TIIE JOURNAL OF BIOLOGKXL CHEMISTRY Vol. 237, No. 8, August 1962

Printed in U.S.A.

The Role of Deoxyribonucleic Acid in Ribonucleic Acid Synthesis

I. THE PURIFICATION AND PROPERTIES OF RIBO?XCLEIC ACID POLYMERASE”

J.J. hRTH,t JERARD HURWITZ,$ ASD ~$ONIKA ANDERS

From the Department of Microbiology, New York University School of Medicine, New York, New York

(Received for publication, January 11, 1962)

It has become evident that several enzymatic reactions exist which lead to the incorporation of the mononucleotide moiety of the ribonucleoside triphosphates into ribonucleic acid (1). The first of these enzymes to be characterized catalyzes the addition of cytosine 5’-phosphate and adenosine 5’-phosphate to the nucle- oside terminal end of soluble ribonucleic acid (2,3). The product of this reaction is a ribonucleic acid, terminated by pCpCpA, and active as an acceptor of amino acid (l-7). It is the purpose of this communication to report the method of purification and the characterization of a second enzyme. This enzyme, obtained from Escherichia coli, catalyzes the synthesis of RNA from the four ribonucleoside triphosphates, and is completely dependent on deoxyribonucleic acid. The other product of the reaction is inorganic pyrophosphate.

A number of reports from different laboratories have demon- strated the wide distribution of this enzyme and have discussed various aspects of the reaction it catalyzes (S-22).

EXPERIMENTAL PROCEDURE

Unlabeled purine and pyrimidine compounds were obtained commercially, as were creatine phosphate, acetyl phosphate, and phosphoenolpyruvate. Myokinase was obtained from C. F. Boehringer and Sons, GMBH, Maanheim, Germany. Strepto- mycin sulfate was obtained from Merck and Company, Inc.; protamine sulfate from Eli Lilly and’company, DNase, RNase, and lysozyme were obtained from Worthington Biochemical Corporation. ATP-8-Cl4 was obtained from Schwarz Bio- Research, Inc.

The isopropylidene derivatives of adenosine, uridine, cytidine, and guanosine used for the preparation of’P32-labeled mono- nucleotides by the procedure of Tener (23) were obtained from Aldrich Chemical Company, Inc. The P32-labeled mononucleo- tides were converted enzymatically to the respective diphosphates by specific kinases and to the triphosphates by nucleoside di- phosphokinase. Nucleoside diphosphokinase was obtained from rabbit muscle (24). Adenosinr-P32-PP was prepared from AMPZ2 using myokinase, creatine phosphate, and creatine phos- phokinase (25). The procedure for the preparation of cytidine- Pa2-PP has been outlined previously (26). UMP kinase was

* This research was supported by a grant from the National Institutes of Health, United States Public Health Service.

t Postdoctoral Fellow of the Sational Institutes of Health, United States Public Health Service.

$ Senior Research Fellow of the Xational Institutes of Health, Unit,ed States Public Health Service.

purified from E. coli by a procedure developed by Dr. A. Bresler.’ GMP kinase was obtained from E. co@ or Ascaris lumbricoides.3

Uniformly labeled C14-nucleotides were obtained by the photo- synthetic fixation of CY402 as described by Ofengand (27). GTP-8-C14 and UTP-2-Cl4 were isolated from red blood cells after incubation of the corresponding bases with red blood cells as described by Lowy, Williams, and London (28). Deoxy- cytidine triphosphate labeled in the a-phosphate was obtained by converting dCMP32, prepared as described by Tener (23), to dCDP by reaction with ATP and CMP kinase (24). Deoxy- guanosine-P32-PP was prepared from dGMP32 with dGMP kinase purified from E. coli infected by T2. Deoxy-CDP and dGDP were converted to the respective nucleoside triphosphate by the action of nucleoside diphosphokinase. Pi3’ was obtained from the Oak Ridge National Laboratory. Before use it was hydrolyzed with 1 N HCl for 1 hour at 100”. P32-labeled pyro- phosphate was prepared by the procedure of Berg (29).

Uridine-PP32-P was prepared with polynucleotide phosphory- lase and nucleoside diphosphokinase as previously described for cytidine-PP32-P (25).

The nucleotides were isolated by chromatography on Dowex 1 (Cl-) with increasing concentrations of HCI and LiCl. The elution pattern was as previously described (25). The appro- priate compounds in the eluate fractions were identified by their distinct.ive ultraviolet absorption spectra, and precipitated as the barium salt by the addition of BaOH, NaHC03, and ethanol. The barium salt was converted to the lithium salt with Dowex 50-Li+.

Calf thymus DNA was prepared by the procedure of Kay, Simmons, and Dounce (30). T2 DNA was prepared by the procedure of Kaiser and Hogness (31). DNA from fMicrococcus lysodeikticus was prepared by a modification of the procedure of Marmur (32). All DNA preparations were characterized by ultraviolet absorption as well as by the determination of their sedimentation constants in the analytical ultracentrifuge. The sedimentation coefficients of DNA of thymus, T2, and dl. lysodeikticus were 14.6 S, 61.4 S, and 18.7 S, respectively. These measurements were obtained from a single run at concentrations of DNA between 0.003 and 0.004 g per 100 ml in a solution of 0.2 RI NaCl containing 0.05 hT sodium citrate, pH 7.0. The

1 Unpublished procedure. * Kindly supplied by Dr. M. J. Osborn. 3 Kindly supplied by Dr. N. Entner. 4 We are indebted to Dr. R. C. Warner for these analyses.

2611

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Role qf DNA in RNA Synthesis. I Vol. 237, No. 8

sedimentation constants were calculated from photographic prints obtained with an ultraviolet absorption optical system.

Soluble-RNA was prepared from yeast by a modification (3) of the procedure of Monier, Stephenson, and Zamecnik (33). Soluble-RNA of E. coli was prepared by direct phenol extraction of the whole cells as described previously for yeast (3). Ribo- somal RNA was prepared from isolated ribosomes by phenol extraction after treatment of the ribosome fraction with DNase. The aqueous phase, after extractions of the digested ribosome suspension with phenol, was treated with 2 volumes of ethanol, dissolved with 0.05 M NaCl, and dialyzed against 0.05 M Tris buffer, pH 7.5, for 24 hours.

A number of other RNA preparations were also used. These included polyriboadenylate and polyribocytidylate (gifts of Dr. S. Ochoa), and RNA of tobacco mosaic virus (a gift of Dr. K. K. Reddi).

Protein was determined by the procedure of Sutherland et al. (34) except when high concentrations of Tris buffer, glycine buffer, or ammonium sulfate were present. In these cases, rather than subtract high blank values, the turbidimetric pro- cedure of Biicher (35) was used.

RNA was determined by the orcinol reaction (36) and Pi by the method of Fiske and SubbaRow (37). Pyrophosphate was determined as Pi after hydrolysis in 1 N HCl at 100” for 10 minutes.

The E. coli W cells used in these experiments were generously supplied by Lederle Laboratories. The conditions of growth have been described previously (3).

DEAE-cellulose (38) was obtained from Brown and Company. This material was treated as follows: 100 g were washed with 0.1 M NaOH + 1 M NaCl until the filtrates were colorless. The DEAE-cellulose was then washed with water until the pH was neutral, and charged by washing with 2 liters of 0.1 N HCl. Excess HCl was removed by washing with water until the fil- trates were neutral.

Assay of RNA Polymerase Activity-RNA polymerase activity was rountinely measured by the following procedure. The reaction mixture, 0.5 ml, contained 40 PM labeled ribonucleoside triphosphate, SO PM each of the other three ribonucleoside tri- phosphates, 2 mM MnClz, 8 mM MgC12, 50 InM Tris buffer, pH 7.5, 2 nnvr mercaptoethanol, calf thymus DNA equivalent to 40 mpmoles of deoxynucleotides, and enzyme. In those instances in which diluted enzyme was used, the dilution was made with a solution containing 2 mg per ml of crystalline bovine serum albumin and 1 mM mercaptoethanol. After incubation for 20 minutes at 38”, the reaction was stopped with 0.2 ml of 7% HC104, and 1.5 mg of albumin were added as “carrier” to insure complete precipitation of the nucleic acid. The acidified mixture was centrifuged for 3 minutes at 10,000 X g and the supernatant solution discarded. The precipitate was washed twice with 3-ml portions of 1 y0 HClO, solution and centrifuged each time. The precipitate was then dissolved in 1.5 ml of 0.2 N NHkOH and the solution decanted into metal planchets and dried. Radio- activity was measured in a windowless Geiger-Muller counter. No correction was made for self-adsorption. Incubation vessels in which DNA, metal, or enzyme was omitted served as controls. A unit of enzyme was defined as that amount of enzyme which catalyzed, with thymus DNA as primer, the incorporation of 1 mpmole of labeled ilTP or UTP into an acid-insoluble product in 20 minutes at 38”. Specific activity was expressed as units per milligram of protein.

RESULTS

Purification of RNA Polymerase-Two purification procedures were developed. With the most purified fraction obtained by either procedure, the reaction was completely dependent on DNA and the four ribonucleoside triphosphates. Since experi- ments were performed with enzyme obtained by both procedures, both methods of purification are given, although the specific activity of the most purified fraction obtained by Procedure 9 is considerably lower than that obtained by Procedure B.

Procedure A

Cells (100 g) were ground for 10 minutes in a precooled mortar with 200 g of Alcoa alumina A-301. The mixture was then sus- pended with 400 ml of a solution containing 0.01 M Tris buffer, pH 7.5, 0.01 M MgC12, and 10e3 M mercaptoethanol, and centri- fuged at 6000 x g. The residue was re-extracted with 200 ml of the solution containing Tris buffer, MgC12, and mercapto- ethanol and discarded. The combined supernatant solutions were then centrifuged for 120 minutes in the Spinco model L ultracentrifuge at 78,000 x g (average). A small wad of glass wool was added to the plastic centrifuge tubes before filling them with solution to trap the pellet when the clear supernatant fluid was decanted. The crude extract, 520 ml, contained 4.77 mg of protein per ml, 12.6 units per ml, and had a specific ac- tivity of 2.64.

The crude extract, 520 ml, was treated with 52 ml of 5% streptomycin sulfate and after 10 minutes at 2” the stringy precipitate was collected by centrifugation. The precipitate was extracted with 265 ml of 0.1 M Tris buffer, pH 8.4, containing 10e3 M mercaptoethanol. After 15 minutes at 2”, the suspension was centrifuged for 20 minutes at 6000 x g and the residue discarded. The Tris bu$er eluate, 245 ml, contained 31 units per ml, 1.1 mg of protein per ml, and had a specific activity of 28.

The Tris buffer eluate, 245 ml, was adjusted to 30% saturation with 41.8 g of solid ammonium sulfate and centrifuged. The supernatant solution, 255 ml, was adjusted to 60% saturation (46.2 g), and after 5 minutes at 2” it was centrifuged. This precipitate, which contained almost all the enzyme, was dissolved in 1O-3 M mercaptoethanol. The ammonium sulfate IA fraction, 9 ml, contained 1182 units per ml, 12.5 mg of protein per ml, and had a specific activity of 95.

To the ammonium sulfate IA fraction, 6.3 pg of crystalline pancreatic DNase were added and the solution was dialyzed for 16 hours against 6 liters of a solution containing 0.01 RI MnC12, 0.0025 M MgC12, 0.01 RI Tris buffer, pH 7.5, and 10e3 M mercaptoethanol. During dialysis a precipitate formed which contained variable amounts of enzyme. This activity could be extracted from the precipitate with 0.1 M imidazole buffer, pH 7.5. The imidazole buffer extract and the dialyzed supernatant solution were combined to yield 18 ml of solution containing 4800 total units and 4.9 mg of protein per ml. This solution was then fractionated with solid ammonium sulfate. The first pre- cipitate was obtained by the addition of 3.5 g of 0 to 35% solid ammonium sulfate. The second fraction containing most of the enzyme was precipitated by the addition of 1.65 g of 35 to 50y0 solid ammonium sulfate. This fraction, ammonium sulfate ZIA, 1.8 ml, contained 2120 units per ml, 16.8 mg of protein per ml, and had a specific activity of 126. It is referred to subse- quently as ASIIA.

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August 1962 J. J. Furth, J. Hurwitx, and iif. Anders 2613

All the enzyme fractions with the exception of the strepto- mycin sulfate fraction could be stored overnight at 2”. Con- centrated enzyme fractions could be stored at 2” for longer periods of time with only a gradual loss of activity. After 3 months at 2”, 70% of the enzyme activity of the ASIIA fraction remained. Repeated freezing and thawing led to inactivation.

Procedure B

Four hundred grams of E. coli W were mixed with 260 ml of a solution containing 0.01 M Tris buffer, pH 7.5, 0.01 M MgClz, and 0.001 M mercaptoethanol in a large Waring Blendor with 1000 g of No. 100 Superbrite glass beads (Minnesota Mining and Manufacturing Company) for 15 minutes at -10”. The glass beads were precooled to -lo”, and during the grinding the temperature of the mixture did not rise above 8”. The mixture was then treated with 500 ml of the solution containing Tris buffer, MgC12, and mercaptoethanol and the mixing continued for 3 minutes. The glass beads were permitted to settle and the supernatant fluid was decanted. The residue was then re- extracted with 400 ml more of the buffer solution by a l-minute mixing in the Waring Blendor, allowed to settle, and the super- natant fluid combined with the first extract. The extract was then centrifuged for 90 minutes at 78,000 X g (average) in the Spinco model L ultracentrifuge in plastic tubes containing glass wool. The supernatant fluid, the crude extract, was decanted and the precipitate discarded.

The crude extract, 1000 ml, was treated with 250 ml of a 0.5% solution of protamine sulfate. After 5 minutes at 0”, the stringy precipitate was collected by centrifugation at 6000 x g in the Servall centrifuge. The pellet was then washed two times by suspension in 500 ml of a solution containing 0.1 M p,

P-dimethylglutarate buffer, pH 7.0, and lop3 M mercaptoeth- anol and recentrifugation. The washings contained negligible amounts of enzyme and were discarded. RNA polymerase was then eluted from the protamine precipitate by washing with 250 ml of a solution containing 0.5 M sodium succinate buffer, pH 6.0, and 1O-3 M mercaptoethanol to yield the protamine eluate.

The protamine eluate, 246 ml, was adjusted to 20% saturation by the addition of 26.3 g of solid ammonium sulfate. After 15 minutes at O”, the precipitate was removed by centrifugation. The supernatant solution, 254 ml, was then adjusted to 35% saturation with 20.4 g of solid ammonium sulfate. This precipi- tate, containing most of the RNA polymerase, was dissolved with approximately 7 ml of 0.1 M Tris buffer, pH 8.4, containing 1O-3 M mercaptoethanol (ASIB fraction).

A column of DEAE-cellulose (20 cm X 3 cm) was washed with 80 ml of a solution containing 0.1 M Tris buffer, pH 8.4, and 10e3 M mercaptoethanol. The enzyme solution (ASIB, 6 ml) was diluted to 60 ml with 0.1 M Tris buffer, pH 8.4, contain- ing 1O-3 M mercaptoethanol and applied to the column. The column was then washed with 250 ml of a 0.5 M Tris buffer, pH 8.4, containing 1OV M mercaptoethanol. This removed approxi- mately 35% of the activity with little purification. The re- maining enzyme was eluted with 125 ml of 0.5 M Tris buffer, pH 7.5, containing 10e3 M mercaptoethanol. Approximately 10 12-ml fractions were collected; Fraction 7 (DEAE-cellulose eluate)

contained 5070 of the initial activity placed on the column. The DEAE-cellulose eluate, 11.4 ml, was treated with 4.1 g

of solid ammonium sulfate. The precipitate obtained after centrifugation was extracted with 5 ml of a solution containing 607. ammonium sulfate, adjusted to pH 7.8 with NH,OH, and

1O-3 M mercaptoethanol. The residue remaining after centrifu- gation was re-extracted successively with 50%, 40%, 30’%, and 207, ammonium sulfate solutions as described above. All the activity was associated with the 40% and 307, ammonium sul- fate fractions. These enzyme fractions had approximately the same specific activity and were combined. This fraction is ASIIB.

The crude extract and ammonium sulfate IB fractions could be stored overnight at 2”, whereas the protamine eluate and DEAE-cellulose eluate fractions lost up to 50% of the initial activity after storage for 16 hours at 2”. The ASIIB fractions lost no detectable activity after 6 weeks at 2”, although freezing and thawing led to marked inactivation.

The purification procedure is outlined in Table I. The RNA polymerase activity has been purified approximately 300-fold with a 20 $& over-all yield.

Properties of Purified Enzyme-Except where noted, similar results are obtained with fractions ASIIA and ASIIB. The results reported previously (12, 13, 19) were obtained with the use of enzyme fraction AS-IIA.

Both of the purified enzyme fractions were free of DNA poly- merase. In Procedure B this enzyme is separated from RNA polymerase during the first ammonium precipitation. RNA polymerase is precipitated with 35% ammonium sulfate, while DNA polymerase remains in solution. DNA polymerase can then be precipitated by adjusting the supernatant solution to 60% saturation with ammonium sulfate.

The purified ASIIB fraction is relatively free of RNase and DNase. RNA formed in the reaction mixture is stable, and incubation of Bacillus subtilis-transforming DNA with 20 units of enzyme for 60 minutes at 38” does not lead to any loss of biological activity.

General Requirements of Reaction-Even with crude extracts, the incorporation of nucleotide with one of the ribonucleoside triphosphates as substrate is stimulated by the addition of the other three ribonucleoside triphosphates and by DNA. With the purified enzyme the requirement for the three ribonucleoside triphosphates and DNA is absolute (Table II). The addition of either DNase or RNase reduces the incorporation of AMP to a barely detectable level. Similar results are obtained when CMP or GMP incorporation is measured.

Essentially complete sensitivity to RNase and DNase is evident only with ASIIB preparation. With the ASIIA prep- aration, only a partial sensitivity to either RNase or DNase is observed. The reasons for this discrepancy are not clear and are currently being investigated.

TABLE 1

Purification of liLYA pol ymerase

Enzyme fraction UMP !incorporation

Specific activity*

total meits zlnilsjmg protein

Crude estractt 60,000 B-10 Protamine eluate 40,000 170 Ammonium sulfate IB I 28,000 440 I)EAE-cellulose eluate.. 15,000 2200 Ammonium sulfate IIB (AS-IIB) 12.000 2800

* See text for definition of units. t From 400 g, wet v-eight,, of Esdwichia coli W.

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2614 Role of DNA in RNA Synthesis. I Vol. 237, Tu’o. 8

With the most highly purified fraction, ASIIB, the reaction proceeds linearly for approximately 40 minutes at 38”. In a typical experiment, using 0.95 pg of protein (2.2 units) and T2 DNA as a primer, the incorporation of C14-AMP into RNA in 10, 20, and 40 minutes was 0.66, 1.19, and 2.26 mpmoles, re- spectively. After 40 minutes the rate of the reaction falls off, and by 60 to 80 minutes the reaction has practically ceased.

The rate of the reaction is proportional to enzyme concentra- tion. In an experiment with T2 DNA, the addition of 1.1, 2.2, 5.5, and 11 pg of protein (2.6 to 26 units) resulted in the incor- poration of 1.61, 3.24, 7.94, and 15.7 mpmoles of AMP, respec- tively, in 20 minutes.

The reaction shows very little change in rate between pH 7.2

TABLE II

12equirenaents of RNA polymerase with T2 DNA as prime?.

The reaction mixture, 0.5 ml, contained: 80 ~CIM labeled ribo- nucleoside triphosphate (CY4-ATP, 1 X lo6 c.p.m. per pmole, or CY-UTP, 5.75 X lo5 c.p.m. per pmole) ; 80 PM each of CTP, GTP, and UTP or ATP; 8 rnM MgC12; 2 rnM mercaptoethanol; 50 rnM Tris buffer, pH 75.; T2 DNA equivalent to 50 mpmoles of deoxy- nucleotide, and 1.1 pg, 2.6 units, of ASIIB enzyme preparation. The reaction was terminated after 20 minutes at, 38” and RNA measured as described in the text.

Additions / AMP. ~. UMP lncorporatlon nxorporation

1. Complete. ................ 2. Omit DNA ................ 3. Complete + RNase (5 fig). 4. Complete + DNase (5 rg) 5. Omit UTP. ............... 6. Omit ATP. ............... 7. Omit CTP. ............... 8. Omit GTP .............. 9. Omit Mg++. ............

2.09 <0.02

0.04 0.06 0.07

0.10 0.07

<0.02

2.14 <0.02 <0.02 <0.02

<0.02 <0.02 <0.02

i <0.02

FIG. 1. The effect of Mn++ concentration. The reaction mix- ture, 0.5 ml, contained: 40~~ CY4-ATP (1 X lo6 c.p.m. per pmole); 80 PM each of CTP, GTP, and UTP; 2 rnn% mercaptoethanol; 50 mM Tris buffer, pH 7.5; thymus DNA equivalent to 50 mpmoles of deoxynucleotide; and 1.2 units of ASIIB enzyme. The con- centration of Mn++ was varied as indicated. The reaction was terminated after 20 minutes, and the amount of acid-insoluble material measured as described in the text.

5 I I 2 4 6 6

S CMg++l mM

FIG. 2. The effect of Mg++ concentration. The conditions were as described in Fig. 1 except that Mg++ was used in place of Mn++.

and 8.6. The rate decreases markedly below pH 7.0 and at pH 6.5 is 50% of the rate at pH 7.5.

Metal Requirements-Initial difficulties with the purification of RNA polymerase with thymus DNA as primer and Mg++ as the only metal cofactor were resolved when Mn++ was added to the reaction mixture. Mn++ markedly stimulated the reaction and made purification of the enzyme activity possible.

The effects of Mn++ and Mg++, added separately, on the rate of AMP incorporation with the purified enzyme are summarized in Figs. 1 and 2. For Mn++ the K, is 2.0 x 10F3 whereas the observed Vmaxwas 1.15mpmoles. WithMg++theK,is4.8 x lop3 M and the observed V,,, was 0.36 mpmole. An extrapolated v max of 2.63 mpmoles for Mn++ and of 0.57 mpmole for Mg+f indicates that high concentrations of metal are inhibitory, Mn++ more so than Mg++.

With thymus DNA as primer, the combination of Mg++ and Mn+f results in approximately the same incorporation as with Mn++ alone, and Mn++ (or Mn++ and Mg++) results in greater incorporation than with Mg++ alone. In contrast, with T2 DNA as primer, Mn++ is not nearly as effective as Mg++, and the combination of Mg++ and Mn++ results in less incorporation than with Mg++ alone. The explanation for this may be related to the appearance of a precipitate during the reaction. The only other DNA preparation which we have found to behave similarly to T2 DNA in the presence of Mn++ is that obtained from the phage X. For these reasons, Mg++ alone was employed in studies with DNA of T2 and X.

-4 number of other metals were found to be inactive. These included Cu*, Ca++, Zn+f, Cd++, Al+++, Co++, and Ni++.

Requirement for Ribonucleoside Triphosphate-The K, for UTP was 6 x lOwE M (Fig. 3). Similar values were found for ATP (8 X low6 M), GTP (6 X 1OV M), and CTP (9 X 1OV M).

Nucleoside diphosphates show little activity, the reaction rate with GDP, for example, being only 5% of that with GTP. There was no detectable utilization of deoxynucleoside triphosphates. The addition of equal amounts of the four deoxynucleoside triphosphates was without effect on the rate of RNA formation. Conversely, the addition of the four ribonucleoside triphosphates and RNA polymerase to a reaction vessel containing the com- ponents of the DNA-synthesizing system had no effect on DNA formation as measured by dCMP32 incorporation. (A compari- son of these two enzyme systems will be published at, a later

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hugust 1962 J. J. F&h, J. Hurwitz, and M. Anders 2615

date.) On the basis of these observations, it would appear that both RNA polymerase and DNA polymerase are specific and, at least in vitro, neither system affects the activity of the other.

DNA Requirement--The requirement for DNA, evident even in early fractions, is absolute with the most purified enzyme preparations. As shown in Fig. 4, half-maximal velocity with thymus DNA is observed at a level of DNA equivalent to 14 mpmoles of deoxynucleotides per ml of reaction mixture. The rate reaches a maximum at a level of DNA equivalent to approxi- mately 100 mpmoles of deoxynucleotides per ml. The theoreti- cal value of V,,, from the LineIveaver-Burk plot (39) is 1.86 mpmoles; this agrees with the observed value and indicates that excess DNA is not inhibitory. The calculated K, for T2 DNA, with Mg++ as the only metal cofactor, was approximately the same.

RNA will not replace DNA. Soluble-RNA of E. co& and yeast, ribosomal RNA of E. coli, tobacco mosaic virus RNA, and polyribocytidylate are inactive.

Nature of Product-The product has the properties of RNA (Table III). An acid-insoluble product containing UMP32 was rendered acid-soluble by RNase or by treatment with alkali; DNase had no effect. Nearest neighbor studies (Table IV) show that all of the 2’(3’)-ribonucleotides isolated after alkaline degradation contain P3*, no matter which cr-Pa*-labeled ribo- nucleoside triphosphate is added.

Evidence for the synthesis de no2ro of RNA is derived from end group analysis, Alkaline degradation of RNA yields the 2’(3’) , B’nucleoside diphosphate from the 5’-phosphate end of the chain, the ribonucleoside from the 3’-hydroxyl end, and 2’(3’)-mononucleotides from the internal ribonucleotides. A product containing V-AMP was obtained from a reaction primed with thymus DNA. After degradation with alkali the mixture was neutralized with HCl and approximately 1 pmole each of 2’(3’) ,5’-adenosine diphosphate, 2’(3’)-AMP, and adenosine were added. The solution was treated with Norit and acidified with HCl. The Norit, containing the adsorbed ribonucleotides, was collected by centrifugation and washed three times with H20. The ribonucleotides were eluted from the charcoal with water-ethanol-ammonia, concentrated, and

5 IO 15 20 25 30 35 40 45 60 120

S CUTPI @l

FIG. 3. The effect of UTP concentration. The conditions were as described in Fig. 1 except that uridine-P32-PP (1 X 10” c.p.m. per mole) was used as the labeled ribonucleotide (its concentra- tion was varied as indicated), thymus DNA equivalent to 100 mpmoles of deoxynucleotides was added, and both 4 mM Mg++ and 2 m&l Mn++ were used.

CTHYMUS DNA] OPTICAL DENSITY AT 260 mp

FIG. 4. The effect of thymus DNA concentration. The con- ditions were as described in Fig. 1 extent that the amount of thymus DNA added was varied ai indicated, both 4 mM Mg* and 2 rnM Mn++ were included, and 2 units of the ASIIB enzyme prepa- ration were used.

TABLE III

E$ect of variozts agents on product

The reaction mixture is similar to that given in Table II, ex- cept that E. coli DNA (equivalent to 75 mpmoles of deoxynucleo- tides) was used in place of T2 DNA, and Mn++ (2 mM) was used in place of Mg++. After 30 minutes, the reaction was terminated with 0.2 ml of 7yo HClOd and the reaction vessel centrifuged. The precipitate was washed three times with 1% HC104, suspended in 1.0 ml of 0.2 M Tris buffer, pH 7.5, and tested as indicated. The control, and the reaction vessels containing RNase or DNase, were incubated 30 minutes at 38”.

Treatment I

Acid-insoluble radioactivity

1. Control (no addition). 2. RNase (1 pg). ......................... 3. DBase (lpg) ........................ 4. NaOH (0.5 M; 12 hr) ................. 5.HC10~(1~;15minat100°). ..........

c.pm. 1475 <20 1470 <20 <20

subjected to paper electrophoresis as described in Table IV. The ultraviolet-absorbing regions were eluted from the paper with 0.01 N HCl. Radioactivity was recovered from 2’(3’) ,5- adenosine diphosphate, 2’(3’)-AMP, and adenosine. No radio- activity was detected from weak ultraviolet-absorbing bands corresponding to 2’(3’)-CMP and 2’(3’)-GMP. As this proce- dure did not adequately separate 2’(3’)-UMP from 2’(3’),5’- adenosine diphosphate, the compounds containing radioactivity were rechromatographed on Dowex 1 (Cl-) columns (1.5 cm x 5 cm). Adenosine, 2’(3’)-AMP, and 2’(3’), 5’-adenosine diphos- phate were eluted with 0.002 M HCl, 0.01 M HCl, and 0.01 M

HCl + 0.1 M LiCl, respectively. The amount of radioactivity associated with these compounds after electrophoresis was quantitatively recovered after column chromatography. The materials eluted from the Dowex 1 (Cl-) were characterized by

their absorption spectra which, in all cases, corresponded to those of the adenine-containing compounds. The compounds, 2’(3’) ,5’-adenosine diphosphate, 2’(3’)-AMP, and adenosine,

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2616 Role of DNA in RNA Synthesis. I Vol. 237, No. 8

TABLE IV TABLE V--Continued PS2 content of 7nononucleotides isolated after alkaline degradation

of en.qmatically synthesized R,VA

The conditions for the synthesis of labeled RNA were as de- scribed in Table II except as follows. Mn++ was included in each reaction mixture, all components were increased 2-fold, and the concentration of labeled nucleoside triphosphate added was as follows: uridine-Pa*-PP, 70 ~LRI (9 X 10” c.p.m. per rmole); adeno- sine-PZ2-PP, 120 ph% (3 X lo6 c.p.m. per pmole) ; cytidine-P3*-PP, 60 p&f (5 X lo6 c.p.m. per rmole); and guanosine-P3*-PP, 16 hi (3 X lo6 c.p.m. per pmole). Five units of RNA polymerase (ASIIB) were added with the exception of the reaction containing GTPaZ in which 10 units of enzyme were added.

supernatant solution was neutralized with KOH, and KC104 removed after 30 minutes at 2”. Aliquots were used to measure orcinol color with AMP as the standard (32). The amount re- ported above has been multiplied by a factor of 2 since only the pnrine nucleotides react in the test.

After precipit’ation with acid and washing, the product was dissolved with 0.5 ml of 0.3 N KOH and incubated at 38” for 48 hours. The mixture was then acidified with HCl and insoluble material removed by centrifugation. Approximately I rmole of each of the follr 2’(3’) mononucleotides was added to each super- natant solution. Norit (0.1 ml of a 30yo suspension) was added, collected by centrifugation, and washed four times with HSO. The ribonucleotides were eluted from the charcoal with water- ethanol-ammonia, (1 part H20, 1 part 95% ethanol, and 0.08 part concentrated NH,OH) yielding approximately 800/, of the total radioactivity in the acid precipitate. After concentration of the solution at room temperature, the nucleotides were applied to paper strips and subjected to paper electrophoresis in 0.05 M formate buffer, pH 3.5. The individual ribonucleotides were quantitatively eluted from the paper with 0.01 N HCl and the amount of radioactivity measured

In Experiment 2 the reaction mixture, 1.0 ml, contained: 0.2 mM C14-ATP (0.94 X lo6 c.p.m. per wmole); 0.4 mM each of UTP, GTP, and CTP; 5 mM MnCl*; 125 rnM Tris buffer, pH 7.5; 5 rnM mercapt,oethanol; DNA equivalent to 75 mpmoles of deoxynucleo- tides; and 20 units of ASIIB. Control tubes lacking metal or en- zyme were run simultaneously. After 90 minutes the reaction was terminated by heating the solutions at 100” for 3 minutes; DNase, 10 rg, was added to each tube and Mn++ to those lacking metal, and the solution incubated for 30 minutes. To 0.05.ml ali- quots from this reaction mixture, 0.2 ml of 770 HClO, and 1.5 mg of albumin solution were added and the precipitate washed three times with 1% HClO(, dissolved in NHIOH, and CWAMP incorpo- ration measured. The remaining material was treated as follows.

1. For optical density increase and orcinol formation, an ali- quot (0.5 ml) from the above reaction mixture was precipitated with 0.2 ml of 7% HClO, and 1.5 mg of albumin. The precipitate was washed with 1 ml of 1% HClOl and combined with the first supernatant solution and saved for Pi and PPi determinations. The precipitate containing albumin and RNA was again washed, suspended in 1.2 ml of 0.5 N HC104, and heated at 100” for 10 minutes. The suspension was then centrifuged, the supernatant solution decanted, and the ultraviolet absorption at 260 ml de- termined. An absorbancy of 10 for 1 pmole of nucleotide was used to calculate the optical density increase. The solution em- ployed for optical densit*y measurement was also used in the or- cinol test. The color intensity at 670 rnp produced in the orcinol test was determined after 40 minutes at 100”. AMP served as the standard.

2. For PPi and Pi measurements, the acid-soluble aliquot ob- tained as described above w-as treated with 0.1 ml of 30% suspen- sion of Norit and centrifuged. The snpernatant solution contain- ing PPi was saved. The Norit was washed once with 1 ml of Hz0 and this washing was combined with the supernatant solution. An aliquot of the combined supernatant solution and washing was made I N with respect to HCl and heated at 100” for 10 minutes. Another aliquot of the combined solution was used for Pi determi- nation. The small amount of Pi found, equivalent to the amount initially present in the ribonucleoside triphosphates added, has been subtracted from the value for total Pi determined after acid hydrolysis t,o obtain the value of PPi formed during the reaction.

Labeled substratl DNA (source)

Uridine-P3*- PP

Adenosine- P32-PP

Cytidine- P32-PP

Guanosine- P32-PP

Thymus M. 1 ysodeik-

ticus Thymus M. 1 ysodeik-

tic21.9

Thymus M. lysodeik-

ticus Thymus ill. lysodeik-

ticus

Amount incor..

porated

mpnroles % % % % 4.38 30.0 26.0 24.0 20.0 2.72 13.4 15.1 29.6 41.9

5.10 18.0 31.8 27.8 22.4 2.54 11.0 14.6 32.4 42.0

3.04 35.1 21.9 23.0 20.0 5.43 18.5 15.5 31.1 35.0

3.80 36.0 31 .o 10.0 23.0 6.33 14.0 14.6 40.2 31.2

Distribution of radioactivity

UMP AMP CMP GMP

T.~BLE V

Stoichiometry of reaction

In Experiment 1, the four separate reaction mixtures, 5 ml each, contained: 40 PM each of ATP, CTP, GTP, and UTP; 8 mnf MgC12; 4 mM MnCl?; 50 mM Tris buffer, pH 7.5; 2 mM mercaptoethanol; DNA equivalent to 250 mpmoles of deoxynucleotides; and 17.3 units of enzyme (ASIIA). The labeled ribonucleotides, where added, possessed the following specific activities (counts per minute per pmole): C14-ATP, 2 X 106; ~I-P~WTP, 0.5 X 106; C14- GTP, 0.5 X 106; and ~I-P~~-UTP, 0.8 X 106. Control tubes were run in which the nucleoside triphosphates were added after the reaction. The reaction was terminated after 30 minutes. An aliquot was removed from each tube and t.he incorporation into an acid-insoluble form was measured as described in the text. The remaining material was precipitated with acid, washed three times wit.h 170 HClOI, and dissolved with alkali (1 ml of 0.5 M KOH). After 18 hours at 38”, the solution was acidified with HCIOl and the precipitate removed by centrifugation. The

Orcinol- %yt-

Incorporation of radioactive nucleotides Optical reacting material

No. ;;:;I

AMP , UMP j CMP / GRIP / Total- iF%z c$&;) phate

~- &mtole jmiole r*mole pmole

1 0.082 2

0.021 0.020 0.017/ 0.017 0.075 0.054

I 1 I (0.182)* 0.220 0.200 0.230

* This value was calculated as described in the text.

contained 2,100, 592.000, and 13,680 c.p.m., respectively.5 The observation that 2’(3’) ,5’-adenosine diphosphate contained radioactivity indicated that at least part, if not all, of the RNA

5 In this experiment, in which the ASIIA enzyme preparation was used, the ratio of adenosine to 2’(3’),5’-adenosine diphos- phate recovered was 6.5. In another experiment in which the ASIIB enzyme preparation was used, the ratio of adenosine to 2’(3’) ,5’-adenosine diphosphate was 1.4, whereas the ratio of 2’(3’)-AMP to 2’(3’) ,5’-adenosine diphosphate was 230.

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August 1962 J. J. Furth, J. Hurwitx, and M. Anders 2617

0.5 1.0 1.5 2.0

S, CPPil mM

FIG. 5. The effect of PPi concentration on the formation of radioactive nucleoside triphosphates. The reaction mixture, 0.5 ml, contained: 80 PM each of ATP, CTP, GTP, and UTP; 4 mM Mg++; 50 mM Tris buffer, pH 7.5; 2 InM mercaptoethanol; thymus DNA equivalent to 48 mpmoles of deoxynucleotides; 2 units of DEAE-cellulose eluate enzyme fraction; and varying amounts of PPi3’ (1.3 X lo6 c.p.m. per pmole). The reaction was terminated after 20 minutes by the addition of perchloric acid, 1.5 mg of albumin were added, and the acid-insoluble material removed by centrifugation. The precipitate was washed with 1 ml of 1% HCIOn and the washing combined with the first supernatant solu- tion. To the combined solutions, 5 pmoles of PPi followed by Norit (0.1 ml of a 3Op/ suspension) were added. The charcoal with adsorbed nucleotides was washed three times with 1% HC104, suspended in 1.5 ml of water-ethanol-ammonia, and the amount of radioactivity determined.

chains were initiated with the added ribonucleoside triphos- phates.

Xtoichiometry of Reaction and Net Synthesis of RNA-The relation of ribonucleotide incorporation to total RNA synthe- sized and to pyrophosphate produced is summarized in Table V. In Experiment 1 the total radioactivity converted into an acid- insoluble product, and the amount of orcinol-reacting material produced are nearly equal. In Experiment 2, only (Y-AMP incorporation was measured. From this value, the total in- corporation (182 mhmoles) was calculated from the base com- position of thymus DNA (A + T/G + C = 1.3). The values for the net increase of RNIZ, as measured by increase in ab- sorbancy at 260 rnp, the increase in orcinol-reactive material, and pyrophosphate formation are nearly equal. In a parallel experiment in which C14-uridine-PP32-P was used, there was stoichiometry between Cl“-UMP incorporated into an acid- insoluble form and PPi3’ produced. The latter was identified as PPi by chromatography (29).

Pyrophosphate Exchange-The enzyme catalyzes the incorpora- tion of PPi” into the ribonucleoside triphosphates. However, the concentration of PPi required for this reaction to occur is approximately loo-fold greater than that required for the forward reaction (the incorporation of ribonucleotides). The K, for PPi (Fig. 5) is lop3 M. At a concentration of lop6 M there is no detectable incorporation of PPi into nucleotide material, and this amount does not inhibit the forward reaction.

The requirements for PPi exchange are shown in Table VI. To demonstrate incorporation of PPi” into ribonucleoside triphos- phates, the reaction components in Table VI, Experiment 3, were increased lo-fold. Approximately 130,000 c.p.m. were converted into Norit-adsorbable material. This material was

eluted from charcoal with water-ethanol-ammonia, carrier nucleoside triphosphates were added, and the solution was chromatographed on a Dowex 1 (Cl-) column. CTP was eluted with a solution of 0.01 M HCl + 0.1 M LiCl and ATP; GTP and UTP were successively eluted in the order given with a solvent containing 0.02 M HCl and 0.2 M LiCl. Each of the ribonucleo- tides was identified by its characteristic ultraviolet absorption spectrum in acid. The distribution of P32 was CTP, 24%; ATP, 25%; GTP, 20%; and UTP, 31%.

The exchange reaction is dependent on the presence of DNA and is greatly reduced by the addition of DNase. It is stimu- lated by RNase addition and by the omission of a single ribo- nucleoside triphosphate (with the exception of UTP). A closer examination of the requirements indicates that the incorpora- tion of PPi into nucleoside triphosphate can occur with only two nucleoside triphosphates, but no clear pattern is apparent.

Pyrophosphorolysis of RNA-The pyrophosphorolysis of an RNA product (prepared in a reaction in which T2 DNA was used as the primer) is shown in Table VII. The rate of pyro- phosphorolysis is very slow and relatively large amounts of enzyme are required. If the enzyme concentration was halved from that shown in Table VII, the rate decreased proportionately.

TABLE VI

Requirements for pyrophosphate exchange

The reaction mixture, 0.5 ml, cont,ained: 80 PM each of ATP, CTP, GTP, and UTP; 8 mM of Mg++; 2 mM mercaptoethanol; 50 mM Tris buffer, pH 7.5; 1 rnM PPiS2 or Pia (both containing 1.35 X lo6 c.p.m. per Mmole); thymus DNA, equivalent to 50 mpmoles of deoxynucleotides; in Experiments 1 and 2, 1.3 and 1.7 units of the DEAE-cellulose eluate enzyme fraction were added; in Ex- periment 3, 17 units of ASIIB were added. After 20 minutes at 38”, the reaction was terminated by the addition of 0.2 ml of 770

HCIO1, 1.5 mg of albumin were added, the mixture centrifuged, and the precipitate washed with 2 ml of 1% HCIO,. The super- natant solutions were combined, and 5 Imoles of PPi and 0.1 ml of a 3070 charcoal suspension were added. The suspension was centrifuged and the charcoal was washed three times with water, suspended in 1.5 ml of water-ethanol-ammonia. and the amount of radioactivity determined

Additions

1. Control (complete). 2. Omit DNA 3. Omit, all ribonucleoside triphos-

phates ._.._.... .._...._ 4. Pi3* in place of PP;“‘. 5. Omit ATP.. _. 6. Omit CTP.. 7. Omit GTP .._.._........._. 8. Omit UTP. 9. Omit ATP and CTP..

10. Omit GTP and UTP.. 11. Omit ATP, GTP, and UTP.. 12. Omit ATP, CTP, and UTP. 13. Omit ATP, GTP, and CTP. 14. Omit CTP, GTP, and UTP 15. Complete + DNase (5 pg) 16. Complete + RNase (5 pg).

PPi incorporated into Norit- adsorbable form

Experi- Experi- ment 1 mentl

0.56 <0.02

<0.02 0.98 1.07 0.87 0.34 0.49

<0.02 <0.02

0.19 0.07

0.13

Experi- ment3

14.1 0.18

<0.02

20.4

1.27 21.7

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2618 Role of DNA in RNA Synthesis. I Vol. 237, No. 8

TABLE VII Pyrophosphorolysis of RNA produced from

TB DNA-primed reaction

The reaction mixture, 0.5 ml, contained: 8 mM MgC12; 1 mM PPiaz (1.0 X 10” c.p.m. per pmole); 50 m&l Tris buffer, pH 7.5; 2 II~M mercaptoethanol; RNA or DNA as indicated; and 20 units of ASIIB. After 20 minutes, the reaction was terminated and PP; incorporation determined as described in Table VI.

Additions PP; incorporation

into Norit- adsorbable form

nzpmoler I ln~nloles

1. RNA product.. 12 0.30 2. E. coli soluble-RN.4 22 <0.02 3. E. coli ribosomal RNA. 25 <O.O2 4. T2 DNA. 22 <0.02 5. RNA product, + RNase (5 rg) 12 <0.02 6. RNA product + Piz2 in place of

PPi32 12 <0.02

If the complementary DNA was added, the rate of PPi3’ in- corporation was inhibited. Under the conditions used, the reaction appears to be specific for RNB produced in the reaction, because soluble-RNA, ribosomal RNA, and DNA in equivalent amounts will not substitute for the reaction product. However, when large amounts of ribosomal RNA are used, there is detecta- ble pyrophosphorolysis.

DISCUSSION

An enzyme, purified 300-fold from E. coli W, catalyzes the net synthesis of RNA from the ribonucleoside triphosphates. The most highly purified enzyme contains no DNA polymerase activity and is relatively free of RNase and DNase. All four ribonucleoside triphosphates are required, and the RNA synthe- sized contains all four ribonucleotides. There is an absolute requirement for DNA and for Mg++ or Mn++.

The high affinity of the enzyme for the ribonucleoside triphos- phates and DNA, and the low affinity for PPi, suggest that the reaction is primarily synthetic. In the presence of an excess of enzyme and limiting amounts of one ribonucleoside triphosphate in an otherwise complete reaction mixture, incorporation proceeds until there is complete utilization of the limiting ribonucleotide. For example, the addition of 2 mkmoles of UTP32, and 20 units of enzyme in an otherwise complete reaction mixture resulted in the incorporation of 1.97 ml.rmoles of UMP into an acid-insoluble form in 20 minutes at 38”.

The products of the reaction are RNA and PPi. The RNA has been shown to begin de novo by the identification of labeled 2’(3’) ,5’-adenosine diphosphate from the product of a reaction in which CY4-ATP was used as a substrate. Assuming that 2’(3’) ,5’-adenosine diphosphate is representative of the be- ginning nucleotide, and AMP is representative of the internal nucleotides, the synthesized RNA would contain about 300 nucleotides and have a molecular weight of approximately 100,000.

PPi has been identified as the other product of the reaction and PPi” will exchange with the pyrophosphate moiety of the nucleoside triphosphates providing the concentration of PPi is relatively high. The exchange reaction requires DNA and is inhibited by DBase. However, unlike the synthetic reaction, the exchange reaction is somewhat stimulated by RNase or by

the omission of ATP, CTP, or GTP (but not UTP). One of the many possible explanations for this is that the RNA formed during the reaction inhibits the enzyme. This explanation is not entirely consistent with all the experimental observations, and further studies on this phenomenon are in progress.

That pyrophosphorolysis of RNA occurs but is a relatively poor reaction (Table VII) is consistent with the reaction being primarily synthetic. Detectable pyrophosphorolysis when large amounts of ribosomal RNA are used may be due to the presence of small amounts of RNA produced by RNA polymerase in oivo in the ribosomal RNA preparation.

The role of DNA in the reaction will be more extensively covered in a subsequent paper, but it should be noted here that the results to be reported are consistent with previously published observations (12, 13). The RNA formed is a complementary copy of the priming DNA, and as theoretical considerations implicate a DNA-like RNA in protein synthesis (40) it is tenta- tively concluded that RNA polymerase synthesizes an RNA involved in protein synthesis. Confirmation of this role for RNA polymerase is indicated by the recent observation that the addition of RNA polymerase and DNA of T2 to extracts of E. coli markedly stimulates amino acid incorporation into protein. This stimulation can be prevented by the addition of DNase6 (41).

SUMMARY

,4n enzyme which catalyzes the net synthesis of ribonucleic acid (RNA) from the ribonucleoside triphosphates of adenine, uracil, guanine, and cytosine has been purified approximately 300.fold from cell-free extracts of Escherichiu coli. For this synthesis, deoxyribonucleic acid and Mn++ or Mg++ are required in addition to the four ribonucleoside triphosphates. The other product of the reaction is inorganic pyrophosphate. The amounts of RNA and pyrophosphate produced are equivalent to the amount of ribonucleotide incorporated into RNA. The reaction has been shown to be reversible although the reaction proceeds more readily in the direction of RNA synthesis.

REFERENCES

1.

2.

3.

4. 5. 6.

7.

8.

9.

10.

11.

12.

DAVIDSON, J. N., International council of scientific unions review. Vol. S. Elsevier Publishing Co.. Amsterdam. 1961. p. 105: ’

- , , ,

PREISS, J., DIECKMANN, M., AND BERG, P., J. Biol. Chem., 236, 1748 (1961).

FURTH, J. J., HURWITZ, J., KRUG, R., AND ALEXANDER, M., J. Biol. Chem., 236, 3317 (1961).

CANELLAKIS. E. S.. Biochim. et Biophvs. Acta. 25, 217 (1957). HERBERT, E:, J. Biol. Chem., 231, 9$5”(1958). ’ ~ ’ ZACKAU, H. G., Acs, G., AND LIPMANN, F., Proc. Natl. Acad.

Sci. U. S., 44, 885 (1958). HECHT, L. I., ZAMECNIK, P. C., STEPHENSON, M. L., AND

SCOTT, J. F., J. Biol. Chem., 233, 954 (1958). SPIEGELMAN, S., in G. TUNEVALL (Editor), Recent progress in

microbioloaw, Charles C Thomas Publisher, Springfield, Ill., __. 1958, p. 81.

._ -

WEISS, S. B., AND GLADSTONE, I,., J. Am. Chem. Sot., 81, 4118 (1959).

HURWI~Z, J., BRESLER, A., AND DIRINGER, R., Biochem. and Biophys. Research Communs., 3, 15 (1960).

STET~ENS, A., Biochem. and Biophys. Research Communs., 3, 92 (1960).

FURTH, J. J., HURWITZ, J. AND GOLDMANN, M., Biochem. and Biophys. Research Communs., 4, 362 (1961).

6 J. J. Furth, J. Hurwite, and E. Hilberman, unpublished ob- servations.

by guest on June 7, 2020http://w

ww

.jbc.org/D

ownloaded from

August 1962 J. J. Furth, J. Hurwitx, and M. Anders 2619

13. FURTH, J. J., HURWITZ, J., AND GOLDMANN, M., Biochem. and Biophys. Research Communs., 4, 431 (1961).

14. WEISS, S. B., Proc. Natl. Acad. Sci. U. S., 46, 1020 (1960). 15. STEVE& A.; J. Biol. Chem., 236, PC 43 (1961). 16. WEISS. S. B.. AND NAKAMOTO. T.. J. Biol. Chem.. 236, PC 18

(1961). ’ I I

17. WEISS, S. B., AND NAKAUOTO, T., Proc. Natl. Acad. Sci. Ii. S., 47, 1400 (1961).

18. GEIDUSCHEK, E. P., NAKAMOTO, T., AXD WEISS, S. B., Proc. Natl. Acak i&i. i?. S., 47, 1405 (1961).

19. HURWITZ, J., FURTH, J. J., ANDERS, M., ORTIZ, P. J., AND AUGUST, J. T., Cold Spring Harbor Symposia Qua&. Biol., XXVI (1962).

20. HUANG. R. C.. MAHESHWARI. N.. AND BONNER. J.. Biochem. and lkophus: Research Cornmu&., 3, 689 (196Oj. ’

21. OCHOA, S:, BURMA, D. P., KROGE~, H., AND WEILL, J. D., Proc. Natl. Acad. Sci. U. S., 47, 670 (1961).

22. BURMA, D. P., KROGER, H., OCHOA, S., WARNER, R. C., AND WEILL, J. D., Proc. Natl. Acad. Sci. U. S., 47, 749 (1961).

23. TENER. G. M.. J. Am. Chem. Sot.. 83. 159 (1959). 24. BERG, b., AND’JOKLIH, W. K., J. bioi. Chek, 2i0, 657 (1954). 25. HURWITZ, J., AND BRESLER, A. E., J. Biol. Chem., 236, 542

(1961). 26. HURWITZ, J., J. Biol. Chem., 234, 2351 (1959).

27. 28.

29. 30.

31.

32. 33.

34.

35. 36. 37.

38.

39.

40. 41.

OFENGAND, E. J., Ph.D. thesis, Washington University, 1959. LOWY, B. A., WILLIANIS, M. K., AND LONDON, I. M., J. Biol.

Chem., 236, 1439 (1961). BERG, P., J. Biol. Chem., 233, 601 (1958). KAY, E. R. M., SIMMONS, N. S., AND DOUNCE, A. L., J. Am.

Chem. Sot., 74, 1724 (1952). KAISER, D. L., AND HOGNESS, D., J. MoEecuZar Biol., 2, 396

(1960). MARMUR, J., J. Molecular Biol. 3, 208 (1961). MONIER, R., STEPHENSON, M. C., AND ZAMECNIK, P. C.,

Biochim. et Biophys. Acta, 43, 1 (1960). SUTHERLAND, E. W., CORI, C. F., HAYNES, R., AND OLSEN,

N. S., J. Biol. Chem., 180, 825 (1949). B~~CIIER, T., Biochim. et Biophys. Acta, 1, 292 (1947). MEJBAUM, W., 2. physiol. Chem., 268, 117 (1939). FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem., 66, 375

(1925). PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot., 78,

751 (1956). LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot., 66, 658

(1934). JACOB, F., AND MONOD, J., J. Molecular Biol., 3, 318 (1961). WOOD, W., AND BERG, P., Proc. Natl. Acad. Sci. U. S., 43,

94 (1962).

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J. J. Furth, Jerard Hurwitz and Monika AndersPOLYMERASE

PURIFICATION AND PROPERTIES OF RIBONUCLEIC ACID The Role of Deoxyribonucleic Acid in Ribonucleic Acid Synthesis: I. THE

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