/. Biochem., 79, 53-60 (1976)
Tsuguo MIZUOCHP and Hirosuke FUKASAWA Department of Biology, Faculty of Science, Kobe University, Rokkodai, Kobe, Hyogo 657 Received for publication, June 12, 1975
The properties of poly(G) polymerase and poly(A) polymerase activities in the DNAdependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower {Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25—28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium.
Poly(A)-synthesizing activity in the presence of DNA has been reported in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6] fraction of procaryotes (1—3) and of a eucaryote (4). There was, however, little poly-
(G)-synthesizing activity. In a previous paper (5), we demonstrated that DNA-dependent RNA polymerase fraction isolated from cauliflower {Brassica oleracea var. botrytis) possesses a high synthesizing activity for poly(G) as well as poly(A). Sasaki ei al. (6) have also re-
1
This study was supported in part by a grant (No. 848003) from the Ministry of Education, Science and Culture, of Japan. * Present address: Department of Biochemistry, Kobe University School of Medicine, Kusunoki-cho, Ikuta-ku, Kobe, Hyogo 650. Abbreviations: Poly(G), polyguanylate; PoIy(A), polyadenylate; Poly(C), polycytidylate; Poly(U), polyuridylate; Poly(dA)-poly(dT), polydeoxyadenylate-polydeoxythymidylate; Poly(dG)-poly(dC)( polydeoxyguanylate-polydeoxycytidylate. Vol. 79, No. 1, 1976
53
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Comparative Studies on Polyguanylate Polymerase and Polyadenylate Polymerase Activities in the DNA-dependent RNA Polymerase I Fraction from Cauliflower1
T. MIZUOCHI and H. FUKASAWA.
54
MATERIALS AND METHODS Chemicals—Radioactive uC-labeled nucleoside triphosphates were purchased from the Radiochemical Centre, Amersham. Unlabeled nucleoside triphospates, calf-thymus DNA (type I), ribonuclease TL [EC 3.1.4.8], poly(A), poly(G), poly(U), poly(C), and yeast RNA (type XI) were obtained from Sigma Chemical Co., yeast transfer RNA and a - Amanitin from Boehringer Co., pancreatic ribonuclease [EC 3.1.4.22] (3000 units/mg) from Worthington Biochemical Co., rifampicin (Rifampin) from Calbiochem, Inc., actinomycin D from SchwarzMann, DEAE-cellulose DE-52 from Whatman Co., and poly(dA)-poly(dT) and poly(dG)-poly(dC) from P-L Biochemicals, Inc. All other chemicals were of reagent grade. Preparation of Enzyme Fraction — aAmanitin - insensitive DNA - dependent RNA polymerase fraction from cauliflower was pre-
pared by the procedures described in the previous paper (13). These procedures yield a greater amount of polymerase I preparation than of polymerase II. All operations were carried out at 2—4°. Assay of Enzyme Activity—Three polymerase activities, RNA polymerase, poly(G) polymerase, and poly(A) polymerase, were assayed in the following two standard reaction mixtures in a total volume of 0.125 ml, unless otherwise indicated. 1) The reaction mixture for RNA polymerase activity contained : 40 mM Tris-HCl, pH 7.5 at 20°, 1 mM MnCl2, 2 mM MgClt, 12 mM 2-mercaptoethanol, 0.4 mM each of ATP, GTP, CTP, and 0.2 mM [UC]UTP (& ^Ci//imole), 40 /^g/ml heat-denatured calfthymus DNA and enzyme (50 i*\). 2) The reaction mixture for poly(G) polymerase activity or poly(A) polymerase activity contained: 40^ mM Tris-HCl, pH 7.5 at 20°, 5 mM MnCU, IS mM MgCU, 12 mM 2-mercaptoethanol, 0.4 mM [UC]ATP for poly(A) polymerase activity or [UC]GTP for poly(G) polymerase activity (each 4 /iCi/^mole), 40 /*g/ml heat-denatured calfthymus DNA and enzyme (50 fi\). The amounts of enzyme protein used were in the range where the activity depended linearly on protein concentration. After incubation for 10min at 40° for RNA polymerase and poly(G) polymerase assays, at 30° for poly(A) polymerase assay), 100 (i\ of the incubation mixture was assayed by the paper-disc method described in a previous paper (5). Enzyme activity was expressed as pmoles of nucleotides incorporated under the respective assay conditions. Corrections of the activity values were made for zero-time controls. Chain-length Determination—Labeled poly(G) or poly(A) was synthesized in the standard reaction mixture. After incubation, the reaction mixture was precipitated with 5% trichloroacetic acid (TCA), washed with 5% TCA to remove unreacted ['^CJGTP or [UC]ATP, dried in vacuo, and hydrolyzed with 0.3 N KOH at 37° for 20 hr. The hydrolysates were spotted on filter paper (Whatman 3-MM) with authentic GMP and guanosine or AMP and adenosine, and developed with 1M ammonium acetateethanol (3 : 7, v/v). The appropriate areas were cut out and counted in a scintillation counter,. /. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/79/1/53/772230 by Leiden University / LUMC user on 16 January 2019
ported that the a-Amanitin-insensitive fraction from cauliflower RNA polymerase has purine ribonucleotide homopolymer-synthesizing activity, and they considered that the activity is intrinsic to enzyme I. Recently poly(G) polymerase as well as poly(A) polymerase was discovered in wheat chloroplasts (7). In Escherichia coli, poly(G) polymerase induced by RNA bacteriophage has been observed in the presence of poly(C) as a template (8, 9), and purine polyribonucleotide polymerase has been isolated from E. coli B (10). Niessing and Sekeris (11, 12) found a ribohomopolymer polymerase including poly(G>forming activity in rat liver nuclei. These poly(G)-synthesizing enzymes require only a ribonucleic acid as a primer. However, cauliflower RNA polymerase used in the present experiment, as described in the previous paper (5), preferentially utilized denatured DNA as a template for the synthesis of poly(G) or poly(A). From comparative investigations on the in vitro synthesis of poly(G), poly(A), and RNA in the polymerase I fraction, it appeared that there were three different mechanisms in these polymerizing reactions and that the synthesis of poly(G) or poly(A) was not due to a purine polyribonucleotide polymerase.
POLY(G)- AND POLY(A)-POLYMERASE ACTIVITIES
The cahin length was determined from the ratio of total counts in nucleoside monophosphate and nucleoside divided by the counts in nucleoside.
Effect of pH on the Enzyme Activity— Figure 1 shows the effect of pH on the activity of poly(G) polymerase, poly(A) polymerase, and RNA polymerase measured in Tris-HCl buffer and Tris-maleate buffer. Different activity profiles were obtained at various pH values for these enzyme activities. In TrisHCl buffer, the optimum pH's of poly(G)-, poly(A)-, and RNA-polymerase activities were 7.0-8.0, 7.5, and 7.0, respectively, though a broad optimum was found for the poly(G) polymerase reaction. In Tris-maleate buffer the pH optimum were similar to those in Tris-HCl buffer. In a parallel experiment, the optimum pH of E. colt RNA polymerase was found to be 8.0-8.5 in Tris-HCl buffer, as described elsewhere. Time Course of Radioactivity Incorporation—The course of radioactivity incorporation
into acid-insoluble material in relation to incubation time at various temperatures is shown in Fig. 2. GTP incorporation was almost linear for the first 10 min, then increased gradually except at 50°. ATP incorporation was almost linear at lower temperatures, but decreased at higher temperatures : 35° and 40°. It was evident that ATP incorporation proceeded even at low temperature ; 5.5°. The optimum temperatures during the first 10 min, shown in Fig. 2-d, were 45° for poly(G) and RNA polymerizations, but 35° for poly(A) polymerization. Thus, the activities of these polymerases were assayed at 40° for RNA synthesis and poly(G) synthesis, and 30° for poly(A) synthesis. The heat stability of the enzyme fraction was investigated. The enzyme was pre-incubated at 35° for 1 to 4 hr and the activities retained were assayed. It was found that poly(A) polymerase activity was always reduced to a greater extent, although the patterns of the decline curves in relation to pre-
600
400
•300
600,
-600
-300
0 10 20 30 Time(min)
Fig. 1. Effect of pH on the enzyme activity. Assays were performed with the appropriate system for each enzyme activity as described in " MATERIALS AND METHODS," except for the pH, which was adjusted at 20°. a) Tris-HCl buffer, b) Tris-maleate buffer, A , RNA polymerase activity; O, poly(G) polymerase activity; • , poly(A) polymerase activity. Vol. 79, No. 1, 1976
60
20 30 A0# 50 Temperature (*C )
Fig. 2. Time course of radioactivity incorporation at various temperatures. Assays were done in the standard reaction mixture except for the incubation time and temperature, which were as indicated in the figure, a) RNA polymerase activity; b) poly(G) polymerase activity; c) poly(A) polymerase activity; d) activities on incubation for 10min. 2-d; • , RNA polymerase activity; O, poly(G) polymerase activity; • , poly(A) polymerase activity.
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RESULTS
55
T. MIZUOCHI and H. FUKASAWA
56
SO
100
01 02 0-3 ["O-NTP conc»ntr*tlon ( mM )
Fig. 3. Effect of 14C-labeled substrate concentration in the low concentration range. The reactions were performed in the standard assay system, except for the concentration of "C-labeled substrates. •, RNA polymerase activity; O, poly(G) polymerase activity; • , poly(A) polymerase activity.
that of poly(A) polymerase. With KC1, small differences were observed at 20 mM, but considerable differences were found at higher concentrations ; RNA polymerase activity was not inhibited, but poly(G) and especially poly(A) polymerase activity was inhibited. It appears that the sensitivity to ionic strength was different in each reaction. The Effect of Metal Ion Concentration—In vitro poly(G), poly(A), and RNA synthesis by the polymerase I fraction requires divalent metal ions. The present experiment was mainly designed to determine what concentration of metal ions would be adequate for each poly-
20
40
60
80
M0
K d (mM)
Fig. 4. Effect of ionic strength on the three polymerase activities. The enzyme from the DEAE-cellulose column was desalted by passage through a Sephadex G-50 column and assayed in the standard reaction mixtures. A, RNA polymerase activity; 1 O, poly(G) polymerase activity; 6 • , poly(A) polymerase activities.c Activity at 100% was as follows. * 153 pmoles; b 176 pmoles; e 678 pmoles. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/79/1/53/772230 by Leiden University / LUMC user on 16 January 2019
incubation time were similar for the three enzyme activities. This was in agreement with the results of the above experiments on the time course of incorporation at various temperatures. The Rate of Synthesis vs. Substrate Concentration—The dependence of the rate of synthesis on substrate concentration was investigated for the poly(G)-, poly(A>, and RNAsynthesizing reactions. l4C-Labeled nucleoside triphosphates were used at various concentrations up to 0.6 mM, and it was found that 0.4 mM was sufficient for the poly(G)- and poly(A)polymerizing reactions, and 0.2 mM of all four nucleoside triphosphates for RNA polymerization. Since some differences in polymerizing patterns could be observed at lower concentrations of the substrates, more refined tests were made in the lower concentration range. As seen in Fig. 3, distinct differences in activity curves were found among the three reactions. It appears that the affinity of the enzyme for the substrate was different in the three polymerizing reactions. The Effect of Ionic Strength—-The effect of ionic strength on the activity of the three polymerases is shown in Fig. 4. In the case of ammonium sulfate, marked differences were observed. The activity of RNA polymerase was slightly stimulated in the presence of 20 mM (NH
Tsuguo MIZUOCHP and Hirosuke FUKASAWA Department of Biology, Faculty of Science, Kobe University, Rokkodai, Kobe, Hyogo 657 Received for publication, June 12, 1975
The properties of poly(G) polymerase and poly(A) polymerase activities in the DNAdependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower {Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25—28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium.
Poly(A)-synthesizing activity in the presence of DNA has been reported in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6] fraction of procaryotes (1—3) and of a eucaryote (4). There was, however, little poly-
(G)-synthesizing activity. In a previous paper (5), we demonstrated that DNA-dependent RNA polymerase fraction isolated from cauliflower {Brassica oleracea var. botrytis) possesses a high synthesizing activity for poly(G) as well as poly(A). Sasaki ei al. (6) have also re-
1
This study was supported in part by a grant (No. 848003) from the Ministry of Education, Science and Culture, of Japan. * Present address: Department of Biochemistry, Kobe University School of Medicine, Kusunoki-cho, Ikuta-ku, Kobe, Hyogo 650. Abbreviations: Poly(G), polyguanylate; PoIy(A), polyadenylate; Poly(C), polycytidylate; Poly(U), polyuridylate; Poly(dA)-poly(dT), polydeoxyadenylate-polydeoxythymidylate; Poly(dG)-poly(dC)( polydeoxyguanylate-polydeoxycytidylate. Vol. 79, No. 1, 1976
53
Downloaded from https://academic.oup.com/jb/article-abstract/79/1/53/772230 by Leiden University / LUMC user on 16 January 2019
Comparative Studies on Polyguanylate Polymerase and Polyadenylate Polymerase Activities in the DNA-dependent RNA Polymerase I Fraction from Cauliflower1
T. MIZUOCHI and H. FUKASAWA.
54
MATERIALS AND METHODS Chemicals—Radioactive uC-labeled nucleoside triphosphates were purchased from the Radiochemical Centre, Amersham. Unlabeled nucleoside triphospates, calf-thymus DNA (type I), ribonuclease TL [EC 3.1.4.8], poly(A), poly(G), poly(U), poly(C), and yeast RNA (type XI) were obtained from Sigma Chemical Co., yeast transfer RNA and a - Amanitin from Boehringer Co., pancreatic ribonuclease [EC 3.1.4.22] (3000 units/mg) from Worthington Biochemical Co., rifampicin (Rifampin) from Calbiochem, Inc., actinomycin D from SchwarzMann, DEAE-cellulose DE-52 from Whatman Co., and poly(dA)-poly(dT) and poly(dG)-poly(dC) from P-L Biochemicals, Inc. All other chemicals were of reagent grade. Preparation of Enzyme Fraction — aAmanitin - insensitive DNA - dependent RNA polymerase fraction from cauliflower was pre-
pared by the procedures described in the previous paper (13). These procedures yield a greater amount of polymerase I preparation than of polymerase II. All operations were carried out at 2—4°. Assay of Enzyme Activity—Three polymerase activities, RNA polymerase, poly(G) polymerase, and poly(A) polymerase, were assayed in the following two standard reaction mixtures in a total volume of 0.125 ml, unless otherwise indicated. 1) The reaction mixture for RNA polymerase activity contained : 40 mM Tris-HCl, pH 7.5 at 20°, 1 mM MnCl2, 2 mM MgClt, 12 mM 2-mercaptoethanol, 0.4 mM each of ATP, GTP, CTP, and 0.2 mM [UC]UTP (& ^Ci//imole), 40 /^g/ml heat-denatured calfthymus DNA and enzyme (50 i*\). 2) The reaction mixture for poly(G) polymerase activity or poly(A) polymerase activity contained: 40^ mM Tris-HCl, pH 7.5 at 20°, 5 mM MnCU, IS mM MgCU, 12 mM 2-mercaptoethanol, 0.4 mM [UC]ATP for poly(A) polymerase activity or [UC]GTP for poly(G) polymerase activity (each 4 /iCi/^mole), 40 /*g/ml heat-denatured calfthymus DNA and enzyme (50 fi\). The amounts of enzyme protein used were in the range where the activity depended linearly on protein concentration. After incubation for 10min at 40° for RNA polymerase and poly(G) polymerase assays, at 30° for poly(A) polymerase assay), 100 (i\ of the incubation mixture was assayed by the paper-disc method described in a previous paper (5). Enzyme activity was expressed as pmoles of nucleotides incorporated under the respective assay conditions. Corrections of the activity values were made for zero-time controls. Chain-length Determination—Labeled poly(G) or poly(A) was synthesized in the standard reaction mixture. After incubation, the reaction mixture was precipitated with 5% trichloroacetic acid (TCA), washed with 5% TCA to remove unreacted ['^CJGTP or [UC]ATP, dried in vacuo, and hydrolyzed with 0.3 N KOH at 37° for 20 hr. The hydrolysates were spotted on filter paper (Whatman 3-MM) with authentic GMP and guanosine or AMP and adenosine, and developed with 1M ammonium acetateethanol (3 : 7, v/v). The appropriate areas were cut out and counted in a scintillation counter,. /. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/79/1/53/772230 by Leiden University / LUMC user on 16 January 2019
ported that the a-Amanitin-insensitive fraction from cauliflower RNA polymerase has purine ribonucleotide homopolymer-synthesizing activity, and they considered that the activity is intrinsic to enzyme I. Recently poly(G) polymerase as well as poly(A) polymerase was discovered in wheat chloroplasts (7). In Escherichia coli, poly(G) polymerase induced by RNA bacteriophage has been observed in the presence of poly(C) as a template (8, 9), and purine polyribonucleotide polymerase has been isolated from E. coli B (10). Niessing and Sekeris (11, 12) found a ribohomopolymer polymerase including poly(G>forming activity in rat liver nuclei. These poly(G)-synthesizing enzymes require only a ribonucleic acid as a primer. However, cauliflower RNA polymerase used in the present experiment, as described in the previous paper (5), preferentially utilized denatured DNA as a template for the synthesis of poly(G) or poly(A). From comparative investigations on the in vitro synthesis of poly(G), poly(A), and RNA in the polymerase I fraction, it appeared that there were three different mechanisms in these polymerizing reactions and that the synthesis of poly(G) or poly(A) was not due to a purine polyribonucleotide polymerase.
POLY(G)- AND POLY(A)-POLYMERASE ACTIVITIES
The cahin length was determined from the ratio of total counts in nucleoside monophosphate and nucleoside divided by the counts in nucleoside.
Effect of pH on the Enzyme Activity— Figure 1 shows the effect of pH on the activity of poly(G) polymerase, poly(A) polymerase, and RNA polymerase measured in Tris-HCl buffer and Tris-maleate buffer. Different activity profiles were obtained at various pH values for these enzyme activities. In TrisHCl buffer, the optimum pH's of poly(G)-, poly(A)-, and RNA-polymerase activities were 7.0-8.0, 7.5, and 7.0, respectively, though a broad optimum was found for the poly(G) polymerase reaction. In Tris-maleate buffer the pH optimum were similar to those in Tris-HCl buffer. In a parallel experiment, the optimum pH of E. colt RNA polymerase was found to be 8.0-8.5 in Tris-HCl buffer, as described elsewhere. Time Course of Radioactivity Incorporation—The course of radioactivity incorporation
into acid-insoluble material in relation to incubation time at various temperatures is shown in Fig. 2. GTP incorporation was almost linear for the first 10 min, then increased gradually except at 50°. ATP incorporation was almost linear at lower temperatures, but decreased at higher temperatures : 35° and 40°. It was evident that ATP incorporation proceeded even at low temperature ; 5.5°. The optimum temperatures during the first 10 min, shown in Fig. 2-d, were 45° for poly(G) and RNA polymerizations, but 35° for poly(A) polymerization. Thus, the activities of these polymerases were assayed at 40° for RNA synthesis and poly(G) synthesis, and 30° for poly(A) synthesis. The heat stability of the enzyme fraction was investigated. The enzyme was pre-incubated at 35° for 1 to 4 hr and the activities retained were assayed. It was found that poly(A) polymerase activity was always reduced to a greater extent, although the patterns of the decline curves in relation to pre-
600
400
•300
600,
-600
-300
0 10 20 30 Time(min)
Fig. 1. Effect of pH on the enzyme activity. Assays were performed with the appropriate system for each enzyme activity as described in " MATERIALS AND METHODS," except for the pH, which was adjusted at 20°. a) Tris-HCl buffer, b) Tris-maleate buffer, A , RNA polymerase activity; O, poly(G) polymerase activity; • , poly(A) polymerase activity. Vol. 79, No. 1, 1976
60
20 30 A0# 50 Temperature (*C )
Fig. 2. Time course of radioactivity incorporation at various temperatures. Assays were done in the standard reaction mixture except for the incubation time and temperature, which were as indicated in the figure, a) RNA polymerase activity; b) poly(G) polymerase activity; c) poly(A) polymerase activity; d) activities on incubation for 10min. 2-d; • , RNA polymerase activity; O, poly(G) polymerase activity; • , poly(A) polymerase activity.
Downloaded from https://academic.oup.com/jb/article-abstract/79/1/53/772230 by Leiden University / LUMC user on 16 January 2019
RESULTS
55
T. MIZUOCHI and H. FUKASAWA
56
SO
100
01 02 0-3 ["O-NTP conc»ntr*tlon ( mM )
Fig. 3. Effect of 14C-labeled substrate concentration in the low concentration range. The reactions were performed in the standard assay system, except for the concentration of "C-labeled substrates. •, RNA polymerase activity; O, poly(G) polymerase activity; • , poly(A) polymerase activity.
that of poly(A) polymerase. With KC1, small differences were observed at 20 mM, but considerable differences were found at higher concentrations ; RNA polymerase activity was not inhibited, but poly(G) and especially poly(A) polymerase activity was inhibited. It appears that the sensitivity to ionic strength was different in each reaction. The Effect of Metal Ion Concentration—In vitro poly(G), poly(A), and RNA synthesis by the polymerase I fraction requires divalent metal ions. The present experiment was mainly designed to determine what concentration of metal ions would be adequate for each poly-
20
40
60
80
M0
K d (mM)
Fig. 4. Effect of ionic strength on the three polymerase activities. The enzyme from the DEAE-cellulose column was desalted by passage through a Sephadex G-50 column and assayed in the standard reaction mixtures. A, RNA polymerase activity; 1 O, poly(G) polymerase activity; 6 • , poly(A) polymerase activities.c Activity at 100% was as follows. * 153 pmoles; b 176 pmoles; e 678 pmoles. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/79/1/53/772230 by Leiden University / LUMC user on 16 January 2019
incubation time were similar for the three enzyme activities. This was in agreement with the results of the above experiments on the time course of incorporation at various temperatures. The Rate of Synthesis vs. Substrate Concentration—The dependence of the rate of synthesis on substrate concentration was investigated for the poly(G)-, poly(A>, and RNAsynthesizing reactions. l4C-Labeled nucleoside triphosphates were used at various concentrations up to 0.6 mM, and it was found that 0.4 mM was sufficient for the poly(G)- and poly(A)polymerizing reactions, and 0.2 mM of all four nucleoside triphosphates for RNA polymerization. Since some differences in polymerizing patterns could be observed at lower concentrations of the substrates, more refined tests were made in the lower concentration range. As seen in Fig. 3, distinct differences in activity curves were found among the three reactions. It appears that the affinity of the enzyme for the substrate was different in the three polymerizing reactions. The Effect of Ionic Strength—-The effect of ionic strength on the activity of the three polymerases is shown in Fig. 4. In the case of ammonium sulfate, marked differences were observed. The activity of RNA polymerase was slightly stimulated in the presence of 20 mM (NH
