Dh'OX YRIBON UCLEIC ACID-DIRECTED SYNTHESIS OF RIBONUCLEIC

ACID BY AN ENZYME FROM ESCHERICHIA COLI*

BY MICHAEL CHAMBERLINt AND PAUL BERG

DEPARTMENT OF BIOCHEMISTRY, STANFORD UNIVERSITY SCHOOL OF MEDICINE

Communicated by Arthur Kornberg, November 17, 1961

l'rotein structure is under genetic control;'-3 yet the precise mechanism by which
DNA$ influences the formation of specific amino acid sequences in proteins is
unknown.  Several years ago, it was discovered that infection of Escherichia coli
with certain virulent bacteriophages induces the formation of an RNA fraction
possessing both a high metabolic turnover rate and a base composition correspond-
ing to the DNA of the infecting vir~s.~-~  The existence of an analogous REA
component in noninfected cells has also been demonstrated; in this instance,
however, the base composition of the RNA resembles that of the cellular DNA.'.
These observations focused attention on the possible role of this type of RNA in
protein synthesis, and some of the evidence consistent with this view has recently
been surnmari~ed.~

Until recently there was no known enzymatic mechanism for a DNA-directed
synthesis of RNA.  Polynucleotide phosphorylasel0, l1 although it catalyzes the
synthesis of polyribonucleotides, does not by itself provide a mechanism for the
formation of RNA with a specific sequence of nucleotides.  The one instance in
which a unique sequence of nucleotides is produced involves the limited addition
of nucleotides exclusively to the end of pre-existing polynucleotide chains. 12-14

Our efforts were therefore directed toward examining alternate mechanisms for
RNA synthesis, and in particular one in which DNA might dictate the nucleotide
sequence of the RNA.  In the present paper, we wish to report the isolation and
some properties of an RNA polymerase from E. coli which, in the presence of DNA
and the four naturally occurring ribonucleoside triphosphates, produces RNA with
a base composition complementary to that of the DNA.  Within the last year,
several laboratories have reported similar findings with enzyme preparations from
bacterial as well as from plant and animal  source^.^^-^^  In the following paper,
the effect of enzymatically synthesized RNA on the rate and extent of amino acid
incorporation into protein by E. coli ribosomes in the presence of a soluble protein
fraction is described.

                  Unlabeled ribonucleoside di- and triphosphates were
purchased from the Sigma Biochemical Corporation and the California Corporation for Biochem-
ical Resenrch.  8-C14-1abeled ATP was purchased from the Schwartz Biochemical Company;
the other, uniformly labeled, C1* ribonucleoside triphosphates were prepared enzymatically from
the corresponding monophosphate derivatives55 isolated from the RN.4 of Chromatium grown on
CL402 as sole carbon source.*6  CTP labeled with P31 in the eater phosphate was obtained by
enzymatic phosphorylation of CMPS* prepared according to Hurwitz.27  The deoxyribonucleoside
triphosphates were obtained by the procedure of Lehman et al.25

                                                     DNA from
Aerobacter aerogenes, Mycobacterium phlei, and bacteriophages T2, T5, T6 was prepared as de-
scribed previously.29                                       Unlabeled
and P32 labeled DNA from E. coli were prepared as previously de~cribed.~'  d-AT and d-GC poly-
mers were prepared according to Schachman et al.32 and Radding et    respectively. Trans-
forming DNA from Ban'ZZus subliZisJ4 was a gift from E. %'. Nester, and DNA from phage 0X

81

Experimental Procedure.-MateriaZs:

Calf thymus and salmon sperm DNA were isolated by the method of Kay et ~1.~8

DNA from Xdg phage was prepared as reported else~here.~a

82 BIOCHEMISTRY: CHAMBERLIN AND BERG PROC. N. A. s.

174 49 was generously supplied by R. L. Sinsheimer.  Double-stranded 0X 174 DNA was syn-
thesized using E. coli DNA polymerasez6 with single-stranded OX 174 DNA as primer.36.   In
this reaction, 2.7 times more DNA was synthesized than had been added as primer.  RNA from
tobacco mosaic virus was obtained from H. Frwenkel-Conrat, and ribosomal and amino acid-
acceptor RNA were isolated from E. coli according to Ofengand et ~1.~~~ 37  Nucleic acid concen-
trations are given as mpmoles of nucleotide phosphorus per ml.

Glass beads, "Superbrite 100," obtained from the Minnesota Mining and Manufacturing
Company, were washed as previously described.26  Streptomycin sulfate was a gift from Merck
and Company, and protamine sulfate was purchased from Eli Lilly Company.  DEAEcellulose
was purchased from Brown and Company.  Crystalline pancreatic RNase and pancreatic DNase
were products of the Worthington Biochemical Co.

     The activities of E. coli-DNA p0lymerase,~6deoxyribonuclease~8 and -DNA dies-
terase, 31 were determined as previously described and ribonuclease activity was measured by the
disappearance of amino acid-acceptor RNA activity.f6 Polynucleotide phosphorylase was
measured by P,32 exchange with ADP as reported by Littauer and Kornberg."  Protein was
determined by the method of Lowry et aLJg

The standard assay for RNA polymerase measures the conversion of either CJ4 or P3z from the
labeled ribonucleoside triphosphates into an acid-insoluble form.  Enzyme dilutions were made
with a solution containing 0.01 M Tris buffer, pH 7.9, 0.01 M MgC12, 0.01 M &mercaptoethanol,
5 X                                                The reaction
mixture (0.25 ml) contained: 10 pmoles of Tris buffer, pH 7.9, 0.25 pmole of MnClz,
1.0 pmole of MgClz, 100 mpmoles each of ATP, CTP, GTP, and UTP, 250 mpmoles of salmon
sperm DNA, 3.0 pmoles of 8-mercaptoethanol, and 10 to 80 units of enzyme.  One of the nucleo-
side triphosphates was labeled with approximately 300 to 600 cpm per mpmole.  After incubation
at 37" for 10 min, the reaction mixture was chilled in ice, and 1.2 mg of serum albumin (0.03 ml)
was added, followed by 3 ml of cold 3.5% perchloric acid (PCA).  The precipitate was dispersed,
centrifuged for 5 min at 15,000 X g, and washed twice with 3.0 ml portions of cold PCA.  The
residue was suspended in 0.5 ml of 2 AT ammonium hydroxide, transferred to an aluminum planchet.
and after drying, counted in a windowless gas-flow counter.

One unit of enzyme activity corresponds to an incorporation of 1 mpmole of CMPaz per hr
under the conditions described above.  The assay was proportional to the amount of enzyme added
up to at least 80 units; thus 6.3, 12.5, and 25 pg of Fraction 4 enzyme incorporated 2.6, 5.1, and
10.0 mpmoles of CMP32.  The rate of the reaction remained constant for approximately 20 min,
and then derreased after this time.

Since the radioactivity incorporated represents only one of the four nucleotides, the observed
incorporation must be multiplied by a factor ranging from 3 to 5 for an estimate of the total amount
of RNA synthesized.

                           E. coli B was grown in
continuous exponential phase culture40 with a glucose-mineral salts medium.41
Cells stored at -20" showed no loss of activity for over six months.  The purifica-
tion procedure and the results of a typical preparation are summarized in Table 1.

Assays:

M EDTA, and 1 mg per ml of crystalline bovine fierum albumin.

The exact factor depends on the composition of the DNA primer used.

Results.-Purification of RNA polymerase:

(1) Cells:

            TABLE 1
PURIFICATION OF RNA POLYMERASE FROM E. coli

Volume Specific activity Total activity

Fraction (ml) (uni ta/mg) (UIlitZi)

1. Initial extract 260 40 370, OOO
2. Protamine eluate 37 1,600 205,000

                                       153 , 000
4. Peak DEAE fraction 2 6,100

3. Ammonium sulfate 5 2,500 200,000

Unless noted otherwise, all operations were carried out at 4" and all centrifugations
were at 30,000 X g for 15 min in an International HR-1 Centrifuge.
       Frozen cells (140 gm) were mixed in a Waring Blendor with 420 gm of
glass beads and 150 ml of a solution (buffer A) containing 0.01 M Tris buffer, pH

(2) Extract:

VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 83

7.9, 0.01 M MgCI2, and O.OOO1 M EDTA.  After disruption of the cells at high
speed for 15 min (maximum temperature lo"), a further 150 ml of buffer A was
added and the glass beads were allowed to settle.  The supernatant fluid was then
decanted and the residue was washed with 75 ml of buffer A.  The combined
supernatant fluid and wash was centrifuged for 30 min and the resulting super-
natant fluid collected (Fraction 1).

(3) Streptomycin-protamine fractionation: Fraction 1 was centrifuged in the
Spinco Model L preparative ultracentrifuge for 4 hr at 30,000 rpm in the No. 30 rotor.
The protein concentration in the supernatant fluid was adjusted to about 12 mg per
ml with buffer A, and /%mercaptoethanol was added to a final concentration of 0.01
M.  To 350 ml of the diluted supernatant solution was added 17.5 ml of a 10%
(w/v) solution of Streptomycin sulfate with stirring.  After 15 min, the solution
was centrifuged, and to 350 ml of the supernatant fluid was added 14.0 ml of a 1%
(w/v) solution of protamine sulfate.  The precipitate, collected by centrifugation,
was washed by suspension in 175 ml of buffer A containing 0.01 M j3-mercapto-
ethanol.  The washed precipitate was then suspended in 35 ml of buffer A con-
taining 0.01 M mercaptoethanol and 0.10 M ammonium sulfate, centrifuged for 30
min, and the supernatant fluid was collected (Fraction 2).

                       To 37 ml of Fraction 2 was added 15.8
ml of ammonium sulfate solution (saturated at 25" and adjusted to pH 7 with
ammonium hydroxide).  The mixture was stirred for 15 min, and the precipitate
was removed by centrifugation.  To the supernatant liquid was added an addi-
tional 16.2 ml of the saturated ammonium sulfate, and after 15 min the precipitate
was collected by centrifugation for 30 min and dissolved in buffer B (0.002 M
KP04, pH 8.4, 0.01 M MgC12, 0.01 M P-mercaptoethanol, and 0.0001 M EDTA)
to a final volume of 5.0 ml (Fraction 3).

                            Fraction 3 was diluted to a
protein concentration of about 3 mg per ml with buffer B and passed onto a DEAE-
cellulose column (10 cm X 1 cm2, washed with 150 ml of buffer B just prior to use)
at a rate of about 0.5 ml per min.  The column was washed with 10 ml of buffer B
and then with enough of the same buffer containing 0.16 M KC1 to reduce the
absorbency of the effluent at 280 mM to less than 0.05.  The enzyme was eluted
from the column with buffer B containing 0.23 M KCl.  The activity appears
within the first five ml of the latter eluant (Fraction 4).

                   The specific activity of enzyme Fraction
4 was from 140 to 170 times greater than that of the initial extract.  The puri-
fication as described here has been quite reproducible, with specific activities in
the final fraction ranging from 5,500 to 6,100.  The enzyme preparation (Fraction
4) has a ratio of absorbencies at 280 and 260 ml of 1.5.

Fraction 4, stored at 0 to 2", retains more than 90 per cent of its activity for up
to two weeks and 40 to 60 per cent of the original activity after one month.  En-
zyme Fractions 1 through 3 are unstable, losing up to 30 per cent of their activity
on overnight storage under a variety of conditions.  Because of the marked in-
stability of these earlier fractions, it is advisable to carry out the purification
without stopping at intermediate stages.

   Contaminating enzymatic activities:  Aliquots (100 pg) of Fraction 4 were
assayed for contaminating enzymatic activities.  This amount cf enzyme cata-

(4)

Ammonium sulfate fractionation:

(5)

Adsorption a,nd elution from DEAE-cellulose:

(6)

Properties of the purijied enzyme:

(7)

84 BIOCHEMISTRY: CHAMBERLIN AND BERG PROC. N. A. S.

lyzed an initial rate of incorporation of 2,000 mpmoles of nucleotide per hr.  No
detectable DNA polymerase was found (< 0.6 mpmole DNA per hr).  DNase
activity was barely detectable under conditions optimal for RNA polymerase.
With either heated or unheated P3* DNA as substrate, no more than 0.13 mpmole
of acid-soluble P32 was released during the course of a 30-min incubation.  There
was only slight RNase activity associated with Fraction 4.  When 100 pg of the
purified enzyme were incubated with 4 pmoles of purified acceptor RNA for 1 hr,
there was no detectable inactivation of leucine-acceptor activity.  Under similar
conditions, 1 mg of enzyme produced a 30 per cent decrease in leucine-acceptor
activity.  With conditions optimal for RNA polymerase, sufficient polynucleotide
phosphorylase activity was present to catalyze the exchange of 6.7 mpmoles of
Pi32 into ADP per hour.

Requirements for the RNA polymerase reaction: With the purified enzyme,
RNA synthesis was dependent on the addition of DNA, a divalent cation, and the
four ribonucleoside triphosphates (Table 2).  In a later section, we shall describe a

TABLE 2 TABLE 3

REOUIREMENTS FOR RNA SYNTHESIS

THE REQUIREMENT FOR RIBONUCLEOSIDE

              Incorporation
                of CMPa*
Components (mpmoles)

Complete syst.em 7.3
minus Mn++ 4.3
minus Mg++ 5.6
minus Mn++ and Mg++ <0.03
minus DNA <0.03

minus enzyme <0.03

minus ATP, GTP, UTP 0.09

The standard system and assay procedure were
used with 7.4 WK of Fraction 4 protein in each tube, ex-
cept that MeCh was omitted from the enzyme diluent.

TRIPHOSPHATES IN RNA SYNTHESIS

              Incorporation
                of CM-Paz
Components (mpmoles)

Complete system 4.6

minus ATP 0.08

minus UTP <o. 03
minus GTP <O. 03
ATP, UTP, GTP replaced by
dATP, dTTP, dGTP 0.05
ATP, UTP, GTP replaced by

ADP, UDP, GDP 0.29

The standard system and assay procedure were used
except that 250 mpmoles of calf thymus DNA were
used as primer.  13 pg of Fraction 4 protein were used
in each assay.  100 mrmoles of each nucleotide were
added to each assay.

reaction in which ATP is converted to an acid-insoluble form in the absence of the
other three triphosphates.  Omission of P-mercaptoethanol from the reactiom
mixture resulted in a 50 per cent loss in activity; however, dilution of the con-
centrated enzyme into solutions not containing a sulfhydryl compound resulted in
as much as 90 per cent inactivation.  The optimal pH for the reaction was between
7.8 to 8.2.  At pH 6.1, 7.0, and 8.9 the activities were 13, 62, and 84 per cent,
respectively, of the maximal value.

(1) Nucleoside triphosphate speci$city: All of the ribonucleoside triphosphates
are required for RNA synthesis (Table 3). The deoxyribonucleoside triphos-
phates do not function as substrates in the reaction, and the ribonucleoside diphos-
phates support synthesis only at a greatly reduced rate.  The observed activity
of the diphosphates may be due to the presence of small amounts of the nucleoside
triphosphates in the diphosphate preparations or to the formation of the triphos-
phates through the action of nucleoside diphosphate kinase.

With CTP32 as the labeled substrate and salmon sperm DNA as primer, vari-
ation of the concentrations of all four ribonucleoside triphosphates as a group pro-
duced a variation in the rate of RNA synthesis.  When the data were plotted ac-
cording to Lineweaver and B~rk,~2 a linear relationship was obtained from which

VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 85

          TABLE 4
THE EFFECT OF DIFFERENT NUCLEIC ACID
  PREPARATIONS ON THE RATE OF RNA
   SYNTHESIS BY RNA POLYMERASE

Incorporation

Source of primer of CMP"*

DNA

Salmon sperm
Calf t.hymus
E. coli
0X 174
B. subtilis
Xdg phage
T2 phage
T6 phage
T5 phage

E. coli amino acid-acceptor
E. coli ribosomal
TMV

RNA

100
43
34
38

27
25
45
30
74

<0.5
<0.5
<0.5

        TABLE 5
THE EFFECT OF DENATURATION ON THE
ABILITY OF DNA PEEPAEATIONS TO
   PRIME FOR RNA SYNTHESIS

Incorporation of CMP"
Native Heated

DNA (mrrmoles)

Calf thymus      2.7 2.3
Salmon sperm     6.1 2.5

E. coli          1.8 1.9

T6 phsge 1.9 0.8

Assay procedure as described reviously, except
that the usual primer waa replacedgy 200 mpmoles of
the DNA to be tested.  7.4 pg of Fraction 4 protein
were used in each amy. The DNA samples were
heated for 10 min at 95 to 99' in 0.05 M NaCl and
rapidly cooled in an ice bath.  The absorbencies of
the heated DNA preparations were 30 to 40 per cent
higher than those of the unheated preparations at 260
mr.

* The incorporation value for salmon sperm DNA
was 5.3 mpmoles and is set at 100 for comparison with
the other primers.
Assay system and procedure as previously de-
scribed, except that 100 mpmoles of each nucleic acid
were used in place of the usual primer.  7.4 pg of Frac-
tion 4 protein were used in each assay.

it was calculated that the rate of synthesis was half maximal when the concen-
tration of each of the triphosphates was 1.3 X  lo-' M.  A similar value (1.4
X    M) was obtained using C14 ATP as a label and calf thymus DNA as
primer.

(2)  The nature of the primer:  All DNA samples tested were active in pro-
moting ribonucleotide incorporation, although the efficiency varied significantly
(Table 4).  Amino acid-acceptor RNA, ribosomal RNA from E. coli, and TMV
RNA did not substitute for DNA.  With the synthetic copolymer d-AT as pri-
mer, only AMP and UMP were incorporated, and only GMP and CMP were in-
corporated in the presence of d-GC polymer (Table 7).  It should be noted, how-
ever, that GTP was incorporated to a considerably greater extent in the latter
case.  A qualitatively similar finding has been reported for the incorporation of
dGMP and dCMP by DNA polymerase with d-GC as primer.a3

Increasing the amount of DNA in an assay mixture over the range 0 to 200
mpmoles resulted in an increase in nucleotide incorporation.  Further increases in
the amount of DNA, up to 400 mpmoles, had no effect on the rate of RNA syn-
thesis.  A similar experiment using calf thymus DNA as primer gave a saturating
value of 250 mpmoles.

The effect of disrupting the DNA double helix by heating43 is shown in Table 5.
It is seen that with several of the DNA preparations there is a significant decrease
in the rate of CMP32 incorporation using the heated DNA while with others the
effect is insignificant.  The ability of single-stranded DNA to function as a pri-
mer for RNA synthesis is further emphasized by the activity of the single-stranded
DNA from $3X 174 phage.

(3) Metal ion requirements: Optimal concentrations for Mn++ and Mg++
when added separately to the reaction mixture were 2 X low3 M and 8 X
                Addition of Mg++ increased the rate at sub-
optimal levels of Mn++; thus, the addition of M Mn++ and 4 X M

M, respectively (Figure 1).

86 BIOCHEMISTRY: CHAMBERLIN AND BERG PROC. N. A. S.

Mg++ to the same reaction mixture gave a rate of incorporation equal to that
found with the optimal concentration of Mn++ alone (2 X
Characterization of the enzymatically synthesized RNA:  (1) Net synthesis of the
RNA product:  With two times the level of the four ribonucleoside triphosphates
and five to ten times the amount of enzyme used in the routine assay, the amount
of RNA formed during an extended incubation exceeded the dbunt of DNA
added to the reaction.                          With most of
the DNA preparations used, the amount of RNA formed was three to five times
greater than the amount of DNA added, while with d-AT copolymer, up to 15
times as much of the corresponding AU polynucleotide was produced (Table 6).
The rate of synthesis decreased after the first 20 min although further synthesis
occurred up to two hr.  Preliminary experiments indicate that this was not due to
enzyme inactivation nor to destruction of the priming DNA, but other possi-
bilities have not yet been investigated in detail.

                          Exposure of the isolated
"net synthesis" product to alkali converted > 98 per cent of the label to acid-
soluble products which were electrophoretically identical with the 2`-(3') nucleo-
side monophosphates.  Treatment with pancreatic DNase or E. coli DNA di-
esterasea1 produced no significant liberation of labeled acid-soluble products.

M).

We will designate this as "net synthesis."

(2)

Enzymatic and alkaline degradation of the product:

VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 87

    TABLE 6
NET SYNTHESIS OF RNA

                 Labeled Calculated Ratio of RNA Method

DNA primer incorporated RNA formed* DNA added isolation

Source of nucleotide amount of isolated to of

(mrmoles)

Calf thymus            81 200           2.0 -4
+X 174 phage          90 510           5.1 A
T2 phage             78 360          3.6 A
T2 phage             72 410           4.1 B

T2 phage             150 460          4.6 C
T5 phage             152 500          5.0 C
d-AT copolymer        155 310         15.0 C

CMP3*

C !`-AM P

* The amount of RNA in the isolated product was calculated from the amount of label incorporated
and the base ratio of the primer DNA.

Sunfhecria;  Each tube contained in a final volume of 0.5 ml:  20 pmoles of Tris buffer,  H 7.85; 8 pmoles
of MgClt; 400 rnpmoles each of ATP, CTP, UTP, GTP; 6 rmolea of &mercaptoethanof; 100 mpmoles of
DNA; and 100 pg of Fraction 4 protein.  When d-AT was used as primer, only 20 mpmoles of primer were
added and CTP and GTP were omitted from the mixture.

           The incubation mixture was heated for 10 min at 60 in 0.4 M NaCI. then di-
alyzed 38 hr against 0.2 M NaCl-O.01 M Tru, pH 7.85.  B.  The reaction mixture waa extracted two times
with phenol and the phenol fractions were washed two times with 0.4 M NaCl.  The aqueous layers were
pooled and dialyzed as in A.       roduct was precipitated from the incubation mixture with a solu-
tion containing 60 per cent ethanol an10.5 M NaCl at OD, washed once with the same solution. and dis
solved in 1 ml of 0.2 M NaCl., then dialyzed aa in A.

The incubation time waa 3 hr at 37".

Product isohfion; A.

C.

The

Treatment of 10 to 20 mpmoles of enzymatically prepared CMP32-labeled RNA
with 0.1 pg of pancreatic RNase for 1 hr liberated 75 to 94 per cent of the P32
label as acid-soluble products.  The amount of acid-insoluble P32 remaining after
RNase treatment varied with different DNA primers and different methods of
product isolation.  Using 10 times the amount of RNase did not appreciably alter
the results.  The significance of this RNase resistant fraction is presently un-
known.

(3) Nucleotide composition: The nucleotide composition of the product was
examined by two different methods.  In the first method, four separate assays,
each containing a different labeled nucleoside triphosphate, were performed with
each DNA preparation, and the molar ratio in which the labeled nucleotides were
incorporated was measured (Method A).  The second method utilized electro-
phoretic separation44 of the mononucleotides resulting from the alkaline degrad-
ation of a "net synthesis" product in which all of the nucleoside triphosphates
were labeled with C14 (Method B).  The distribution of the label among isolated
nucleotides wdtherefore a measure of the composition of the newly synthesized
RNA.  The results (Table 7) indicate that the gross composition of the product
at all stages of synthesis was Complementary to that of the primer within the ac-
curacy of the method.  For double-stranded DNA, this complementary relation-
ship becomes one of identity, since in the priming DNA adenine equals thymine
and guanine equals cytosine.  However, in the case of single-stranded @X 174
DNA (Table 8), the composition is indeed complementary to that of the DNA,
and in this instance the amounts of AMP and UMP incorporation and of GMP
and CMP incorporation are not equal.  Furthermore, when double-stranded @X
174 DNA is used, the nucleotide composition of the resulting RNA is again iden-
tical to that of the DNA primer.

(4) Sedimentation telocity of the isolated product: The sedimentation velocity
of the isolated RNA product was determined in the Spinco Model E analytical
ultrac.entrifuge using ultraviolet optics. Values obtained in 0.2 M NaCl-

88

DNA primer

d-GC polymer
d-AT copolymer
d-AT copolymer
T2 phage
T5 phage
E. coli
dl. phlei
A. aerogenes

BIOCHEMISTRY: CHAMBERLIN AND BERG

PROC. N. A. s.

           TABLE 7
NUCLEOTIDE COMPOSITION OF THE RNA PRODUCT

Method                             Primer*
of -Nucleotide Composition--  E
analysis AMP UMP GMP CMP   G + C

(mpmoles)

A <0.03  <0.03    1.90 0.23 -
B 21.7   20.0     -
A 20.8    22.2   <0.03 <0.03 -
B 7.7    7.4    4.3 4.3 1.76
B 4.8    5.0    3.6 3.4 1.56

- -

A 1.8    1.9    1.9    2.0    1.01
A 3.7    4.0    7.9    8.5   0.48
A 1.8    1.7    2.3    2.2   0.80

Product  Product
A+U
      A+G
G+C   u+c

- -

1.76   1.03
1.40    1.00
0.95   0.95
0.47    0.93
0.78    1.05

* The values given for the ratio A + T/G + C in the priming DNA are those found by Jome et a1.a" except in
the case of phage T5 DNA.Ja

Method A:  For each DNA sample. four separate incubations were used. each containing a different ClLlabeled
nucleotide.  The amounts of DNA used in the various tests were as follows:  20 mpmoles of M. phlei, 50 mpmoles
of A. aerooenea, 180 mpmoles of E. coli, 20 mpmoles of d-AT 20 mpmoles of d-GC.  12.5 pg of Fraction 4 enzyme
were used in each incubation; all other conditions were tho& given for a standard assay.

Mefhod B:  The synthesis of the C24-labeled RNA was carried out under the following conditions.  The reaction
mixture (0.5 ml) contained:  20 pmoles of Tris buffer, pH 7.85, 0.5 pmole of MnCL. 2 pmoles of MgCk, 6 pmoles of
8-mercaptoethanol, 100 mpmoles each of CicATP. CicUTP, CiLGTP, CicCTP. 100 mpmolea of DNA, and 180
pg of Fraction 4 enzyme.  Where d-AT primer was used, 20 mprnoles of primer were added and no CTP or GTP
were added.  After 180 min at 37", the product was precipitated and washed with cold 3 per cent PCA and incubated
in 0.3 M KOH for 18 hr at 37'.  An aliquot to which carrier nucleotides had been added was subjected to paper
electrophoresis at pH 3.5 in 0.05 M citrate buffer.  The individual nucleotides which were visualized with a UV
lamp were eluted in 0.01 M HCI and counted.  Recovery of the CIClabel in the eluted fractions was >95 percent.
180 mpmoles, 140 mpmoles, and 200 mpmoles of polyribonucleotide were produced in the reactions primed with T2
DNA, T5 DNA, and d-AT, respectively.

TABLE 8


RNA SYNTHESIS

COMPAR4TIVE BEHAVIOR OF SINGLE- AND DOUBLE-STR4NDED

0x

174 DNA AS PRIMER FOR

                               --Nucleotide Composition of RNA-
State of DN.4 AMP UMP GMP CMP
used as primer (per cent)

Si ?!le-stranded Predicted * 32 8 24 6 18 5 24 1
           Found by method A 32 0 24 1 19 5 24 3
           Found by method B 35 0 24 6 19 3 21 1

Double-stranded Predicted* 28 7 28 7 21 3 21 3
            Found by method B 28 9 29 1 20 9 20 9

     'I
'I ',

1L Ll

Method A:  Conditions as given in Table 7.  32 mpmoles of single-stranded PX 174 DNA were used in
each incubation with 8 pg of Fraction 4 enzyme.

Method B:  Conditions as given in Table 7. With single-stranded DNA as primer, 25 mpmoles of, priming
DNA were added, 71 mpmoles of RNA were produced in a 60 min incubation with 80 pg of Fraction 4 en-
zyme.  For the double-stranded DNA, 26 mamoles of priming DNA were added;  32 mpmoles of RNA
were produced in a 60 min incubation with 40 pg of Fraction 4 enzyme.

* The predicted values were calculated on the assum tion that the single-stranded 0X 174 DNA would
yield RNA with a composition complementary to tEe composition reported by Sinsheimer.'9  Upon
replication of 0X 174 DNA with DNA-polymerase it was aaaumed that the uroduct (presumably double-
stranded DNA) had a base composition which is the average of the composition of the oriainal and of the
newly synthesized strands.

That this is a reasonable assumption is shown by unpublished studies of M. Swartz. T. Trautner, and
A. Kornberg. When @X 174, DNA was used to prime limited (<30 per cent) or extensive (600 per cent)
DNA synthesis. the composition of the newly formed DNA was:

                  dAMP TMP dGMP dCMP
Limited synthesis 31.0 24.1 20.1 24.5

Extensive synthesis 29.4 26.9 22.3 21 .s

0.01 M Tris, pH 7.9, ranged from 6 to 7.5 for 2- to 15-fold [[net synthesis" products
prepared by phenol extraction or by salt-ethanol precipitation.
                        As pointed out earlier, REA
synthesis, as measured by the incorporation of either labeled CTP, UTP, or GTP
did not occur in the absence of the other three nucleoside triphosphates or, in fact,
in the absence of any one of the nucleoside triphosphates.  It was therefore sur-
prising to find that purified fractions of RNA polymerase catalyze the conversion

DNA-dependent formation of polyadenylic acid:

VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 89

of C14-ATP to an acid-insoluble form in the absence of the other three ribonucleo-
side triphosphates.

The ratio of the activities

AMP incorporated in the absence of UTP, CTP, GTP

AMP incorporated in the presence of UTP, CTP, GTP

increased from 0.5 to 10 as purification of the enzyme progressed.

(1) Requirements for polyadenylic acid formation: Polyadenylic acid formation
from ATP occurred only in the presence of DNA, a divalent cation, and the pur-
ified enzyme (Table 9).  Note that addition of unlabeled ADP produces only a

TABLE 9 TABLE 10


FORMATION RNA POLYMERASE

REQUIREMENTS FOR POLYADENYLIC ACID

INCORPORATION OF SINGLE NUCLEOTIDES BY

Incorporation
of AMP

System (mpmoles)

Complete (with ATP as the only

nucleoside triphosphate) 9.9

minus DNA <O. 03
minus Mn++ 2.5
minus Mg++ 8.7
plus RNase 6.9
plus DNase 0.3
plus ADP 7.6

The-reaction mixture contained, in a final volume of
0.25 ml: 10 pmoles of Tris buffer, pH 7.85; 0.5 pmole
of MnClt; 2 pmoles of MgClz: 3 pmoles of @-mer-
captoethanol; 100 mpmolea of ClLATP; 280 mp-
moles of calf thymus DNA:  and 3 pg of Fraction 4
RNA polymerase.  Where indicated, 25 fig of pancre-
atic RNase, 25 pg of pancreatic DNase, and 100
mpmolea of ADP were added.  The incubation time
was 10 min at 37".

Nucleotide Nucleotide

CI4-ATP 23
CId-UTP 0.90
C"GTP 0.09
CTP3* 0.07

added incorporation

(mpmoles)

The conditions were the same as those described in
Table 9. except that ATP was replaced where indi-
cated by an equal amount of each of the other nucleo-
side triphosphates.  13 pg of Fraction 4 enzyme were
added.

small dilution of the incorporation of label from C14-ATP.  The rate of incorpor-
ation was directly proportional to the amount of enzyme added; 1.8, 3.6, and 7.2
pg of Fraction 4 enzyme catalyzed the incorporation of 4.0, 8.2, and 17.5 mpmoles
of CI4-AMP in a standard 10 min assay.  The rate of incorporation remained
constant up to over 75 per cent utilization of the added ATP.

There was no incorporation of CMP or GMP when the corresponding nucleo-
side triphosphates were added singly to the reaction, although UMP incorporation
occurred to a small, but significant, extent (Table 10).

   The DNA requirement for polyadenylic acid formation:  The ability of
various nucleic acid preparations to support polyadenylic acid synthesis is shown
in Table 11.  Note that neither RNA nor polyadenylic acid itself replaced the
DNA requirement.  To test whether DNA might be necessary only to initiate
polyadenylic acid synthesis, an experiment was performed in which the priming
DNA was destroyed after some polyadenylic acid formation had already occurred.
It can be seen that destruction of the DNA by DNase blocked further synthesis
of the polyadenylic acid (Table 12).  This implies that the DNA is required not
only for the initiation of polyadenylic acid synthesis, but also for the continued
formation of the polynucleotide.

(3)  The e$ect of the other ribonucleoside triphosphates on polyadenylic acid for-
mation:  The addition of the other ribonucleoside triphosphates resulted in an
inhibition of the rate of CL4 AMP incorporation (Table 13).  It can be seen, for

(2)

90 BIOCHEMISII'BY: CHAMBERLIN AND BERG PRnc. N. A. S.

          TABLE 11
PRIMING EFFICIENCY OF VARIOUS NUCLEIC
ACID PREPARATIONS FOR POLYADENYLIC ACID
          FORMATION

  AMP
incorporation

Primer (mpmoles)

Calf thymus DNA 10
Salmon sperm DNA 7.7
T2 phage DNA 4.9

d-AT copolymer <0.03
Amino acid-acceptor RNA 0.35
Polyadenylic acid 0.07

The Conditions of the incubation were as described
in Table 9, except that the following amounts of
nucleic acid were added: 300 mpmoles of salmon
*perm DNA; 100 mpmoles of T2 phage DNA; 12
mprnoles of d-AT; 110 rnrmoles of amino acid-
acceptor RNA and 5 mpmoles of polyadenylic acid.
3 p~ of Fraction 4 enzyme were added.

        TABLE 12


ADDITION DURING POLYADENYLIC ACID
        SYNTHESIS

THE EFFECT OF DEOXYRIBONUCI.E.4SE

  AMP
incorporation

Tube Treatment (mpmoles)

1 5-min incubation 7.1
2 10-min incubation 16.2
3 10-min incubation 6.9

The reaction mixtures were as described io Table 9.
exce t that 6 pg of Fraction 4 waa used.  Tube 1 was
incugated for 5 min, heated for 3 min at 100", and
then assayed as usual.  Tube 2 was incubated for 10
min before assaying.  Tube 3 was incubated for 5 niin
and heated as in the case of tube 1; 25 pg of pancreatic
DNase were then added and the mixture incubated
for an additional 5 min.  At this time, 6 pg of fresh
RNA polymerase were added and a third 5min incii-
bation was allowed.

                       TABLE 13
EFFECT OF THE OTHER RIBONUCLEOSIDE TRIPHOSPHATES ON POLYADENYLIC ACID FORMATION

Component

Complete system

DlUS CTP

bluZ UTP
plus GTP
dus CTP. UTP

  AMP
incorporation
(mpmoles)
  26

6.0

5.2
2.0
1.3
06

plus CTP; GTP
plus UTP, GTP 0.5
plus UTP, GTP, CTP 2.2

Complete system as in Table 9, except that 6 pg of Fraction 4 protein were added.

Where indicated, 100

mprnoles of each nucleoside triphosphate were added.

example, that in the presence of any two of the other triphosphates the amount of
polyadenylic acid formed is less than 5 per cent that of the control in which only
ATP was added.  As has been previously shown, in the presence of all four tri-
phosphates, AMP is incorporated into a product having a base composition deter-
mined by the DNA primer, and hence under these conditions polyadenylic acid
synthesis does not appear to occur.

(4) Characterization of the polyadenylic acid product: Preliminary characteri-
zation of the product is consistent with its identity as a polyadenylic acid.  The
addition of pancreatic RNase to the assay system lowered the rate of incorporation
only slightly (about 30%).  Treatment with 0.5 M KOH for 18 hr at 37" con-
verted the product to an acid-soluble form.  Of the C14 in the hydrolysate, 97 per
cent was associated with 2'-(3') AMP on paper ~hromatography~~ and paper
electroph~resis,~~ less than 1.5 per cent with adenosine, and less than 1.5 per cent
was found in a region corresponding to adenosine 3'4' diphosphate.  This im-
plies that the minimum chain length of the polyadenylate is in the order of 60 to
70 nucleotide residues.

Discussion.-There is a striking similarity between the reactions catalyzed by the
RNA polymerase described here and E. coli DNA polymerasezs  Both use only
the nucleoside triphosphates as nucleotidyl donors, and both display absolute
requirements for a divalent cation and a DNA primer for polynucleotide synthesis.$
In both cases, some ambiguity exists as to the relative efficiency of single as com-
pared to double-stranded DNA for priming of polynucleotide synthesis.  In each

VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 91

reaction, both forms of DKA are active as primers, but a meaningful comparison
between the two with regard to the mechanism of priming must await a more de-
tailed physical and chemical characterization of the different DNA preparations,
and further purification of the enzymes involved.
The product formed by RNA polymerase, as in the analogous case of DNA
polymerasez9 has a base composition which, within experimental error, is comple-
mentary to that of the priming DNA.  This finding, which is in agreement with
the results obtained by othersl6v 17, 21, supports the view that the nucleotide se-
quences in the DNA direct the order of nucleotides in the enzymatically synthesized
RNA.  A more critical test of this hypothesis involves a comparison of the nucleo-
tide sequence of the priming DNA and the newly synthesized RNA.  In this re-
gard, Furth et al.19 have shown that the repeating sequence of dAMP and dTMP
in d-AT copolymer is faithfully replicated by the RNA polymerase in the form of
an alternating AMP and UMP sequence.  More recently Weiss and Nakam~to~~
have shown that RNA synthesized with an RNA polymerase from M. lysodeikticus
contains the same frequencies of dinucleotide pairs as occur in the DNA primer.
Additional  experiment^^^ which demonstrate the formation of a DNA-RNA com-
plex after heating and slow-cooling48 suggest that the homology of nucleotide se-
quences may occur over relatively long regions.

Does RNA polymerase copy the sequence of only one or both strands of DNA?
This question is relevant not only to an understanding of the enzymatic copying
mechanism, but also to any speculations as to the mechanism of information
transfer from DNA to RNA.  The fact that with double-stranded DNA primers
the base composition of the newly made RNA is essentially identical to the over-all
composition of both strands of the DNA already suggests that each strand can
function equally well.  An alternative hypothesis is to suppose that only one
strand can be copied, and that the "primer" strand has, in the case of every DNA
studled, a base composition identical to the average composition of both strands.
Using the double-stranded form of @bx 174 DNA3S* 36 in which it is known that the
base compositions of the two strands differ,49 it is possible to test this question di-
rectly.  The results show that both strands of the duplex Serve to direct the com-
position of the RNA product.

This result still leaves open the question of whether both strands are copied in
one replication cycle or whether only one strand is copied at a time and the choice
between strands is random.  When considering the relevance of this finding to
information transfer, one must bear in mind that the existence of artificially pro-
duced ends in an isolated DNA preparation may allow RNA formation to proceed
from both ends of the double strand.  This, however, may not occur with the DNA
as it exists in the genome; that is, in vivo some structural feature in the chromo-
somal DNA may cause RNA synthesis to proceed in a unidirectional manner and
therefore copy the sequence of only one of the two strands.

The formation of DNA-RNA complexes has been described by several groups of
workers14*~ *I  although only limited information is available concerning their
chemical structure and their metabolic and chemical stability.  The fact that in
the enzymatic reaction net synthesis of RNA occurs argues against the formation
of a stable, stoichiometric complex of RNA and DNA.  A further argument
against the formation of such a complex is the finding that most of the DNA re-

92 UIOCHEAfISTIiY: CH.4AfBEKLI.Y -1.YD BERG PROC. N. A. s.

mainirig at the end of the reaction appears to be identical to the DXA added, and
no component containing both DKA and newly synthesized RKA was detectable
on CsCl gradient ccntrif~gation.~~  Whether some transient complex is formed as
an intermediate is somewhat more difficult to assess.

The formation of polyadenylic acid in a DNA-dependent reaction is significant
in view of the fact that none of the other ribonucleoside triphosphates, takeii
singly or even in groups of three, are utilized to any appieciable extent for poly-
nucleotide synthesis.  An exceptioii to this is, of course, the situation where the
DKA dictates the incorporation of only one or two nucleotides (e.g., with poly
dT," d-AT," or d-GC).

Three possibilities which could account for polyadeiiylic acid synthesis are that
it results from (a) a special feature of RNA polymerase itself, (b) a separate poly-
adenylic acid polymerase, or (c) polynucleotide phosphorylase.  The last pos-
sibility is least likely because of the absolute requirement for DNA in the initiation
and continuation of synthesis, the failure of ADP to give a significant dilution of
the incorporation from ATP, the low amounts of polynucleotide phosphorylasc
activity found in the enzyme preparation as measured by 1',32 exchange, the in-
ability of Mg++ alone to support maximal rates of synthesis, the lack of polymeri-
zation of the other nucleoside triphosphates, and the marked inhibition of poly-
adenylic acid synthesis by any one or all four of the triphosphates  The question
of whether polyadenylic acid synthesis is catalyzed by RSA polymerase or by
another enzyme cannot be resolved at the preseiit time.

With regard to the mechanism of the DNA-dependent polyadenylic acid for-
mation, two aspects deserve specific comment.  The first concerns the mechanism
of the inhibition of polyadenylic acid synthesis by any one or all of the other tri-
phosphates.  It should be recalled that polyadenylic acid synthesis does not occur
in the presence of all four ribonucleoside triphosphates (< 3y0), since under thcsc
conditions the base composition of the newly synthesized RNA is very close to that
predicted by the composition of the DSA primers.  The second notable feature of
the reaction is its complete dependence on DXA, and the failure of d-AT to prime
polyadenylic acid synthesis.  One way to account for these findings is to assume
that a sequence of thymidylate rcsidues in the DNA, which does not occur in d-AT,
can prime the formation of a corresponding run of AMP residues and, by subsequent
"slippa>' of one chain along the other, lead to a DNA-dependent elongation of the

The introduction of any other iiucleotidc into the growing

     lic acid chain.

polyadenylic acid chain.

Summary.-An RNA polymerase has been isolated from E. coli which in the pres-
ence of the four ribonucleoside triphosphates, a divalent metal ion, and DNA
synthesizes RNA with a base composition complementary to that of the priming
DNA.                             Thus, while single-
stranded @X 174 DNA yields RNA with a base composition complementary to that
of the single-stranded form, double-stranded $JX 171 DXA (synt hcsized with DNA
polymerase) primes the synthesis of RATA with a base composition virtually the
same as that in both strands of the DNA.  A novel feature of the RXA polymerase
preparations is their ability to catalyze a DNA-dependent formation of polyadenylic
acid in the presence of ATP alone.  Neither UTP, GTP, nor CTP yields corre-

t block or inhibit the sliding process and thereby terminate the growing

Both strands of DNA can prime new KXA synthesis.

VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 93

sponding homopolymers; the DNA-dependent formation of polyadenylic acid is vir-
tually completely inhibited by the presence of the other nucleoside triphosphates.

* This work was supported by Public Health Service Research Grant No. RG6814 and Public

t Pre-doctoral Fellow.

f The abbreviations used in this paper are:  RNA and DNA for rib+ and deoxyribonucleic
acid, respectively; poly dT for polydeoxythymidylate; d-AT for the deoxyadenylate-thymidylate
copolymer; d-GC for the deoxyguanylate-deoxycytidylate polymer:  AMP, ADP and ATP for
adenosine-5'-mono-, di-, and triphosphates, respectively.  A similar notation is used for the cyti-
dine (C), guanosine (G), and uridine (U) derivatives and their deoxy analogues (dA, dC, dG, dT) .
Pi is used for inorganic orthophosphate, TMV for tobacco mosaic virus, and DNase and RNase
for deoxyribo- and ribonuclease activities, respectively.

Q Under certain conditions DNA polymerase preparations will, in the absence of DNA, produce

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