Competition between RNA polymerase and DNA polymerase for the DNA template

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COMPETITION AND Paul

Berg,

BETWEEN

RNA

POLYMERASE

DNA

POLYMERASE

FOR

Roger

D. Kornberg,

Harriet

Fancher

Stanford

University

Department

of Biochemistry, &lo

Alto,

THE

DNA

TEMPLATE* and Marianne School

Dieckmann

of Medicine,

California

Received January 29, 1965 A gap in our understanding synthesis, mation

particularly about

form

Are

of unique

there

binds

exist,

is their

in vivo,

of the DNA

specific

nucleotide

the enzyme what

as it occurs

how transcription

terminated.

of the mechanism

and from

which

nature

results

from

template

“start-stop”

sequences

of DNA-directed

it dissociates

of infor-

is initiated

signals,

or structural

a lack

and

perhaps

in the

conformations, ? And if

and how does the enzyme

RNA

at which

“signals

recognize

” do

and respond

to them? One approach RNA

polymerase

deduced

from

to this

and the DNA kinetic

inhibit

Tissieres, shown

to inhibit

(Krakow

and Ochoa,

Initially

these

which

1963;

Fox,

Chamberlin,

1963;

1962;

Stevens

and Henry,

1964) and inhibitor

Menten *

This Public

DNA,

were

(competitive

work

was

Health

interpreted inhibition)

supported

as the findings effective

and Evans,

RNA

kinetics

in part

(Fox

by grants

that

Service. 932

DNA

templates 1964).

single-stranded (Hurwitz,

Fox and Weiss,

of classical

from

1964);

and Weiss,

template

et al.,

and Ochoa,

homopolymer

(Wood and Berg,

in the context

was

and synthetic

(Krakow

Haselkorn

is a more

Anders

that natural

Fox and Weiss,

from

between

information

in vitro

1963;

Robinson,

as well

observations,

DNA at low concentrations

stranded

showed

and Gros, transcription

the interaction

The earliest

DNA transcription

Bourgeois

was also

is to examine

template.

experiments

polyribonucleotides 1963;

problem

1964;

Furth, 1964;

1964) than

double-

MichaelisFox and Weiss,

the United

States

Vol. 18, No. 5-6, 1965

1964;

BIOCHEMICAL AND 8lOPHYSlCAL RESEARCH COMMUNICATIONS

Wood and Berg,

volved:

instead

1964).

But the situation

of a readily

polynucleotide,

dissociable

the interaction

(or RNA)

produces

a relatively

inhibition

is actually

is actually

more

between

enzyme

complex

of RNA

polymerase

and DNA

poorly

dissociating

complex,

“pseudo-competitive”

inand

template so that

the

(see note to Wood and Berg,

1964). Evidence erase

and DNA

Bremer DNA

for

virtually

irreversible

has been obtained

and Konrad molecule

(1964)

was under

copying

were

unable

clusion

is reinforced

(Berg,

unpublished)

but produces

little

inhibition

of RNA

inhibition

nucleoside

polyphosphates

of the lack

of appreciable the newly

at maximal ternary

rate

complex

of another

DNA.

several

RNA,

for only

a short

of DNA,

RNA

from

explains

of

studies

the non-competitive

polyadenylate

synthesis

1964).

Indeed,

from

the transcription

and RNA

by

because

template

reaction

the resulting

polymerase

mixing

The low dissociation

1964).

with

dAT

of template,

after

made

of the enzyme

time

con-

poly

addition

minutes

and Berg,

dissociation

This

communication)

was also

of DNA-directed

in the

inhibits

before

also

(Chamberlin

formed

engaged

minutes

polynucleotides

of a particular

molecules

private

(Weiss,

polym-

of experiments.

completely

conclusion

of RNA

from

(“high-efficiency”)

and from

several

A similar

polymerase

DNA

if it is added

properties

types

dC (Chamberlin,

it is added

of the inhibitory

that

of RNA

transcription

transcription

when

and template.

once

enzyme

by the finding or poly

several

that

those

to initiate

transcription

enzyme

from

showed way,

associations

DNA

in vitro

proceeds

accumulation (Bremer

of a

and Konrad,

1964). The nature stoichiometry,

the sites

the interactions present

of the putative

study

which

replication binding

we show that

to DNA,

inhibits

exonuclease

II (Lehman

(Richardson,

Lehman

E . coli

at which

complex,

enzyme binding,

polymerase

binds

preventing

template.

and Kornberg,

in particular

binding

strongly

Moreover,

RNA

of DNA

exonuclease

(Cunningham,

Roussos 933

by

I (Lehman,

1962).

the

initiating

polymerase,

and de Garilhe,

and Pratt,

In the

from

but does not affect

Catlin

and

at or near

1964) and exonuclease 1964),

its

occurs,

is not known.

DNA polymerase

and Richardson,

I (Lehman,

DNA

strong

the action

nuclease

endonuclease

such

RNA

DNA,

of the same

of micrococcal

on the DNA produce

ends of double-stranded

enzyme-

1960),

III the activity 1956) and

Vol. 18, No. 5-6, 1965

BJOCHEMICAL AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

,dNA

synthesis l

15 30

Legend

to Figure

The or

DNA

found

data

with

7 m4,

by itself.

of

of calf

thymus

and

DNA

of RNA

polymerase

(Fraction

of

polymerase

(hydroxylapatite

each

indicated of ATP,

GTP,

10 mpmoles umole)

or

at 37O, were

each all ml and

of

PCA

aliquot

Cab-o-sil scintillation

was and

IV

of cold the

added the

contained

fraction

C14,

(5 to

dCTP

or

nucleoside

Dounce,

spectrometer.

934

either

dTTP-

three

then

pre-

(1962). 1962))

10 pg

and

et al.,

POP-

C l4

1964)).

or

x 106cpm/

(4.5

After

and

in

POPOP were

2 pg

100 mumoles

times

dissolved

isotopes

buffer,

10 x 106cpm/pmole),

(PCA)

washed

both

1952)

triphosphates. acid

and

or

of Tris varying

(Richardson

contained

in

triphosphates.

20 pmoles

and

DNA

synthesis

ribonucleoside

Berg,

to a toluene-ethanol-

P32,

DNA

and

was

by centrifugation

four

8

0

of 2-mercaptoethanol,

perchloric

precipitate

8 -

RNA

to that

0 -

(Chamberlin

dGTP,

3.5%

synthesis;

Kornberg

a-P32CTP

of either

enzymes,

and

UTPand

eight

two

by Aposhian

mixtures

above

RNA

Polymerase.

rates

of the

Simmons

reaction

of dATP,

of the

added

aliquots An

1.5

as described

the

ml)

(Ray,

with

Where

the

by RNA

of initial

0

2 pmoles

treated

DNA

DNase

ratio

polymerase;

(0.3

MgC12,

Synthesis

0 -

of RNA

mixtures

135 “0, I65 Is0

presence

polymerase

reaction

2 pmoles

quantities

as the in the

presence

of RNA

The pH

enzyme

in the

of DNA

expressed

observed

each

presence

Inhibition

are

synthesis

synthesis the

1.

45 60 75 90 to5 no LThymus DNA],,uM

-

0.5

mg

with 1.5

of albumin

1.5-ml N

mixture

measured

20 min.

NH40H. containing in a Tri-Garb

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

Vol. 18, No. 5-6, 1965

RESULTS

The template

original

(Chamberlin

DNA

polymerase

1964)). influence

polymerase

DNA

synthesis

presence

by RNA

but

DNA

amount

Kornberg,

enzyme the

is unaffected

had

effect

the

by the

no at

presence

triphosphates.

By

polymerase

at all

more

is

DNA

not of

pronounced

DNA

in the

concen-

levels.

necessary

The

for

synthesis absence

of

contrast,

at low

at higher

inhibition

given

is that

amount inhibition.

templates

DNA

synthesis.

(or

would

inhiat low

of the

ribo-

Since

of helical results

DNA from

level

As

replication

more

DNA

its

it strong

was

not

of

DNA

synthesis

very

DNA

template.

935

at or

blocked;

synthesis

near

likely

the

that

more

initiating

replication

inhibition near

polymer-

of fresh

for

initiates

at or

with

sites

is added

sites

3).

affected

addition

new

amount

polymerase

by RNA

polymerase

possible

association

the

(Figure

appreciably

polymerase.

to provide

seems

of dAT

DNA

are

polymerase

chains,

rate

RNA

sites)

be expected

the

to

upon

of RNA

of

strongly

concentrations

concentration

quantities

inhibition

binds

depends

template

example,

dAT

As

2).

synthesis

14 PM of dAT

the

polymerase.

at low

(Figure

dAT

triphosphates)

DNA

marked

increasing

in the

then

by

levels

on the

is initiated.

molecules

polymerase

and

polymerase

replication

dAT

For

increase

RNA

copolymer

of dAT

and

for

of ribonucleoside

is quite

of dAT,

explanation

DNA

dAT

at high

polymerase

One

absence

of inhibition

by up to a five-fold

DNA

formation

and

, 1962),

triphosphates

inhibition

added

greater

ends

hardly

(in the

reversed

6 pg of RNA

the

at limiting

(by RNA

DNA

each

&.

by RNA

in fact

the

polymerase

template

strikingly

of the

primers,

any

where

of template

deoxynucleoside

is even

replication

The

ase

affect Aposhian

synthesis

ribonucleoside

completely

produce

RNA

four

polymerase

the

of RNA

of RNA

would

(Hurwitz

if,

triphosphates.

inhibits with

1962)) Schildkraut,

is inhibited

concentrations

to determine

striking.

polymerase;

RNA

With

the

was

synthesis

levels

is

is inhibited

DNA

nucleoside

Berg,

that

and

of four

primer

and

activity

1 shows

DNA

of

concomitant

of template

Figure

but

other’s

levels

trations

the

at saturating

on the

limiting

experiment

(Richardson,

Although

bition

of the

concentrations,

polymerase (by

plan

the

by RNA ends

of the

at

Vol. 18, No. 5-6, 1965

Legend

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to Figure The

synthesis absence 20 pmoles

2.

data

Inhibition

are

in the

expressed

presence

of RNA

polymerase.

of

buffer,

Tris

2-mercaptoethanol, copolymer

dAT

of RNA

pH

synthesis

was

mixture

measured

Pblymerase.

rate

of dAT

observed

in the

(0.3

ml)

contained

of MgC12,

of dATP

Radding,

(where

initial

to that

2 pmoles each

by RNA

of the

reaction

7.4,

Adler,

ratio

polymerase

The

polymerase

Replication

as the

10 mpmoles

(Schachman,

10 ng of RNA

of dAT

and

Lehman

indicated)

and

as described

2 pmoles of 14 dTTPC , dAT

and 0.5

in Fig.

Kornberg,

pg of

DNA

1960), polymerase.

1.

RNA polymerose,pg Legend

to Figure

3.

Inhibition

of dAT

Concentrations The those

conditions

described

of the

in Figures

of RNA

reaction 1 and

Replication

2.

936

and

at Different

Polymerase

method

of assay

and were

dAT. essentially

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 18, No. 5-6, 1965

According on

DNA

DNA

to this

at the

3’- hydroxyl

replication

be inhibited

the

exonuclease 1964)

(micrococcal (Lehman

effects

of RNA

III

were

but

which

has

on the

to Figure

1956)

et al.,

Figure

examined.

Effect

4.

of RNA

The degradative absence

data

are

process of the

20 pmoles

expressed in the

and

of Tris

presence The

buffer,

pH

2-mercaptoethanol,

variable

(4 x 105cpm/~mole)

and

not

more

30 min. and

than at 37O.

0. 5 ml

fugation napthalene-

30% of the

of cold

an aliquot POP-

At

the

7.4,

ratio

an amount

end

reaction

POPOP

of dAT Richardson,

by two

endonucleases endonuclease

4 shows

that

the

-

I

activity

e o Q Q 0

-

on Endo-

and

of

of each

initial

polymerase (0.3

of MgC12, 14 C -labeled of the

rates

of each

to the

rate

ml)

2 pmoles d.AT

enzymes

listed

to an acid-soluble

of the

incubation

50 pg of calf.thymus

supernatant

fluid in the

937

Tri-

were was Garb

added. counted

of

copolymer

converted

acid

in the

contained

was

solvent

by

coli

mixture

2 moles

3. 5% trichloracetic of the

DNA.

E.

of the

of RNA

quantities

dAT

also

Action. as the

polymerase.

of

and

and

Polymerase

Exonuclease

levels

in

should

degradation

1964)

attack

implicated

1963),

II (Lehman

et al.,

their

been

at high

Endonuclease I Micrococcal nucleaseExonuclease RI ExonuckaseZ Exonuclease t

Legend

initiate

Kornberg,

not

exonuclease

(Cunningham

1962))

Hal.,

end

polymerase

(Richardson

which

and

at low

1960),

nuclease

the

Schildkraut

polymerase

exonuclease

enzymes

terminus,

I (Lehman,

and

other

(Richardson, by RNA

Accordingly,

model,

After

so that form

in

DNA centri-

in a dioxane-

scintillation

spectrometer.

Vol. 18, No. 5-6, 1965

of micrococcal

nuclease

polymerase each

at any

of the

levels

of dAT

and

It has

been

level

only

is

so poorly

polymerase

for

view

concentrations

inhibition

of

DNA

DNA

Henry, one

sense. sites

from

synthesis

since

It seems

rather

equivalent

by RNA

polymerase

1962;

as an RNA

nucleotide

Berg, the

1964). ability

to reverse

(Fig.

Fox

enzyme-DNA

that

and

DNA

for

interaction

comparing

native

“affinity”

of DNA

(Wood

an experiment and

the

the

DNA

model, at low

et al.,

describe

per

of denatured

a greater

But

cannot

by the

concentrations.

(Hurwitz

1964).

in double-stranded

comes

has

by RNA

polymerase

dAT

polymerase

combining

than

this

increasing

and

by RNA

at higher

double-stranded

dissociable

more

in single-stranded Support

RNA

unaffected

as predicted

inhibited

Michaelis-Menten

finds

But,

inhibited

that

for

I is virtually

substrate.

slightly

Stevens

in the

“affinity”

endonuclease

of dAT

stated

1964;

AND 8lOPHYSlCAL RESEARCH COMMUNICATIONS

is markedly

than

Weiss,

complex

and

exonucleases

single-stranded and

BIOCHtMICAl

5).

Per

of

the nucleotide

DNA

:

g.0 0 5” IO 15 ” 20 25 ” 30 35” P [Thymus DNA],,uM 0 Legend

to Figure

The described sample 0.01 was

5.

Difference RNA

Polymerase

Calf

Thymus

experimental in

of calf M Tris,

Fig.

pH

7.4,

DNA for

that

and

either

(heated 10 min.

”

45

50

Inhibition Using

DNA

conditions

1 except

thymus

in the

40

as

of DNA Denatured

and

Native

of assay

were

those

thymus

DNA

or

calf

at a concentration in boiling

used.

938

by

Templates.

method

native

Synthesis

water

of 75 kg/ml and

cooled

a heated in

rapidly)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 18, No. 5-6, 1965

denatured

residue, the

inhibition

calf

by RNA

thymus

DNA

polymerase

is far

than

more

effective

is native

calf

in overcoming

thymus

DNA.

DISCUSSION

The forms

present

a poorly

polymerase

is bound for

complex

is not

binds

probably

amounts

of DNA

triphosphates, of

DNA

replication

the

of dAT only

calf

same

time

would

predict,

way

the

cation

fact

single-stranded is

made

of the

Berg,

1963),

for

1964)

and

Hayashi that

RNA

mentary

the

the

which DNA

by

reaction

binding

sites

to RNA

Relatively

at any

measure

inhibition

(with

dATP

and

after,

level of

dTTP

is observed

whether

or

at the

experiments).

DNA

to the

that

deoxynucleoside

before,

DNA

or

activity

unpublished

RNA

terminus.

complex.

polymerase

enzyme

of

to mean

in contrast

of inhibition

(Berg,

polymerase

that

there

than

in helical

does

the

are

or

also

As

polymerase inhibits

a given

of RNA

one

is under

further

present

repli-

of the 1964; does

Therefore

not

unit

1962;

8X

ring

174

Sinsheimer require

DNA and

3’- hydroxyl

it seems

939

reasonable

more

copolymer

and

(Wood

of the

1963;

structure

Lawrence,

require-

Stevens, (Bassel,

1964) to initiate

to suppose

and

inhibitory DNA

Berg,

ends

are

Fox

single-stranded and

nucleotide,

polymerase

by neutralization the

This

of DNA

et.,

(Chamberlin

in

measurements

of dAT

Moreover,

polymerase

comment.

of RNA

(Hurwitz

case

synthesis

per

whether

of transcription

polymerase.

Spiegelman,

that

DNA,

RNA

some

amount

synthesis

for

merits

showing

double-stranded

transcription

copying.

DNA

saturates

as in the

polymerase

more

by studies

polyadenylate

and

with

to which replication

3’-hydroxyl

without

thymus

replication

by inhibition

of RNA

ment

RNA

amount

copolymer

after

kinetics

1964),

activity

dAT

DNA than

Weiss,

same

is mixed

suggested

single-stranded effectively

or

by calf

the

interpret

dissociable with

with

DNA

polymerase

unpublished).

The

conclusion

a readily

for

we

RNA DNA

polymerase,

copolymer

of RNA

(Berg,

DNA

that

Helical

the

in experiments

even

addition

that

near

Moreover,

DNA

as the

at or

interfere

substrates),

thymus

DNA.

which

polymerase,

do not

template.

as the

forms

notion

as a template

action,

to note

the

with

available

strongly

It is interesting

large

supports

exonuclease

polymerase

polymerase,

further

dissociable

as a substrate RNA

study

that

indicate complethe

Vol. 18, No. 5-6, 1965

enzyme DNA

can

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bind

to any

molecules

1964)

“frayed”

or

and

therefore

provide

sites

that

sequence

RNA

(Krakow

argues

and

against

as the

polymerase

the

unique

site

whether

in

“native”

pairing

between

the

two

RNA

polymerase

ruptions

might

regions

where

only

could

adjacent

bases

regard

unstable DNA are

or

and

bind

intrastrand

that

shown

denaturation (bzarus

of RNA

but

and

the

can

physiologic

polymerase

interto

keto-amino between bases.

is

In

relatively

synthesis

with

clearly

further

nature

of the

and

at

be restricted

paired

dAT

Quite

significance

to DNA

Such

copolymer

direct

to

sites

interactions

AT

led base-

providing

between

1964).

Swartz,

where

of the

still

Fox*.,

regions

and

d6-methyl

to

we are

in length

bonding

even

Rather,

synthesis.

hydrophobic

been

or

1964;

RNA

modifications

polymerase.

of a specific

thereby

initiate

other

RNA

Weiss,

short

of helical

personal

existence

interrupted,

hydrogen

to assess

exist

nucleotides

or

and

the

ends

to dAT

attachment.

interstrand

to thermal

polymerase

binding

can

destabilize

it has

needed

is

binding

Fox

there

strands

methylation

functions

this

DNA

The (Zimm,

binds

assuming

of enzyme

be several

for

1963;

for

DNA.

unpaired

readily

Ochoa,

need

wonder

which

of the

probably

fact

homopolymers

segment

are

communication) The

unpaired

studies strong

templates.

SUMMARY

RNA relatively

polymerase

poorly

dissociated

as templates

fo.r

exonucleases

I,

coccal to mean terminus

or

of E. RNA

with

by

DNA

although

coli

endonuclease

DNA

and Such

denatured

DNA

complexes

are

polymerase

III,

polymerase

of helical

native

complexes.

reelication II,

nuclease that

reacts

they

binds

are

or readily

strongly

at or

unavailable

as substrates degraded

We interpret

I.

to give

near

for by micro-

these the

findings

3’- hydroxyl

chains.

ACKNOWLEDGMENTS Some cance

of RNA

of these polymerase

collaborations 1963;

notions

Chamberlin,

with

William 1963;

regarding

binding B.

the

to

DNA

Wood

and

Chamberlin

and

940

nature,

were Michael Berg,

location

developed Chamberlin 1964).

and

signifi-

in earlier (Wood,

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

Aposhian, Bassel,

H. A., Sci.,

Bremer,

H. 5l-,

V.

Chamberlin, 48,

Hayashi, M. and Wash. 52, 796. and

Fox,

C.

Hurwitz,

~a~,

(1963).

Ph. D.

Dissertation,

M.

and

Berg,

P.

(1962).

P~oc.

and

Berg,

P.

(1964).

J.

Catlin, L., Sot. 78,

and

R.

J. 49,

Lehman,

B. W. 4642.

M.,

S.

B.

J. J., 3752.

S.

3,

and

and

Chem.

(1964).

237,

Proc.

Nat.

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