enzymatic synthesis of ribonucleic acid - jbc.org · reaction has been studied by several groups...

THE JOURNAL OF Broxvxca~ CHEMISTRY Vol. 239, No. 1, January 1964

Printed in U.S.A.

Enzymatic Synthesis of Ribonucleic Acid

II. PROPERTIES OF THE DEOXYRIBONUCLEIC ACID-PRIMED REACTION WITH MICROCOCCUS LYSODEIKTICUS RIBONUCLEIC ACID POLYMERASE*

C. FRED Fox? AND SAMUEL B. WEISS

From the Argonne Cancer Research Hospitalf and the Department of Biochemistry, The University oj Chicago, Chicago 37, Illinois

(Received for publication, June 3, 1963)

The discovery of enzymes which catalyze the deoxyribonucleic acid-dependent synthesis of ribopolynucleotides (l-6) stimulated an immediate inquiry into the role played by DNA in thisunique reaction. The speculation that DNA might serve as a template for the polymerization of ribonucleotides into a complementary ribonucleic acid molecule was confirmed by studies from a number of different groups (7-15). The evidence for this may be summarized as follows: (a) when different DNAs (either natural or synthetic) which varied widely in their respective guanine, adenine, cytosine, and thymine content were used, the base composition of the RNA product always resembled that of the particular primer used; (b) the nearest neighbor frequencies for the 16 possible base pairs in the RNA formed were remarkably similar to the frequencies of the same base pairs in the DNA4 primer; and (c) specific RNA-DNA hybrids could be formed only by anneal- ing the purified RNA product with the DNA which served to direct its synthesis.

Evidence has now been provided, from three independent sources, that the reaction in z&o results in the formation of RNA molecules which are complementary to both DNA strands (12- 15). Furthermore, the synthesized RNA is self-complementary, and is unique among ribopolynucleotides in that it may form a DNA-like highly ordered secondary structure (13). This infor- mation is included in the scheme for RNA synthesis (Fig. 1). At the present time, the question of whether one or both strands of DNA may serve to prime RNA synthesis in z&o is not re- solved.

Although the studies in vitro on RNA polymerase have eluci- dated the function of the primer and some of the physicochemical properties of the product, very little is known about the polymerization mechanism itself. A study of the properties of the reaction is important as an initial step in understanding the nature of this complex problem.

Two separate DNA-primed reactions have been detected with RNA polymerase: (a) in the presence of all four ribonucleoside triphosphates, an RNA product is formed which is complementary in base composition and sequence to its DNA primer (4- NTP reaction), and (a) in the presence of a single triphosphate, a ribopolynucleotide forms which contains only a single base

*This investigation was supported in part by funds from the Helen and Joseph Regenstein Foundation.

t Predoctoral Fellow of the National Science Foundation. $ Operated by The University of Chicago for the United States

Atomic Energy Commission.

(single-NTP reaction). The formation of RNA by the 4-NTP reaction has been studied by several groups (l-15), and the single-NTP reaction has also been observed by several investigators (15-17). We have confirmed and extended the observation of the DNA-primed synthesis of an RNA homopolymer. In this paper we shall describe some of the properties associated with the two types of DNA-primed reactions when catalyzed by the partially purified RNA polymerase prepared from Mierococcus lysod&tik.s.

EXPERIMENTAL PROCEDURE

Materials and Alethods-The enzyme preparations were prepared from lysed extracts of Micrococcus lysodeikticus by Proce- dure A as detailed in the preceding paper (18). The enzyme preparation corresponding to Fraction VI was used in all experiments described here. The assay methods for RNA synthesis and the experimental procedures were as in the previous report (18) unless otherwise stated.

All nonlabeled ribonucleotides were of commercial origin. Calf thymus DNA was purchased from the Sigma Chemical Com- pany. Rat liver DNA was prepared by the method of Kay, Simmons, and Dounce (19). Phage T4 DNA was a gift from Dr. L. Grossman. Phage T2 and T7 DNA’were isolated by phenol extraction (20) from the respective phages. M. Zysodeikticus DNA was prepared by the procedure of Marmur (21) as modified by Grossman.* DNA from crab testis was a gift from Dr. M. Rabinowitz. All other DNAs used were generously supplied by Dr. E. P. Geiduschek. DNA samples were dissolved in a solution of 0.01 M NaCl-0.01 M Tris buffer, pH 7.5. Heat-denatured DNA was prepared by heating for 10 minutes at 100” and rapidly quenching in an ice bath.

Ultraviolet-treated DNA was prepared by irradiation with a G15T8 Westinghouse St&lamp by the procedure of Grossman (22) except that no Vycor glass filter was employed.

Crystalline pancreatic RNase and DNase were purchased from Worthington Biochemical Corporation. The DNase was further purified by chromatography on DEAE-cellulose as suggested by Dr. S. Spiegelman. Exposure of turnip yellow mosaic virus RNA to the cellulose-treated DNase resulted in no apparent alteration of the sedimentation rate during sucrose gradient centrifugation. We are grateful to Dr. R. Haselkorn for per- forming these assays and for providing the viral RNA.

* L. Grossman, personal communication.

175

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176 Enzymatic Synthesis of Ribmucleic Acid. II Vol. 239, X0. 1

nATP I,...! 1 I \ / t---a .A u.

T I 1 nCTP g---c

+ nUTP

+

Mll++ +

nGTP I I c---g

I I

DNA Complementary

RNA

FIG. 1. Reaction scheme for complementary RNA synthesis

Spermine-HCl and spermidine phosphate were purchased from Mann Research Laboratories, Inc;; cadaverine-HCl and putres- tine-HCl from California Corporation for Biochemical Research; poly+lysine-HBr, molecular weight 110,000, from Pilot Chemi- cals, Inc.; protamine sulfate from Nutritional Biochemicals Corporation; and calf thymus histone from Worthington Bio- chemical Corporation. The polyamines were made up in water and neutralized with NaHC03.

Inorganic phosphate was determined by the method of Gomori (23). Inorganic pyrophosphate was estimated by the increase in Pi released after hydrolysis in 1 N HCl for 20 minutes at 100”. Labeled 3’PPi was prepared by the method of Jones et al. (24).

RESULTS

Requirements for .J-NTP Reactain-The formation of RNA complementary to its DNA primer by RNA polymerase requires all four ribonucleoside triphosphates, a divalent metal ion, and DNA (Table I). Maximal synthesis requires the presence of spermidine, but other polyamines also stimulate the reaction. The addition of either RNase or DNase results in no appreciable product formation. In the presence of all four deoxynucleoside triphosphates no reaction occurs, although substitution of dTT3*P for UT32P in a complete system results in the incorporation of measurable quantities of label into the acid-insoluble fraction. The ribonucleoside diphosphates cannot substitute for the triphosphates in this system.

In the absence of a single ribonucleotide, little or no reaction takes place. Fig. 2 shows the dependence of RNA synthesis on each ribonucleotide and the concent,rations required to achieve

TABLE I

Requirements for four-nucleotide DNA-primed reaction The complete reaction mixture, 0.50 ml, contained 0.10 M Tris-

HCl, pH 7.5, 0.4 mu each ATP, UTP, and GTP, 0.4 mM CT3*P (2.8 X lo6 c.p.m. per pmole), 2.5 mM MnCl,, 2.0 mM spermidine phosphate, 100 pg of calf thymus DNA, and 6 units of enzyme. The reaction was incubated for 20 minutes at 30” and then assayed for RNA formation (18).

System I

CMSP incorporated

Complete Omit ATP. ............. Omit UTP. ............. Omit GTP. ............. OnAt Mn++ ............. Omit DNA. ............ Omit spermidine. ....... Add ribonuclease ....... Add deoxyribonuclease.

10.3 <0.2 <0.2

0.5 <O.l <O.l

7.6 0.4 0.2

saturation. The calculated K, values for the respective triphosphates range from 3 to 6 X 10-G M and are similar but slightly higher than the values reported for Eschetihiu coli RNA polymerase (24).

pH Optimum-The reaction is optimal between pH 7 and 8.5, and falls sharply above or below these values.

Metal Ion Requirement-As mentioned previously, a divalent metal ion is required for RNA synthesis. For M. lysodeikticus “polymerase,” Mn++ is the most effective metal ion tested, although Co++ and Mg++ also stimulate synthesis (Fig. 3). At concentrations higher than 3 X lo-3 M, both manganese and cobalt inhibit polymerization, whereas inhibition with magnesium occurs at concentrations higher than lop2 M. Other metal ions such as Ni”, Al*, Fe++, Zn*, and Ca++ are not active. The metal ion requirements for this enzyme and the enzyme prepared from E. coZi (15,25) are similar except for the activation by Co++ observed with the X. lysodeiktieus preparation.

Linearity and Temperature-The polymerization reaction is linear only for a relatively short time (Fig. 4). After the initial reaction, polymerization proceeds at a slower rate and may continue for several hours. The time at which the reaction levels off varies with different DNAs or with different preparations of the same DNA. In this regard, RNA synthesis with T7 DNA as primer levels off significantly later than in incubation mixtures containing calf thymus DNA. The rate of RNA formation is slightly more rapid at 40” than at lower temperatures. With T7 DNA, the rates observed when assayed at various temperatures were not as different as one might expect. However, greater differences have been observed with other DNA primers (calf thymus) as well as RNA primers (polycytidylate). The effect of temperature on the reaction kinetics with RNA primers is presented in the following paper.

The addition of ribonucleotides, DNA, or both after the reaction has reached maximal synthesis does not further stimulate polymerization. The addition of more enzyme, however, results in another burst of RNA synthesis. The exact meaning of this phenomenon is unclear.

Net Synthesis and Stoichiometry of 4-NTP Reaction-The net synthesis of complement,ary RNA may be readily demonstrated with M. Zysodxikticus RNA polymerase (Table II) (4). The amount of alkali-sensitive material released after hydrolysis of the acid-insoluble ribopolynucleotide formed during the reaction compares rather favorably with the amount of ribonucleotide polymerized as calculated from the 32P incorporation data. Analysis of the acid-soluble fraction after synthesis indicates that the quantity of inorganic pyrophosphate released is nearly equivalent to the total number of micromoles of substrate incorporated. Our criterion for the presence of PPi is based on the increased amount of Pi which appears after acid hydrolysis. The Pi which is present before acid hydrolysis arises from the small amount of Pi normally found in commercial preparations of the nucleotides employed. The relatively high quantity of Pi released after acid hydrolysis is probably due to incomplete adsorption of the acid-soluble nucleotides onto charcoal. Be- cause of this, the analytical error for our PPi determination may be relatively high.

Native and Heat-denatured DNA Priming (.J-NTP Reactionb- In the 4-NTP reaction, both native and heat-denatured DNA can serve as primers for RNA formation (Fig. 5). Saturation with respect to primer is achieved with denatured DNA at


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January 1964 C. F. Fox and 8. B. Weiss 177

! =3.-r x 10-5 2 4 6 6 IO

$104 j ~~=6,13x10-6 2 4 6 E IO

I I I I I I

!

-I I !

~~~5~10-6 2 4 6 6 IO ~~~5~10-6 2 4 6 6 IO

$104

I I I I I 0 I 2 3 4 5 0 I 2 3 4 5

NIJCLEOSIDE TRIPHOSPHATE M x 10-4

FIG. 2. The reaction mixture, 0.50 ml, contained 0.10 M Tris- HCl, pH 7.5, 2.5 mM MnC12, 0.8 mM spermidine phosphate, 100 pg of calf thymus DNA, 3 units of enzyme, 0.80 mM each ATP, GTP, CTP, and UTP, and either 0.80 mM GT32P (6.60 X lo6 c.p.m. per Mmole) or 0.80 mM CTazP (1.21 X lo6 c.p.m. per @mole) as the labeled substrate. For demonstrating the dependence of the reaction on ATP, a combination of CTP, UTP, and GTazP was

used; for CTP, a combination of ATP, UTP, and GT32P; and for UTP, a combination of ATP, GTP, and CT3ZP. The ribonucleotides under study were added at various concentrations, and the mixtures were incubated for 10 minutes at 30”. The reactions were stopped with acid, and the acid-insoluble material was analyzed for radioactivity.

Metal Ion Concentration M x 10m3

FIG. 3. The reaction conditions were as for Fig. 2 except that MnC12, CoC12, and MgC12 were tested at the concentrations indicated above and 5 unit,s of enzyme were used.

Raoctlon lime (Minuter)

FIG. 4. The reaction conditions were as for Fig. 2 except that GTS*P served as the label and 50 ag of T7 DNA were used as primer. The vessels were incubated at the temperatures and time periods indicated.

approximately one-tenth the concentration observed for native DNA. Furthermore, under saturation conditions for both primers, native DNA is considerably more efficient in stimulating RNA synthesis than heat-denatured DNA. We have found similar results with different DNA primers.


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178 Enzymatic Synthesis of Ribonucleic Acid. II Vol. 239, No. 1

TABLE II

Stoichiometry of DNA-primed RNA synthesis (4.NTP reaction)

The reaction mixtures, 6.5 ml each, contained 0.10 M Tris-HCl, pH 7.5,0.4 mM each GTP, UTP, and CTP, 0.4 mM ATa*P (1.09 X lo5 c.p.m. per Mmole), 0.4 mru spermine, 600 rg of M. lysodeikticus DNA, and 50 units of enzyme. In addition, one vessel contained 2.5 mM MnClz. The reaction was stopped after 60 minutes by the addition of lOO/*moles of Versene, and the volume was adjusted to 7.0 ml. A l-ml aliquot was removed for the determination of AM3*P incorporation into RNA. The total RNA formed was calculated from the AM32P incorporation data by assuming that the thymidine content of M. lysodeikticus DNA was 16 mole To. A 5-ml aliquot was treated with HCIOI, and the acid-insoluble material was collected and washed three times by solution in 0.2 M NaOH and precipitation with acid. This material was then hydrolyzed in 0.5 M KOH at 37” for 18 hours, and the release of ultraviolet-absorbing material was determined at 260 rnp after acidification with HCIO, and removal of the precipitate. An EM of 10.3 X lo-$ mM was used to calculate the number of micromoles released from the acid-insoluble fraction by alkaline hydrolysis. Norit (200 mg) was added to the acid-soluble fraction, mixed and removed by centrifugation. This procedure was repeated once more. One aliquot was taken for Pi determination directly, and another aliquot was adjusted to 1 N HCl with concentrated acid, hydrolyzed for 20 minutes in a boiling water bath, and cooled, after which the Pi content was determined.

I Acid-insoluble fraction Acid-soluble fraction

system Nucleotides Total incorporated

K”~uc~$&is’ T;?l Total Pi p, released befk after

HCl releakd Net,

h$%- h’;s~‘- hyk- Pi/2

Total Net Total Net ysis ysis

Complete.. 1.178 1.177 1.50 1.29 1.65 13.30 11.65 1.26 OmitMn++l 0.001~ IO.21 1 ( 1.22 1 10.35 19.13 1

Requirement for Polyamine (4-NTP Reaction)-Optimal rates

and extension of RNA synthesis with M. lysodeikticus RNA polymerase require the presence of polyamines. The stimulation observed with added polyamines has ranged from 1.2 to 2.0 times that obtained in the absence of any organic cations. The formation of RNA in the presence and absence of spermidine with different DNA primers is presented in Table III. In each case, the addition of spermidine results in a significant enhance- ment of polynucleotide synthesis although the absolute amount of RNA formed varies slightly for the different DNAs used.

Spermine and putrescine are equally as effective as spermidine for satisfying the polyamine requirement in this system (Table IV). Optimal synthesis with each of these organic cations occurs at different molar concentrations. Table IV also indicates that calf thymus histone, protamine, and polylysine do not stimulate synthesis. At concentrations higher than those indicated in Table IV, most polyamines inhibit the reaction since they induce precipitation of the DNA primer.

So far, the requirement for polyamines has only been observed in the PNTP reaction where helical DNA serves as primer. With native DNA and in the absence of spermine, RNA synthesis appears to level off earlier than when spermine is present (Fig. 6). If, after 60 minutes of incubation in the absence of any polyamine, spermine is added, the rate and extent of RNA synthesis are increased. The exact interpretation of this

phenomenon is not clear. With heat-denatured DNA, the addition of spermine results in slightly elevated levels of label incorporation. This stimulation is smaller than that obtained with native DNA. With native DNA, the amount of polyamine required for optimal RNA synthesis varies with the concentration of DNA employed.

Znhibition of DNA-dependent Synthesis by RNA-The DNA- primed synthesis of RNA can be markedly inhibited by the addition of various RNAs to the polymerizing system. Syn- thetic RNAs as well as natural ribopolymers can inhibit the reaction. An example of this type of inhibition may be readily demonstrated by the addition of polyribocytidylate to a DNA- primed reaction in which AT32P serves as the labeled substrate (Fig. 7). As the polycytidylate concentration is increased, the incorporation of AM32P diminishes for each concentration of DNA used (Fig. 7A). A Lineweaver-Burk plot of the inhibition data indicates that for each level of polycytidylate tested, the extrapolated curves intersect at the same point on the ordinate, suggesting that the inhibition is of a competitive nature (Fig. 7B). This implies that polycytidylate can combine with the enzyme at sites along the enzyme surface where DNA normally binds. We can also demonstrate the reverse situation, that is, the DNA inhibition of the RNA-directed synthesis of RNA by the same enzyme. These findings will be reported in a succeeding paper. Krakow and Ochoa have reported similar results with A zotobacter RNA polymerase (29).

Effect of Ultraviolet Zrradiation of DNA Priming-Geiduschek has reported that under appropriate conditions of nitrous acid treatment, DNAs can be made which are fully reversible after

7- l Natlvr

6-

b j- I . :

2 I hat

2 *- ./*

’ Denatured

I I I I I 2 4 6 6 IO

17 DNA GM x lO’5 per ml.

FIG. 5. The reaction conditions were as for Fig. 2 except that native and heat-denatured T7 DNAs, at the concentrations indicated above, were used as primers. The reaction mixtures were incubated at 30” for 10 minutes.


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January 1964 C. F. Fox and S. B. Weiss 179

TABLE III

Eflect of spermidine with various DNA primers (4-NTP reaction)

The conditions for the reaction were similar to those described in Table I except that 50 pg of each DNA and 3.5 units of enzyme were used. Spermidine (2.0 mM) was added where indicated, and the reaction time was 60 minutes. The total polynucleotide formed was derived by dividing the number of millimicromoles of labeled substrate incorporated by the mole per cent of the substrate reported for each DNA (26-28).

Primer DNA

Calf thymus

Rat liver

Bacteriophage T2

Bacteriophage T4

Salmon sperm

Sea urchin sperm

Escherichia coli

Serratia marcescens

Pseudomonas jluorescens

Aerobacter aerogenes

Micrococcus lysodeikticus

Spermidine

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

Polynucleotide synthesized

nt~WZOlL?S

31.5 21.5 33.3 24.2 35.4 22.3 16.0 10.9 42.9 32.8 28.1 21.7 34.6 25.0 33.0 24.2 42.9 30.2 37.2 27.4 31.1 22.1

TABLE IV

Effect of various polyamines on RNA synthesis The reaction mixture and conditions used were similar to those

described for Table III.

Additions GM**P incorporated

m@?mles

None................................. 7.80 Spermidine (0.4 pmole). 10.35 Spermidine (1.0 rmole). 11.30 Spermine (0.1 pmole) 9.78 Spermine (0.2 pmole). 14.30 Putrescine (2.0 pmoles). 8.96 Putrescine (10.0 pmoles) 13.50 Cadaverine (2.0 pmoles) 8.75 Cadaverine (10.0 pmoles) 9.56 Calf thymus histone (20 p.(p). 7.98 Protamine (20 pg) 7.84 Polylysine (10 pg). 7.34

heat denaturation (30). He has concluded that such treatment, in part, results in cross-linking between the double strands of DNA. DNAs so prepared are relatively poor primers (31). Ultraviolet irradiation of DNA has also been reported to cause partial denaturation and cross-linking (32, 33), and therefore one might expect to find an impaired priming ability of DNA

exposed to this treatment as well as to nitrous acid. Fig. 8 demonstrates that exposure of DNA to ultraviolet irradiation rapidly reduces its ability to prime for RNA synthesis. Further- more, native DNA is considerably more sensitive to this treatment than heat-denatured DNA. The effects of ultraviolet irradiation on nucleic acids are complex (32). An extensive study of irradiated primers and the products formed in enzymatic systems of this type may help to clarify the complex changes which occur.

Requirements for the Single-NTP Reaction-Chamberlin and Berg first described the DNA-primed synthesis of polyadenylate catalyzed by E. coli RNA polymerase (15). M. Zysodeilcticus “polymerase” also catalyzes a reaction of this nature, and some of the requirements for the formation of two different homopolymers are given by Table V. In the presence of ATP, manganese ion, and calf thymus DNA, RNA polymerase catalyzes the vigorous synthesis of polyadenylate. Other homopolymers may be formed with different nucleotides or with different DNAs, e.g. the formation of polycytidylate in the presence of Pseudomonas jluorescens DNA. The extents of homopolymer formation for these two separate reactions are not the same. Moreover, the addition of other nucleotides markedly inhibits this synthesis. In the presence of four ribonucleotides, the amount of RNA formed is considerably lower than the ribopolymer assembled when only ATP is present. The relatively poor 4-NTP reaction is due primarily to the absence of polyamine from the reaction mixture. With P. jZuorescens DNA, the 4-NTP reaction is less extensive than usual, owing to the omission of polyamines, but it is still more active than ribopolymer formation with CTP alone.

In the single-NTP reaction, the addition of spermidine does not stimulate homopolymer formation. In fact, we have usually observed some inhibition when polyamines are present. DNase addition partially inhibits polyadenylate and polycytidylate

IO -

9-

x 6-

& 4 5-

‘0 I I I , , , I , , , , ,

20 40 60 SO 100 120

Minutes of Incubation

FIG. 6. The reaction conditions were similar to those described in Table I except that 0.2 mM spermine, 56 pg of native or heat- denatured calf thymus DNA, and 4.3 units of enzyme were used. ATaeP (specific activity, 0.30 X lo6 c.p.m. per #mole) was used as the labeled substrate.


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180

3.0

E 5 2.5

5

k g 2.0

z

x a 1.5 I Q

& 1.0

!.i

z * 0.5 E

0

3.0 -‘;; -

II 0:

E

z 2.0

--v)

!3

z

% I.C

C

Enzymatic Synthesis of Ribonucleic Acid. II Vol. 239, No. 1

- 5pg Poly C/ml.

/ ./

$5 - 4Opg Poly C/ml.

. * 100/q Poly C/ml

i’i

ip- @ .

L I I I 1 I

IO 20 30 40 50 /bg of DNA

/

IOOpg Poly C/ml.

4Opg Poly C/ml

I I I , I I

2 4 6 6 IO

FIG. 7. The reaction conditions were as in Table I except that no spermidine was present and ATasP was the labeled substrate. Four sets of vessels were prepared to which were added 0, 5, 40, and 100 pg of polyribocytidylate. To each set was added a different amount of calf thymus DNA as indicated. The vessels were incubated for 10 minutes at 30”, and the amount of AMazP incorporated into RNA was determined. The reciprocal values of the DNA concentrations and the millimicromoles of AMaSP incorporated were used for the plots in Part B. Poly C, poly- cytidylic acid.

synthesis, whereas RNase inhibits only polycytidylate formation* The synthesis of significant amounts of homopolymer even in the presence of added DNase is probably due to DNA fragments which are sufficiently large to serve as primers.

In the absence of manganese or DNA, little or no product forms. Fig. 9 illustrates the dependence of RNA formation on the concentration of DNA. The K, values for DNA in the C-NTP and single-NTP reaction in the absence of spermidine have been determined, and are approximately 5.9 x 1W6 and 3.8 x 10-‘J g per ml of reaction, respectively (Fig. 9, A and B). The similarity in the values suggests that in both cases the affinity of the enzyme for the primer is approximately the same.

Metal Ion Requirement (Single-NTP Reaction)-The single- NTP DNA-primed reaction, with M. Zysodeikticus RNA polymer-

ase, requires manganese ion for synthesis (Fig. 10). Optimal synthesis occurs at approximately 2 x 10m3 M MnCl, and is similar to the optimal concentration required for the 4-NTP reaction. Although Mg++ can partially substitute for Mn++ in the 4-NTP reaction, the single-NTP reaction is almost completely inactive in the presence of magnesium ion.

that Denatured DNA

Native DNA

01 I I I I 0 2 4 6 6

Minutes of Irradiation

FIG. 8. The reaction conditions were as in Table I except that 55 rg of native and heat-denatured calf thymus DNA were used. The method for ultraviolet inactivation is described under “Ex- perimental Procedure.” One hundred per cent GMa*P incorporation with native and heat-denatured DNA represents 4.0 and 1.9 mpmoles, respectively.

TABLE V Requirements for single-nucleotide DNA-primed reaction

The basal mixture, 0.50 ml, contained 0.10 M Tris-HCI, pH 7.5, 2.0 mM MnCle, 0.8 mM ATa2P (1.4 X lo6 c.p.m. per pmole) or 0.8 mM CTa*P (7.14 X 106 c.p.m. per rmole), 50 pg of calf thymus or P. jkorescens DNA, and 18 units of enzyme. GTP, UTP, CTP, and ATP (0.8 mM each), 2 mM spermidine phosphate, and 10 erg of RNase or DNase were added to the complete system where indicated. After 60 minutes at 30”, the reaction was stopped with acid and the insoluble fraction was assayed for radioactivity.

Primer: calf thymus DNA Primer: P.~%uoresccns DNA Substrate: AT=P Substrate: CT”eP

Experiment 1 Experiment 2

System system CMazP incor-

,orated -

Basal mixture 63.7 Basal mixture Add GTP 32.3 Add ATP Add GTP, CTP 1.6 Add ATP, GTP Add GTP, CTP, UTP 24.5 Add ATP, GTP, UTP Add spermidine 44.8 Add spermidine Omit Mn++ 1.3 Omit Mn++ Omit DNA 0 Omit DNA Add ribonuclease 74.8 Add ribonuclease Add deoxyribonuclease 20.7 Add deoxyribonuclease

npnolcs

5.2 1.3 0.6

27.6 3.9

<O.l 0

<O.l 0.7


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January 1964 C. F. Fox and S. B. Weiss

Subrtrote: ATP. UTP, CTP. GTP=

.w /

x10-6 2 6 $X105

IO

I 1 , I ,

Substrate: ATP=

Km= 3.8 x 10-6 2 6 IO

I CALF THYMUS DNA GM x lO-5 PER ML

181

FIG. 9. In Part A, the conditions for the reaction were as in Fig. 2 except that various quantities of calf thymus DNA were tested at the levels indicated and no spermidine was added. All four ribonucleoside triphosphates were present at 0.8 mM each. The labeled substrate was GT32P. In Part B, the conditions were identical with Fig. 3A except that AT32P was the only nucleotide present.

Stoichimetry of Single-NTP Reaction-The polymerization of labeled AMP into an acid-insoluble polymer is accompanied by the release of an equivalent amount of inorganic pyrophosphate (Table VI). Alkaline hydrolysis of the acid-insoluble fraction results in the release of acid-soluble material which has an absorption spectrum identical with that of AMP, and is approximately equivalent to the amount of labeled substrate incorporated.

Single-NTP Reaction with Various DNAs-The RNA polymerase-catalyzed synthesis of ribohomopolymers with various DNA primers is shown in Table VII. With all DNAs tested, except for M. lysodeikticus DNA, a significant incorporation of AM3*P and UM32P was found for the single-NTP reaction. In general, DNAs which are rich in adenine and thymine were some- what better primers for polyadenylate and polyuridylate synthesis than those with a high content of guanine and cytosine. For polycytidylate synthesis, DNAs rich in guanine and cytosine appeared more efficient than those high in adenine and thymine. Significant GM32P incorporation could only be demonstrated with T2 DNA. On the whole, polyadenylate synthesis was more vigorous with each DNA tested than the formation of any other homopolymer. The inability of iv. Zysodeikticus DNA to prime for homopolymer synthesis is not understood. In addition, Table VII shows that each DNA functioned as an efficient primer for complementary RNA synthesis in the 4-NTP reaction. These results differ from those reported by Goldberg, Rabinowitz, and Reich (16) with rega.rd to the synthesis of polyuridylate and polycytidylate in the presence of M. lysodeikticus and calf thymus DNA with E. coli “polymerase.”

Linearity and DNA Secondary Structure (Single-NTP Reactim) -The DNA-primed synthesis of polyadenylate with RNA

polymerase is linear for periods exceeding 1 hour (Fig. 11). Furthermore, heat-denatured calf thymus DNA functions as a better primer for AMP polymerization than native DNA. This agrees with the report by Stevens (17). Both these properties for the single-NTP reaction are distinctly different from those described for the 4-NTP reaction. Heat denaturation of all DNAs tested has given similar increases in RNA synthesis for the single-NTP reaction, except for polyuridylate formation with P. jlucrescens DNA (Table VIII). Significant quantities

0 , .y; I I

0 2 4 6 6 IO

Met01 Ion Concentration M X IOm3

FIG. 10. The reaction mixture, 0.50 ml, contained 0.10 M Tris-HCl, pH 7.5, 0.8 mM ATz2P (1.56 k lo6 c.p.m. per rmole), 100 pg of calf thymus DNA, 4 units of enzyme, and various concentrations of MnClz or MgC12. The mixtures were incubated for 10 minutes at 30” and then analyzed for AMasP incorporation into the acid-insoluble fraction.

TABLE VI

Stoichiometry of DNA-primed polyadenylate synthesis (single-NTP reaction)

The reaction mixtures, 6.5 ml, and conditions of incubation were as indicated in Table II except that GTP, UTP, and CTP were omitted and 600 Irg of calf thymus DNA were used as primer. The nrocedure was identical with that described in Table II.

I Acid-insoluble fraction I Acid-soluble fraction

system AM=P

incorporated KOHA-ased

T;tal bef&

TP aft&

TP’ rele:sed Net

HCl HCl Pi/2 Total Net Total Net bydrol- hydrol- hy!&-

ysis ysis ysis I I I I I

#?nole pm&

0.47 1.01 /

2.48 1.47 0.55

Omit Mn++ 0 0.83 1.20 1 0.37 1 ,


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182 Enzymatic Synthesis of Ribonucleic Acid. II Vol. 239, No. 1

TABLE VII Single- and $-NTP reaction with various DNAs

The conditions for the 4-NTP reaction were as in Table I except that 1.2 mM spermidine phosphate was used and the reaction was stopped after 10 minutes. The single-NTP reaction mixture, 0.50 ml, contained 0.10 M Tris-HCl, pH 7.5, 2.5 mM MnCl2, either AT32P, UTa2P, CT32P, or GT32P employed individually (1.2 mM each) with specific activities of 8.97 X 105, 9.4 X 105, 13.2 X 105, and 13.7 X lo6 c.p.m. per pmole, respectively, 50 pg of DNA, and 6 units of enzyme. The reaction was incubated at 30” and assayed for the incorporation of label into an acid-insoluble fraction after 60 minutes.

Source of primer DNA

Bacteriophage T4. Bacteriophage T2. Sea urchin sperm. Salmon sperm. Bacillus subtilis Rat liver.......... Calf thymus.. Bacteriophage T7. E. coli B. A.aerogenes....... S. marcescens. P. jluorescens. . M. lysodeikticus

)NA has ratio,*

A + T): (G+ C)

1.90 1.85 1.70 1.43 1.36 1.35 1.29 1.08 0.96 0.74 0.70 0.59 0.39

e-

1:

-

Total ribopolynucleotide formed

I-NTP wction, 1 minutes

mpioles

5.2 13.2 19.2 26.0 14.1 21.7 24.9 18.2 17.0 20.8 22.8 26.6 28.3

Single-NTP reaction, 60 minutes

ATa*P 1 UTa*P 1 CT=P 1 GT=P

qmioles

33.90 8.32 25.10 5.64 23.10 6.62 16.55 3.19 39.40 8.10 14.92 4.56 20.70 3.19 3.23 1.11

18.62 3.50 14.78 4.06 13.00 2.69 7.42 2.27 0.21 0

I

0.47 0 0.32 0.32 0.29 0.04 0.26 0.01 0.89 0 0.23 0.09 0.23 0.10 0.46 0.02 0.98 0.03 1.24 0 1.48 0.04 1.59 0 0.20 0

* Base ratios as reported by Chargaff (26), Belozersky and Spirin (27), and Sinsheimer (28).

6 60

0 IO 20 30 40 50 60 70 60 90

Incubation Time ( Minutes 1

FIG. 11. The conditions were as in Table VII for the single- NTP reaction, except that the reaction mixtures contained only ATazP, 100 pg of native or heat-denatured calf thymus DNA, and 15 units of enzyme. The mixtures were incubated at 30” for the periods shown.

of polyguanylate may be formed with heat-denatured M. lysodeikticus or T2 DNA.

It should be mentioned that the K,,, values for native and heat- denatured calf thymus DNA in the single-NTP reaction were the same. Therefore, it appears unlikely that the activity observed with native DNA as primer for polyadenylate formation was due to the presence of contaminating denatured DNA.

Pyrophosphorolysis-Furth, Hurwitz, and Anders (25) reported that E. coli RNA polymerase catalyzed the pyrophosphorolysis of RNA when the latter was prepared in vitro with T2 DNA as primer. These workers also report that the reverse reaction is very slow and requires large quantities of enzyme, and that relatively small amounts of 32PPi were converted to a Norit-adsorbable form. So far, we have not been able to demonstrate the pyrophosphorolysis of any ribopolynucleotide with M. Zysodeikticus RNA polymerase. Incubation of mixtures containing highly radioactive inorganic pyrophosphate, RNA polymerase, Mg++, and synthetic or natural RNAs, in the presence of DNA, have resulted in insignificant amounts of Norit-adsorbable counts.

Pyrophosphate Exchange-The exchange of “PPi with ribonucleoside triphosphates is catalyzed by RNA polymerase (Table IX). The exchange reaction requires DNA for optimal activity, a divalent metal ion, ribonucleoside triphosphates, and enzyme. The reaction is linear with respect to enzyme at concentrations up to 120 pg per ml, and with respect to time for periods exceeding 1 hour. The K, for ATP and GTP was 3 X lop4 M. PPi saturation occurred at approximately 1 X lop3 M.

Optimal exchange occurs with 3 to 6 x low3 M Mg*; higher levels result in inhibition. No exchange was detected when 3’Pi was substituted for 32PPi.

Table IX shows that the exchange reaction occurred with a variety of DNAs. In the presence of all four ribonucleotides, no significant differences were observed between native and heat- denatured DNA. Isolation of the ribonucleoside triphosphates by chromatography on Dowex l-Cl after the reaction showed that labeled 32PPi had exchanged with each nucleotide. If one compares the exchange data in Table IX with the incorporation of ribonucleotides into RNA (e.g. Table III), it is apparent that the PPi exchange reaction occurs at a much slower rate than the forward reaction. In the absence of added DNA, the relatively small conversion of 32PPi to Norit-adsorbable counts was significant and reproducible. This exchange is probably related to the “unprimed” synthesis of RNA which we have detected with

TABLE VIII Single-NTP reaction with native and denatured DNA

The conditions for the single-NTP reaction were as for Table VII except that 10 units of enzyme were used. Heat-denatured DNA, 50 pg, replaced native DNA where indicated and was prepared as described under “Experimental Procedure.”

Source of primer DNA

Calf thymus .......... ATP P. jIuorescens. ........ ATP M. lysodeikticus. ...... ATP

Calf thymus .......... UTP P. jluorescens. ........ UTP

P. jluorescens. ........ CTP M. lysoofeikticus ....... CTP

Bacteriophage T2. .... GTP M. lysodeikticus. ..... GTP

-

-

-

Labeled substrate

Ribopolynudeotide formed

Native Heat- denatured

mjLmoles

38.10 52.60 8.24 15.58 0.18 2.44

3.65 4.10 2.65 0.73

2.04 2.29 0.16 2.90

0.22 0.28 0.03 0.23


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.January 1964 C. F. Fox and S. B. Weiss 183

TABLE IX

Pyrophosphate exchange reaction

The reaction mixture, 0.50 ml, contained 0.10 M Tris-HCI, pH 7.5, 5.0 rnM MgC12, 0.83 mM 3ePPi (2.0 X lo6 c.p.m. per pmole), 50 rg ,of the respective DNAs except for crab d-AT, which was 1.72 fig, 0.8 mM each ATP, GTP, CTP, and UTP where indicated, 0.8 mM spermidine phosphate where indicated, and 15 units of enzyme. Heat-denatured DNA was prepared as described under “Experi- mental Procedure.” The reaction mixture was incubated at 30” for 30 minutes except for the vessels containing no DNA and crab d-AT copolymers (see below). At the end of this time, 1 mg of carrier albumin was added and the reaction was stopped with 50?& trichloroacetic acid containing 10-a M PPi. The precipitate was removed, and 30 mg of Norit were added and mixed with the acid-soluble fraction. The Norit was collected by centrifugation and washed four times with the acid-PPi solution. The Norit was subjected to hydrolysis in 1 N HCl for 20 minutes at 100” and cooled. The volumes in each vessel were adjusted to 2.0 ml with water and centrifuged, and l-ml aliquots were dried and assayed for their radioactive content. Control ‘*PPi standards were treated in a similar manner.

Treatment of DNA

Experiment 1 Native Native Native Native Nat,ive Native Native

Experiment 2 Native Heat-denatured Heat-denatured Heat-denatured Heat-denatured Heat-denatured

ATP, GTP, CTP, UTP ATP, GTP, CTP, UTP (omit Mg++) ATP, GTP , CTP , UTP , spermidine

ATP GTP CTP UTP

ATP, GTP, CTP, UTP ATP, GTP, CTP, UTP

ATP GTP CTP UTP

*Exchange reaction incubated for 80 minutes.

&f. Zysodeikticus “polymerase” (34). Other studies suggest that in the presence of DNA, the “unprimed” reaction does not occur.

When various ribonucleoside triphosphates are tested singly, only the purine nucleotides actively participate in the PPi exchange. This striking phenomenon was found for each of the DNAs tested, and occurred with either native or heat-denatured deoxypolynucleotides. With E. coli RNA polymerase, Furth et al. (25) reported a similar observation. With heat-denatured M. lysodeikticus DNA, we noted a slight exchange with CTP alone.

M. Zysodeikticus RNA polymerase catalyzes at least two separate DNA-dependent reactions. With respect to the nature of the product formed, one reaction results in the synthesis of complementary RNA, and the other gives rise to an RNA homopolymer. Although these reactions are similar in some respects, they differ in many ways.

Qualitatively, another pattern appears to develop from these experiments. Exchange reactions carried out with DNAs rich in adenine and thymine exchange better with ATP than with GTP. With DNAs of high guanine and cytosine content, the reverse appears to follow. The exchange reaction with crab d-AT copolymer occurred in the presence of all four ribonucleotides, and with ATP alone. No other single triphosphate gave a significant exchange. This finding is contrary to a report by Goldberg, Rabinowitz, and Reich (35), which demonstrated a marked exchange with UTP alone and a negligible reaction with ATP. It should be mentioned that the exchange reaction shows different quantitative results by the Norit adsorption assay method when different mixtures of each ribonucleotide are present. The experiments reported here for the single nucleotide exchange reactions were performed with commercial preparations of ribonucleoside triphosphates which had been purified by chromatography on Dowex l-C1 before their use.

Both reactions require ribonucleoside triphosphates as sub- strates, and synthesis occurs with the elimination of inorganic pyrophosphate. The release of inorganic pyrophosphate, in each case, is stoichiometric with the amount of ribonucleotide polymerized. Native and heat-denatured DNA can prime in either reaction.

The synthesis of complementary RNA requires all four ribonucleotides, and either Mn++, Co*, or Mgff may satisfy the divalent metal ion requirement. On the other hand, the formation of a ribohomopolymer requires the presence of only one ribonucleotide, and only Mn++ has been found to fulfill the metal ion requirement effectively. In agreement with Chamberlin and Berg (15), we find that the latter reaction is inhibited by the presence of more than one nucleotide in the system.

Another apparent difference between the 4-NTP and single- NTP reactions is observed when denatured DNA replaces native DNA. In most instances, heat-denatured DNA is more effective than helical DNA for RNA synthesis in the single-NTP reaction (Fig. 11). With one DNA, we observed the reverse situation. Although other instances of this reverse phenomenon may exist,

Cofactors added

Norit-adsorbable ‘*PPi after reaction with various DNAs

-

0.52 <O.Ol

0.21 <O.Ol <O.Ol <O.Ol

2.98 1.39 1.32 1.07 1.58 <O.Ol <O.Ol <O.Ol <O.Ol <O.Ol

3.04 1.38 1.53 1.20 1.67 0.86 2.04 1.58 0.10 0.35 0.08 0.25 0.20 0.13 0.58

<O.Ol <O.Ol <O.Ol <O.Ol <O.Ol <O.Ol <O.Ol <O.Ol <O.Ol <O.Ol

1.17 1.17 1.28 1.53 1.14 0.95 1.54 1.14 2.10 1.92 0.40 0.50 0.70 0.73 3.77 0.77

<O.Ol <O.Ol 0.21 <O.Ol <O.Ol <O.Ol 0.03 <O.Ol

0.15 <O.Ol

0.38 0.02

<O.Ol <O.Ol

DISCUSSION


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184 Enzymatic Synthesis of Ribonucleic Acid. II Vol. 239, TNO. I

only with heat-denatured M. lysodeikticus and T2 DNA could we observe any polyguanylate formation. There was no significant difference between the K, values for helical and denatured DNA primer in the single-NTP reaction.

The efficiency of priming with native and denatured DNA was completely reversed in the 4-NTP reaction. When denatured DNA replaced native DNA, both the rate of complementary RNA synthesis and the concentration required for DNA saturation were markedly reduced (Fig. 5). This finding is in general agreement with other reports (11, 15, 25, 29), although some investigators have found little or no change in the priming efficiency of certain DNAs subjected to heat denaturation (15). One possible explanation for the consistent differences in RNA synthesis which we have observed between ordered and disordered DNA primers may lie in the addition of polyamine to the polymerizing system.

Recently, we found that when denatured DNA serves as primer in the 4-NTP reaction, the RNA which forms is largely resistant to RNase degradation. Furthermore, equilibrium density gradient centrifugation in CsCl results in the appearance of labeled RNA where DNA usually bands. These observations are quite different from those previously reported by Geiduschek et al. (12, 13) when native DNA primed for ribopolynucleotide synthesis. The assembly of RNA with single stranded DNA as template appears to result in the formation of an RNA-DNA complex which does not readily separate, and could account for the reduced rate of polynucleotide synthesis observed. A complete description of these experiments will be reported elsewhere. A report by Warner et al. (36) on the formation of DNA-RNA hybrids when denatured DNA serves as primer with Azotobacter vinelandii RNA polymerase is in agreement with the observations reported here.

One of the most striking and as yet least understood properties of the 4-NTP reaction is the polyamine stimulation obtained when native DNA is used for RNA synthesis. Priming with heat-denatured DNA, in either the 4-NTP or the single-NTP reaction, does not show this marked polyamine stimulation. In fact, inhibition occurs when spermidine is added to the single- NTP reaction. The stimulatory effect of polyamines varies slightly with different DNAs, and this variation appears to be associated with the DNA preparation itself, rather than with the enzyme. For this reason, we have only obtained consistent assays with M. Zysodeikticus RNA polymerase in the presence of polyamines.

Tabor, Tabor, and Rosenthal (37) have recently reviewed some of the effecm of polyamines in various systems, and their interactions with nucleic acids. Organic cations such as polyamines bind readily with polynucleotides, as do divalent metal cations. These studies suggest that both types of cations facilitate the formation of highly ordered structures, primarily DNAs high in adenine and thymine (38, 39). In view of the polyamine stimulation with primer M. Zysodeikticus DNA (72 mole y0 guanine and cytosine) by spermidine, some other explanation may be required for the polyamine effect observed in this report. Others have reported that spermine and spermidine partially protect nucleic acids from the hydrolytic action of RNase and DNase (37). This protective action could account for the results observed here; however, the enzyme preparations and DNAs used in this work have been shown to contain only trace quantities of these degradative enzymes. Moreover, the addition of spermidine to RNA-DNA-directed reactions and to

the DNA-primed synthesis of polycytidylate shows no stimulatory effect. Although polyamines may protect either the primer or the product from partial degradation, it is also possible that organic cations serve another function in RNA synthesis when primed by native DNA. The concentrations required to achieve optimal synthesis with various polyamines are not the same. This difference may be related to the different affinities of various polyamines toward nucleic acids as well as to steric effects im- posed on the polymerizing system.

The formation of homopolymers by the single-NTP reaction may occur with a variety of different DNA primers. There appears to be some qualitative correlation between the base composition of the primer and the ability to form certain homopolymers. These results support the suggestion of Chamberlin and Berg (15) that repeating sequences of a single base in DNA can prime the synthesis of a complementary homopolymer. One would expect that the probability of consecutive sequences of a single base occurring in DNAs rich in a particular nucleotide or nucleotides would be relatively high. It is noteworthy that even with DNAs relatively poor in thymidylate, an active polyadenylate synthesis was found. Repeating sequences of thymidylate may not, therefore, be restricted only to DNAs rich in this deoxynucleotide. In view of the less active homopolymer synthesis found with UTP, CTP, and GTP alone, it is important to know whether thymidylate sequences have a univer- sal meaning in terms of biological function.

The results obtained in the single-NTP reaction suggest certain inconsistencies with accepted base-pairing theory. Since every run of thymidylate sequences in one DNA strand should be matched by an equivalent run of adenylate sequences in the opposing strand, it is difficult to account for the quantitative differences in polyadenylate and polyuridylate synthesized for any of the DNAs tested (Table VII). One possible explanation might be that the 32P-polyuridylic acid product was incompletely precipitated by cold trichloroacetic acid (40). On the other hand, the priming mechanism could be more complex than our current data suggest. In the single-NTP reaction, heat denaturation of the DNA may allow certain consecutive sequences to prime more effectively.

Bessman et al. (41) have shown that for DNA polymerase, the rate of PPi exchange with deoxynucleoside triphosphates was of the same magnitude as the rate of deoxynucleotide incorporation into DNA. In the absence of all four triphosphates, these workers showed a reduced but measurable pyrophosphorolytic reaction. With the enzyme under examination in this report, the rates of PPi exchange are much slower than the synthetic rates observed, and no significant pyrophosphorolysis could be demonstrated. The observation that all four ribonucleoside triphosphates were labeled after ‘*PPi exchange, when a full complement of nucleotides was present, suggests that a reversal of the synthetic reaction had occurred. Under the conditions at which pyrophosphate exchange reactions were conducted, the forward reaction still occurs. In a review on pyrophosphorolytic reactions (42), Kornberg points out that the free energy associated with the release of pyrophosphate is relatively small and that the synthetic direction of many pyrophosphate- releasing reactions is demonstrably reversible. The difficulty encountered in demonstrating a reversible reaction in this system as well as the poor reversal found with the E. coli system (25) may not be a simple representation of the synthetic nature of RNA polymerase, but rather a reflection of the complex order


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January 1964 C. F. Fox and S. B. Weiss 185

in which ribonucleotides are polymerized. The exchange reaction described in this report is interesting because virtually no reaction occurs with UTP or CTP alone, but does occur with ATP and GTP alone. Assays with a variety of DNAs show the same result.

Bt the present time, the interpretation of the results obtained by the single-NTP reaction and the PPi exchange data should be regarded with caution. Because of certain differences in both of these reactions between the enzyme studied here and the enzyme isolated from E. coli (16, 35), one must consider the possibility that the enzyme itself may impart certain specificities to the polymerization reaction.

SUMMART

Various species of deoxyribonucleic acid may serve to prime ribonucleic acid synthesis catalyzed by Micrococcus lysoo?eih&us ri-

bonucleic acid polymerase. Two separate reactions have been studied: one which results in the formation of complementary RNA and one which gives rise to an RNA homopolymer. The properties of these reactions have been examined with respect to cofactor requirements, stoichiometry, primer-enzyme affinity, rates and extent of reaction, heat denaturation of primer, and inorganic pyrophosphate-nucleotide exchange reactions.

The DNA-primed synthesis of complementary RNA requires all four ribonucleoside triphosphates, a divalent metal ion (Mn++, Co*, Mg++), a DNA primer, and enzyme. A polyamine is required for maximal synthesis, and heat-denatured DNA is less effective for synthesis than native DNA.

The DNA-primed synthesis of an RNA homopolymer requires only one ribonucleoside triphosphate, a divalent metal ion (Mn++), a DNA primer, and enzyme. Homopolymers containing adenylic, uridylic, cytidylic, and guanylic acid have been formed. Polyamines and the addition of more than one nucleotide inhibit synthesis, and in general heat-denatured DNA is more effective than native DNA.

Both reactions result in the net synthesis of product and occur by the elimination of inorganic pyrophosphate. Pyrophosphate exchange reactions have been demonstrated and they require DNA, at least one ribonucleotide, a divalent metal ion, and enzyme. Spermidine and heat-denatured DNA have no effect on the exchange reaction, and in the presence of single triphos- phat.es only the purine nucleotides exhibit a significant exchange.

1. 2.

3.

4.

5.

6.

7.

8.

9. 10.

11. 12.

13.

14.

15.

16.

17.

18.

19.

20.

21. 22. 23. 24.

25.

26.

27.

28.

29.

30. 31.

32.

33.

34.

35.

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C. Fred Fox and Samuel B. WeissMICROCOCCUS LYSODEIKTICUS RIBONUCLEIC ACID POLYMERASE

DEOXYRIBONUCLEIC ACID-PRIMED REACTION WITH Enzymatic Synthesis of Ribonucleic Acid: II. PROPERTIES OF THE

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