see Figure h). Purification of mu polymerase from 2. 22.1.9. through the Peak DEAR-Cellulose Fraction has relilted in a 160 fold increase in specific activity with a 20$ recovery of activity. ZLoperties of 601 was agate Eaction 2; losses of 50$ or more of the activity of RNA polymerase in the 60$. Ammonium ailfate Fraction II were observed upon storage at “ca for two weeks. No activity was detected after 2? freezing the fraction to -20°C and thawing to the assay temperature. The enzyme fraction could, however, be quickly frozen in a dry ice-acetone bath and stored in liquid nitrogen (-196°C) with only asall losses in activity over a two month period. Repeated freezing to 496°C and thawing to 0°C did not significantly effect the activity of the enzyme. Neither RNase nor DNase activities were observed in the 601 Ammonium Sulfate Fraction II as asayed according to the procedures described in Haterials and Hethods. The possibility that there are asll amounts of these nucleasss still associated with the enzyme fraction cannot be ruled out on the basis of this assay. Comments Concerning ‘- the mification mcedure Further purification following the Streptomycin Precipitate (step 3) proved to be difficult. The aural f procedures arch as ammonium sulfate precipitation or DEAE- cellulose chromatography did not work in the presence of the high concentrations of nucleic acids found in the Streptomycin Precipitate fraction. Following the degradation of DNA by DNase, further purification proved possible. The remlts of DEAD-cellulose chromatography are clown in Figure 1. Nearly all of the activity. could be eluted from the column in one 10 ml fraction. The total recovery of absorbency at 280 mm from the column was 851. The ratio of the absorbency at 280 mp compared to that at 260 :91 was 1.3 for the peak fraction. Using the data of Narburg and 28 .cohacea mes 59,330.33!" egg—Hound dun. .u R an :35: n 2:. coups—35 .58 .33 .8 an}: mm ma. «83095 on» no as +3.0 cenasunoo ennga floapoeon no as .n seem .8355 «830.39 uo Ha\ue.noa an a: nodpcnoauoona $8 anaconda." can: v.33 suds 83.3 mg .la omm as aqua—Ho on» no honepnoane on» 09.3.25." can: 6216.0 .3: 85.3 when .copoednoo one: 33300.3 H! o." .oeoazaaeonmdfln no H .3305 epsuasm £53252 mownon no manage—song an enema 29 ABSORBANCY, 280 mp <--------) -— 5.0 c. o 0. 0. . l IODN W 9'0 IODN W Q'O IODN IN 3'0 IODN W 'I'O L l l I O O O O (D m «u- N IOO -— ( ) . (nus/sown: flw) OBlVHOdUOONI dWO Figure 1 FRACTION NUMBER 30 Christian (19%), such a ratio indicates that the contamina- tion with nucleic acids was less than 1.3%. when DEAR-cellulose chromatography was performed in the absence of Hg“ or 2-mercaptoethsnol or both, recovery of activity was poor. Kinetics of the mthesis of RNA by DNA-Dependent RNA 0 Eerase The kinetics of the DNA-dependent synthesis of RNA by RNA polymerase (60% Ammonium ailfate Fraction II)are presented in Figure 2. RNA synthesis was not observed when assayed in the absence of a DNA template. In the presence of exogenous DNA, RNA synthesis was linear for 5 minutes or less. By 30 minutes of incubation RNA synthesis had nearly stopped. Effect of RNA zolnerase concentration onRate of Reaction The rate of incorporation of GNP into RNA was linearly dependent upon enzyme concentration (Peak DEAD-Cellulose Fraction) within the range tested (Figure 3). Effect of DNA Concentration on Rate of Reaction The concentration of DNA in the reaction nixture strongly effects the initial rate of RNA synthesis (Figure 10). For these experiments approxinately 220 )ig of gh-l DNA per ml of reaction mixture gave the maximum synthesis of RNA. Higher concentrations of DNA in the reaction mixture lowered the rate of RNA synthesis. The possibility that the apparent decrease in RNA synthesis at high concentrations of DNA was caused by a 31 Figure 2. Kinetics of the synthesis of RNA by DNA-dependent RNA polymerase. 601 Ammonium Sulfate Fraction II (260 pg) was added to the standard 0. 5 ml reaction mixture containing either no exogenous DNA (O-—O) or 1&5 opg of gh-l DNA (0——-O). During incubation at 37 C, 0.1 ml samples were withdrawn for radioactive analysis. 32 5.0 — _ _ _ o. o. o o. 4. 3 $22: 1.5 owadmoamooz arc 20 IO TIME (min) Figure 2 35 Figure 1+. Effect of gh-l DNA concentration on the rate of GNP incorporation. 60% Ammonium ailfate Fraction II (2}: )1“) was added to a reac- tion mixture containing moles Tris-H01, pH 8.0; 0.1} mole MgCl ; 0.1}; mole MnCl; 0. 3 mole -mercaptoet anol; 0001 mol; E3; 3 0.0“3 )1 mole each of ATP, GTP, pUTP and CTP; and varying concentrations of gh-l DNA in a total volume of 0.1 ml. Incubation was 5 minutes at3 c. The results are plotted as GNP incorporation3 in mm moles/m1 of reaction mix- ture versus gh-l DNA concentration in )ig/ml. 36 200 300 400 oh-I DNA (us/ml) Figure I} ‘ IOO _ _ _ k 6 4 . 2 estate as. amadmoamooé aso 37 self-adsorption phenomena was considered. It would be expected that the self-adsorption of radioactivity in a precipitate would increase with increasing amounts of material. However no decrease in radioactivity was observed when up to 500 pg of gh-l DNA was mixed with [33] uracil labeled RNA, precipitated with cold 10$ TCA, and assayed as described in Naterials and Methods. Effect of ER on RNA gmthesis The effect of pH on the rate of incorporation of 0111’ into RNA has been studied using Peak DEAN-Cellulose Fraction enzyme (Table II). The reaction has a broad pH Optimum between pH 8 and 9 in the Tris-ROI buffer. when assayed below pH 7.0, no RNA synthesis was observed. Effect of Tsperature on m mthesis The kinetics of RNA synthesis using the 60$ Almonium Sulfate Fraction II enzyme at various reaction temperatures are presented in Figure 5. A lag in RNA synthesis observed at 25°C and 20°C was not observed at 30°C. 37°C or 1.2%. Once the leg was over, the rate of mu synthesis at 20°C was half that at 30°C. The extent of RNA synthesis was greater at 30°C through 30 minutes of incubation than at any other incubation temperature uud. Preincubaticn of the 601 Ammonium Sulfate Fraction II with the reaction mixture minus ribonucleoside triphosphates for 11 minutes at 25°C before initiation of the reaction with the ribonucleoside triphosphates, eliminated the lag that occurred at 25°C. 38 Table II. Effect of pH on the rate of GNP incorporation. Standard 0. 5 m1 reaction mixtures modified to contain 20 )i moles of Tris-RCl buffer at various pH values were prepared. Each reaction mixture contained Peak DEAR-Cellulose Fraction enzyme (10.5 mg) and T DNA (15 pg). Samples (0.2 ml) were remov d at 5 and 10 minutes as the mixtures were incubated at 37°C. The TCA insoluble radioactivity was determined. iififi 39 TABLE II. Effect of pH on the Rate of GNP Incorporation pH of Reaction CNP Mixture . Inggragizzion 6.5 0.0 7.0 0.01 7.5 0.34 7.9 0.62 8.5 0.69 8.9 0.61 9.1 0.56 40 Figure 5. Effect of temperature on rate of GNP incorporation. The standard 0.5 m1 reaction mixtures containing 95 pg of gh-l DNA and 10 g of 605 Ammonium Sulfate Fraction I were incubated at 20°C (e—e); 30 C “—0); 37 C or h2°C (o—o). no reaction mixture used to obtain the curve at 25 C (H) was modified to a final volume of 0.75 ml. The concentration of the components were identical to the 0.5 m1 reaction mixture. Samples (0.1 ml) were removed at various times for radioactivity analysis. 1+1 JA d dc _ _ A _ 0 8. 6. L O 0 $22.. as omadmoamooz. $20 20 IO TIME (min) Figure 5 42 Effect of fig“ and an“ gogcentration on RNA Synthesis Table III shows that Hg” or Nn'H' stimulate RNA synthesis. In the absence of divalent cation no synthesis of mm was observed. The addition of both Mg” and Mn“ to the reaction mixture at the concentrations used by Chamberlin and Berg (1962) was more effective in the stimulation of RNA synthesis than either of the two cations individually. at... o; Substrate Qonogntration on RNA mthesis The results presented in Table I? show that maximal RNA synthesis was observed when the concentration of each triphosphate was 10.3 x 10"lb N. Inhibition of RNA synthesis was observed when concentrations of substrate greater than #3 x 10"“ N were used. ggect of Mona PEA Templates on g" gathesis When DNA from various sources, at approximately the same concentration, was used as the template for RNA polymerase (Peak DEAN-Cellulose Fraction), differences in the total incorporation of monophosphates into RNA were observed (Table V). The total monophosphate incorporation into RNA was calculated using the CMP incorporation observed and the publimied (EC-content of each of the templates. Gh-l DNA functions 2 fold better than either Ta or z. putida mm and over 5 fold better than salmon sperm DNA. Kinetics of Mpho abate Formation by RN‘ gglflerase The kinetics of pyrophosphate formation are presented in Figure 6. When RNA polymerase (60$ Ammonium Sulfate Fraction “3 Table III. Effect of Mg“ and an” concentration on the rate of GNP incorporation. Peak DEAF- Cellulose Fraction was dialyzed 15 hours against buffer A containing 0.005 N hmercaptoethanol to remove traces of Mg‘H' and Mn . This enzyme (21 mg) was added to the standard 0. ml reach tion mixture modified to give the Ng and Mn concentrations shown. Each reaction mixture contained 68 pg of salmon sperm DNA. Samples (0.1 ml) were withdrawn periodically as the reaction mixtures were incubated at 37 . TCA insoluble radioactivity was determined. TABLE III. #0 Effect of Mg“ and Mn“ on the Rate of GNP Incorporation Concentration of Concentration of GNP Ng++'in the Mn in the Incorporated Reaction Mixture Reaction Mixture C(mpnmolesfiml)a (my moleswml).. ,, ” (my.moles) 0.0 0.0 0.0 4.0 0.0 0.25 0.0 1.0 0.20 4.0 1.0 0.30 45 Table IV. Effect of mibstrate concentration on the rate of GNP incorporation. The reac- tion mixture was that of Figure 4 only each was modified to contain the various aibstrate concentrations shown and 19.8 pg of gh-l DNA. The reaction mixtures contained 2 pg of 60$ Ammonium Sulfate Fraction II. Each mixture was incubated 10 mirmtes and the TCA insoluble radioactivity was determined. 1&6 TABLE IV. Effect of Substrate Concentration on the Rate of GNP Incorporation Concentration of Rate of Incorpora- Each substrate in tion of cup into mu Reaction Mixture (N) . (r91 moles/10 min) . ,. 0.0 0.0 0.86 r 10'“ 0.13 2.15 x 10-“ 0.25 11.30 x 10"“ 0.118 10.1 x 10"“ 0.16 h. 2°e1 I 10- 0.07 “7 Table V. Effect of various DNA templates on the rate of synthesis of RNA. The standard 0.5 m1 reaction mixture contained 10.5 pg of Peak DEAR-Cellulose Fraction and the stated concentration of each of the various DNA tmnplates. Samples (0.1 ml) were withdrawn at various times from the geaction mixtures which were incubated at 37 . TCA insoluble radioactivity was measured. From the CMP incorporation and the GC-content of the DNA template, the total nucleotide incorporation was calculated. #8 TABLE V. Effect of Various DNA Templates on the Rate of RNA Synthesis by RNA Polymerase Source of DNA CNP % GC Factor Nucleo- DNA Concentra- Incorpora- tides Template tion ted Incorpora- (pa/m1) (up moles) ted . . , A _ (191 moles). Salmon sperm 135 0.15 111.2 11.85 0.73 ;. Eutida 11.0 0.50 63.0 3.17 1.6 '1“. 118 0.32 35.0 5.72 1.7 311-1 129 1.1 - 57.0 3.50 3.8 ’49 Figure 6. Kinetics of pyrophosphate formation by RNA polymerase. To the standard 0. 5 m1 reaction mixture was added 99 pg of gh-l DNA and 25.1 pg of 60% Ammonium Sulfate Fraction II (curve a). The complete reaction mixture enzyme (curve b); minus DNA (curve c); minus ribonucleoside triphosphates (curve (1). The reaction mixtures were incubated at 30°C during the assay. 50 0 9 $29: as: Samoa .. TIME (min) Figure 6 51 II), curve b; gh-l DNA, curve c; or ribonucleoside triphos- phates, curve (1, are not included in the reaction mixture, little synthesis of pyrophosphate was observed. The kinetics of pyrophosphate formation in the complete reaction mixture are presented in curve a. The synthesis of perphosphate is linear for 30 minutes at 30°C. fect c Pol erase Concentration on the Rate of 0 ha e rma on The'rate of perphosphate formation by RNA polymerase was linearly dependent upon the enzyme concentration within the range tested(Figure 7). nfect of InhibitorsI RNase or DNase on mgphogghate F032 tion Actinomycin D, nogalamycin or DNase (Figure 8, curve c) inhibit the formation of pyropho sphate by RNA polymerase. RNase stimulated the formation of pyrophosphate (curve a). The extent of RNase stimulation of pyrophosphate forma- tion by RNA polymerase varies with the concentration of RNase (Table VI). The greatest observed stimulation of pyrophosphate formation occurred with RNase at a concentration of 2 pg/ml of reaction mixture. gmarison of the Radioactive A33; and the Spectrophotgetg‘g A22! Figure 9 presents the kinetics of the formation of perphosphate as followed by the spectrOphotometric assay and as calculated from the GC-content of the DNA template and the CNP incorporation observed when followed by the radioactive assay. pr I ' . . a . v ‘ 1 I I h o ‘1“ . . ' . . u‘ I O .' f . ‘ . g . -. I ‘ ‘ ,5 e . . -7 . . a . . I. g . I! , e. ‘ . , . a‘l .. s t " . | | ,' c . .. i. ' ‘ v . ; O i , ‘ v 'P n ‘ v . 5- e ‘1 ‘ ‘ Q . A ' - t I g. . . .. . . . . e . b ‘ A A . t 52 Figure 7. Effect of RNA polymerase concentra- tion on the rate of pyrophosphate formation. The standard 0.5 ml reaction mixture contained 99 pg of gh-l DNA and various concentrations of 60% Ammonium Sulfate Fraction II. RNA polymerase was added following a 1&0 minute o preincubation of the reactionomixture at 30 C. The assay was performed at 30 C. 53 _ _. P 5. 0. 5 . .l I O 35.28. as. summon .aa l0 PROTEIN (no/ml) Figure 7 51} Figure 8. Effect of inhibitors, RNase or DNase on pyrophosphate formation. The standard 0.5 ml reaction mixture contained 99 pg of gh-l DNA and 25.1 pg of 60% Amonium Sulfate Fraction II (curve b). The results of the addition of 2.511(1g of nogalamycin or 2.0 pg of actinomycin or 2 pg of DNase are presented in curve c. The remilt of the addition of 2 pg of RNase to the complete reaction mixture are shown in curve a. reagtion mixtures were incubated 1&0 minutes at 30 C before addition of the oRNA-polymerase. The assay temperature was 30° C. 55 _ 0 9 “mgoE 1.5 music“. in 20 30 TIME (min) I0 Figure 8 56 Table VI. Effect of ribonuclease concentra- tion on the rate of pymphosphate formation. The standard 0.5 m1 reaction mixture was prepared with 99 )lg/ml of gh-l DNA and 5 pg of 60% Ammonium Sulfate Fraction II. RNase was added to the mixtures at the stated concentration. Following 1&0 minutes of preincubation, RNA polymegase was added. The assay was conducted at 30 . 57 TABLE VI. Effect of Pancreatic Ribonuclease Concentration on Rate of Perphosphate Formation Concentration of Pyrophos- Pancreatic Ribo- phate nuclease in the Formation Reaction.Hixture (1 on (pgflml) ,. . _ -, ... a . . . Control) 0.00 100 0.01 113 0.10 122 1.00 130 2.00 142 20.0 113 58 Figure 9. Couparison of the formation of pyropho sphate as measured by the spectrophotomet- rio assay ( ) with the calculated formation of perphosphate as determined by the radio- active assay (0-—-0). Two standard 0.5 ml reaction mixtures containing the same concentra- tion of each component were prepared. Each mixture contained 99 pg of gh-l DNA and 10 ug of 60% Ammonium Sulfate Fraction II. The mixture which was used for the 3R mination. of GNP incorporation contained f CTP. reaction mixtures were preincubated 40 minut:a before addition of the RNA polymerase fraction. The assays were conducted at 30°C. Pyrophos- phate formation as observed by the spectro- photometric assay and calculated from the GO- content of gh-l DNA and the observed GNP incorporation were plotted. 59 60 4o ,‘ TIME (min) 80- 0 0 6 4 ., case as 82%... .mm . 20t— 20 Figure 9 60 It is observed that the initial rate of the radioactive assay is greater, but within a factor of 2, than the steady state rate of the spectrophotometric assay. The extent of perpho sphate formation is much greater than that calculated from the radioactive assay. The total amount of radioactivity detected after 40 minutes of reaction decreases. This is reflected in the lowered calculated quantities of pyrophosphate. flmterization of the EA Synthesized by m zolnense It is observed in Table VI that the RNA product from the in 2212. reaction with RNA polymerase is completely resistant to DNase degradation and is greater than 96$ susceptible to KOH digestion. The product RNA was only 80$ susceptible to the action of RNase. However, treatment of the reaction mixture following the synthesis of RNA with we as described, resulted in RNA which was approximately 96$ susceptible to RNase. 61 TABLE VII. Characterization of the RNA Synthesized by DNA-Dependent RNA Polymerase Treatment the 3a RNA Susceptible to the . Treatment. Heat treated reaction mixture (1) RNase, 1 hour, 1 pg/ml (2) RNase, 3 hours, 10 pg/ml (3) DNase,0 .5 hour, 1 pg/ml moo oooo ' e e O\OO\ EDS treated reaction mixture (1 RNase, 0. 5 hour, 25 pg/ml 96.4 (2 KOH, 30 3 haura, 0e 3" 96e5 D1 800 831011 This investigation has been concerned with the purifica- tion and characterization of DNA-dependent RNA polymerase from Pseudomonas BEE-.21.: A.3.12. The enzyme has been purified to a specific activity of 5,590 mp moles of CTP converted to TCA insoluble form per hour per mg of protein. The increase in specific activity was 160 fold over that of the Initial Extract and the recovery of activity was 20$. The ‘280u260 ratio of the Peak DEAR-Cellulose Fraction indicated that there was l.3$ (w/w) contamination by nucleic acids. The kinetics of RNA synthesis exhibited by DNA-dependent RNA polymerase from 3. 221129. were of the type described by Bremer and Konrad (1964) for the g. 32;; enzyme. In the absence of exogenous DNA there was no RNA synthesis. In the presence of exogenous DNA, RNA synthesis occurred. For both the g. 22;; and g. pg'tida enzymes RNA synthesis was linear for a relatively mort period of the assay. RNA synthesis stopped because of the mispected formation of a complex [DNA-enzyme-RNA] which inhibited further reaction. It was observed that the kinetics of the reaction changed during the purification. The early fractions catalyzed the synthesis of RNA in a linear manner for nearly 15 minutes and the synthesis of RNA continued for 1.5 to 2.0 hours. It is possible that purification of the enzyme removed a factor which prevented or postponed the formation of the suspected complex. Such factors have been Crown to exist. Revel and 62 63 Gros (1967) have found _i_._n_ 2123.9. DNA-dependent RNA synthesis to be stimulated by ribosomes when a protein factor was present. Some RNA was observed to have been released from the template in the presence of this factor. A second possibility is that the exogenous DNA template which had been purified by phenol extraction did not have the some properties as did the endogenous 2. 2212.9. DNA used for assay of the early fractions. The effect of DNA concentration on the rate of m1 synthesis was investigated. It was observed that at high concentrations of DNA there was a marked inhibition of RNA synthesis. Theoretically, the binding of RNA polymerase to DNA should be favored at high DNA concentrations. The rate of synthesis of RNA would increase until all of the available enzyme was bound to DNA and was functioning. Any further addition of DNA to the reaction mixture would not cause an increased rate of RNA synthesis. The possibility that 14g” and Nn'H' were removed from the reaction mixture by binding to nucleic acids, thus preventing tho synthesis of RNA was considered. Binding of divalent cations to DNA has been demonstrated by equilibrium dialysis experiments md conductivity studies (Zubay and Doty, 1958) . It is thought that binding involves the phos- phate group and that each phosphate may bind one divalent cation. when gh-l DNA was added at 390 pg/ml of reaction mixture, the molar concentration of nucleotide phosphate was 61} about 11.8 x 10'“ N. The nclar concentration of Mg“ and Mn“ was 1&0 x 10'“ and 10 x 10"“ respectively. If all of the DNA phosphate was capable of binding Hg“ or Mn“ cations, there would still be an excess of free cations to perform other functions. However, Mg” and Mn“ can be chelated by the nucleoside triphosphates (Lowenstein, 1958). The concentration of the nucleoside triphosphates in the reaction nixtures used for detersining the effect of DNA was 17.2 x 10"“ M. Asaming each triphosphate was capable of chelating a ug‘H’ or Mn“ cation, the total calcu1ated concentration of bound divalent metal ions would be 29 x 10"“ N (11.8 x 10"“ plus 17.2 x 10"“). This brings the total bound netal ion concentra- tion to within a factor of 2 of the divalent cation concentra- tion initially added to the reaction mixture. However, not all of the triphosphates or DNA phosphates may bind as“ or Nn‘H'. The inhibition RNA synthesis at high ribonucleoside triphosphate concentrations was observed. This phenomenon may also be explained on the basis of as“ and Mn“ binding to both DNA phosphate and the tripho sphates. The concentra- tion of DNA phosphate in these reaction mixtures was 7 x 10"” N. The concentration of the triphosphates at which inhibition was first observed was #0 x 10"“ N. Since the concentration of divalent metal ions was only 50 x 10"“ 1!, it must be suspected that the as“ end Nn'H' concentrations were inmfficient and rate limiting. When the 60% Anoniun Sulfate Fraction II was assayed at temperatures below 30°C, a lag phase in RNA synthesis was lil‘ll llas‘ ls‘[[ l ‘1‘ l {[I‘l‘ll‘lll[{[[[[l 65 observed. Zillig, Fuchs and Nillette (1966) have observed a lag in the synthesis of RNA when the g. 921; enzyme was assayed at 20°C. When the 3. 211133 enzyme was preincubated with DNA before addition of the triphosphates, the lag phase was reduced. These results suggest that either the binding or the initiation reaction was affected by low temperatures. Richardson (1966) has observed that binding of It DNA to g. 32;; RNA polymerase was rapid and complete at O to 10°C. Therefore, the initiation reaction is the process which is probably affected by temperature. g LE9. synthesis of RNA does not proceed equally with each DNA tasplate. This observation may be explained on the basis that there are physical as well as chemical differences among the DNA templates. r“ and gh-l mu have ssaller _i_n_ 313 molecular weights than does 3. 2932-29. and probably salmon sperm DNA. It is expected that the large DNA molecules would be more maceptible to fragaentation during the isolation procedure. Fragmentation of gh-l DNA does not occur during its purification. Furthermore, the purification of the bacteriophage DNA yielded a template having a greater A260: A230 ratio than did g. m. Both 1'“ and gh-l DNA are linear. However, Th contains the unusual gluco sylated hydroxymethyl cytosine, whereas, gh-l DNA contains no unuaial bases (Lee and Boezi, 1966). The assay employed for the measurement of the rate of perpho sphate formation is unique. Its development was aided by the fortuitous facts that none of the enzyaes in the assay have a pH optimum far from that of RNA polymerase and that 66 none of the caponents in the assay of RNA polymerase inhibit the coupling enzymes at the concentrations employed in the assay. The assay permits the kinetic study of the formation of pyropho sphate in the spectrOphctometer where the results are observed and plotted as an infinite series of points- ' Using the spectrophotometric assay it was shown that the RNA polymerase catalyzed formation of pyrophosphate was DNA dependent and required the nucleoside triphosphates for activity. Furthermore, perphosphate formation was town to be inhibited by nogalamycin and actinomycin D, two specific inhibitors of the synthesis of RNA. when DNase was added to the reaction mixture, no pyrophosphate fonation was observed. Finally, the (rate of pyrophosphate formation was linearly dependent upon the concentration of the enzyme. The advantage of the spectrOph’ctometric assay in the study of RNA polymerase is that the reaction may be studied under conditions much that neither RNA nor perphosphate accumulate, i.e. in the presence of RNase. When the exper- iment was performed,“ was found that RNase stimulated the reaction by up to 1.10 fold. Krakow measlred FZPPi] release from ‘6 -32P labeled ribonucleoside triphosphates during the reaction of RNA polymerase. It was observed that when pancreatic ribonuclease and T1 ribonuclease were added to DNA-dependent mu polymerase reactions, there was a stimula- tion of [32133] release and its formation was linear for relatively longer periods. The results were consistent with the hypothesis that RNA formed during the course of the 6? reaction inhibited the reaction. It was apparent from the comparison of the two methods of assay that pyrophosphate fonation was linear for longer periods and was of greater extent than was the observed RNA synthesis. Two possible explanations are considered. The first implicates the action of RNase to explain the remilts. That RNase is present in the 60% Ammonium Sulfate Fraction II was demonstrated by the reduction in TCA insoluble radic- activity following #0 minutes of reaction. A steady state between synthesis and degradation of RNA probably existed at 1&0 minutes of reaction. As the RNA polymerase reaction began to stop, the RNase degraded more RNA than was synthesized. The total RNA in the reaction mixture decreased as was observed. The spectrophotometric assay, however, measured the formation of pyrophosphate. The presence of RNase, as has been shown, stimulated the reaction and prevented the formation of the complex which would step further RNA synthesis. Thus, pyrophosphate would occur long after. the steady state rate of mu synthesis had been achieved. It has been demonstrated that high RNase activities do not exist in the 60$ Ammonium ailfate Fraction II. Therefore, the question of whether or not sufficient RNase activity is present to account for the above explanation must be answered. The second possibility suggests that following the forma- tion of the couplex which prevents further synthesis of RNA, the nucleoside triphosphates continue to be cleaved to the 68 monOpho sphates and pyropho sphate by a perpho sphorylase-like activity of the bound enzyme. The bound enzyme, in effect, would be uncoupling RNA synthesis from perpho sphate fana- tion. A choice between the two possibilities presented above awaits studies on more highly purified fractions which can be demonstrated to be free of RNase. Further purification and characterization of DNA-dependent RNA polymerase will be performed by this investigator as a part of his Ph. D. program. 1. 2. 3. 1+. 5. 6. 7. 8. 9. 10. 11. 12. 13. ll}. 15. 16. REFERENCES Abelson, J., and Thomas, C. A., Jr., J. No___1_. _B___iol., l__8_, 262, (1966). 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