Identification of the nucleoside monophosphate end-group on the product of the polynucleotide phosphorylase reaction

Vol. 23, No. 4, 1966

BIOCHEMICAL

IDENTIFICATION

OF

AND BIOPHYSICAL

THE

RESEARCH COMMUNICATIONS

NUCLEOSIDE

MONOPHOSPHATE

END-GROUPONTHEPRODUCTOFTHEPOLYNUCLEOTIDE PHOSPHORYLASEREACTION Richard Institut

de Biologie

Received

April

A. Harvey

the

polymerization

ing

linear

3’-5’

phosphorylase

synthesis

preparation

cr -phosphate

predicts

that

polynucleotide

units

bridges

(Grunberg-Manago, occurs

primers

that

the chain

proposed

group

a polymer

polymers

is discussed. Polynucleotide

-3’-phosphate-5’-pyrophosphate

polymerase This

with ppNpNp.

structure

, however,

an unspecified

provides

phosphorylase

was

448

. . NpNpN

that

phosphorylase

than a 5’-pyrophosphate. on the mechanism of the purified

and Grunberg-Manago (ppUp>

would nucleoside

evidence

by polynucleotide rather findings

by a 5’-

with

the

, in a (Maitra,

synthesized

terminate

; NDP

and the

mechanism

of NDP

synthesized

of Williams

bond formation

the polymerization

nucleoside paper

RNA

The simplest

diphosphate

1965).

would is,

Escheri-

molecule

of the polymer

terminate with a 5-monophosphate The possible significance of these

by the methods

for

the 5’-end

to yield

involves

of nucleoside

and Hurwitz,

that

by

In the case

from

1964).

reaction

Baltimore

. present

enzyme

of one diphosphate

molecule

to that

yield-

the participation --de novo without may possibly contaminate the enzyme

initiation group

an unspecified

ribonucleotide

enzyme

(France)

linked

1962).

by the

and Grunberg-Manago,

group;

be expected diphosphate) The

acid

phosphorylase

pyrophosphate (N being

Paris

to catalyze

diphosphates

initiation

analogous

shown

of nucleoside

of a second

Novogrodsky,

been

of ribonucleotide

the 3’ hydroxyl

reaction

Curie,

composed

(Williams for

has

of polyadenylic

chain --chia coli, of polynucleotide mclchanism

Pierre

of a number

polymers

phosphodiester

between

rue

7, 1966

Polynucleotide

of the

and M. Grunberg-Manago

Physico-chimique,

and uridine-3’

from --E. coli (196Q.Uridine ,5’-diphosphate

Vol. 23, No. 4, 1966

(pup)

BIOCHEMICAL

were

ADP

synthesized

and (p-32

nucleoside

1956).

Company

prepared

using

Five

times

measurements

were in

catalyzed , Ortiz

ribonuclease

either group

Tri-carb

background

counter.

on the product

phosphorylase reaction degradation of polyuridylic

tri-

Chemical Radioactivity

a Packard low

and

and

of Sigma

respectively.

of the 5’-end

(B-32P) unlabeled

reaction

products

or a Tracerlab

nature

Pi and

exchange

recrystalized

made

counter

polynucleotide by alkaline

from

BioResearch,

were

scintillation The

the

(1964).

(Grunberg-Manago

diphosphate

and Schwarz

RESEARCH COMMUNICATIONS

to Michels2on

phosphorylase

uridine

1JDP. Table

were

diphosphate,

Ochoa, tiated

according

P> UDP

by polynucleotide

AND BIOPHYSICAL

of the

was first investigated acid synthesized from

A reaction medium containing doubly labeled UDP (see I> was incubated at 37”. Aliquots (0.15 ml> were removed

6 and 60 minutes

after

of a solution sodium

of incubation

containing

dodecyl

and were

0.01

M ammonium

The

product

sulfate.

added

to 1.5

acetate-O.

ml

001 M

of the reaction

,polyuridylic

acid,was separated from the low molecular weight components of the reaction mixture by gel filtration on Sephadex G-50 equilibrated

with

the polymer

was

The

alkaline

unlabeled

solution pup

The

components

using

stepwise

and KCl.

0.01

M ammonium

hydrolyzed was

and ppUp

with

added

with

yields

exclusively

2’(3’)

-be the

expected

:mer with

neutralized were

elutions alkaline

.terminated

M KOH

of the hydrolysate

Since

products

and ppUp. incubation; were

however,

strongly

labeled

It is unlikely

end group

radioactivity

degradation is

not

during

appreciably

Alternatively,

the uridine-3’ with

cleaved

bond

Up and U would acid

in contrast, would

yield

found

a polypup,

Up dispup

in uridine-3’-

6 or 60 minutes

,5’-diphosphate

of

fractions

tritium.

the absence

the alkaline

the terminal

either

2,

of HCl

I shows the observed phosphorous in Up, was

after

that

markers. on Dowex

if the polyuridylic group;

digestion. Table and radioactive

Negligible

,ppUp,

at 37’.

50 (H+)and

concentrations

mononucleotides

phosphate-5’-pyrophosphate

Dowex

of the phosphodiester

a 5’-pyrophosphate

and U on alkaline tribution of tritium

lyophilyzation

18 hours

separated

increasing

a 5’-monophosphate

After for

as chromatographic were

cleavage

hydrolysis

with

acetate.

in 0.3

of ppUp

hydrolysis under

the

monophosphate

449

is due to chemical since

conditions group

authentic

ppUp

employed. might

be explained

Vol.

23,

No.

4, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE COMPOSITlON

Dowex fraction

Isotope counted

HYDROLYSATE ACID

15 ml

Counts/min/O.

6 min

COMMUNICATIONS

I

OF ALKALINE POLYURIDYLIC

OF

RESEARCH

incubation

incubation

60

medium

min

incubation

--------------------------------------------

615,000

3,

1,060,000

UP

25

=P

-------------------------------------------

30

3,200

3,

4,500

PUP 32P

20

10

PPUP

The reaction mixture (0.5 ml> contained 25 pmoles of Tris (pH 8.2)) 10 pmo1e.s 06 doubly labellgd UDP (&32P, 3H) ‘fit activity 8.1 x 10 and 1.8 x 10 counts/min/pmoles P and 3H, respectively), 5 pmoles MgC12 and 19 pg of polynucleotide phosphorylase (specific activity 340). HCl

by

the

presence

nucleotide minating the

enzyme

nascent

under

specific

for

identical

pg/ml) 8.2;

newly upon

To

was

added

UDP, 460;

20

products

due

detectable the

an

pmoles;

2 ml.

polynucleotide

polyuridylic

incomplete

polymer

the

the

of

a phosphatase

of

a ribonucleotide

the

5’

in

which

end

of present

reaction. as

450

or

polymer-

(Tris-HCl

50

j enzyme

30

pmoles

pg , specific

conditions,

immediately

by

the

pup

ribonuclease

these

degradation

was

in

the

hydrolyzed

phosphorylase;

course

phosphatase

effect

> . Under is

poly-

employed

medium

acid

the

ppUp,

2 pmoles

MgC12,

from to

at

performed incubation

volume

during of

to

final

synthesized release

was

no

possible

linkage

experiment

of

including those

the

poly-

a 5’-mono-

since

exhibited

to

eliminate

with

unlikely

compounds,

the a conta-

pyrophosphate acid

preparation of

in such

terminal

seems

a pyrophosphate an

activity

the polyuridylic

possibility

conditions

activity

Conceivably

hydrolyze

a variety

reaction.

polymer,

phosphatase

yielding This

toward

ization

group

could

phosphorylasc

activity

pH

extraneous preparation.

group.

nucleotide

pppU

an

polymer,

phosphate

(10

of

phosphorylase

no oligonucleotide ribonuclease Thus,

a mononucleotide

were the

5’ terminal during

the

Vol.

23,

No.

4, 1966

BIOCHEMICAL

synthetic

reaction

attack

and,

matogram

in parallel

found

with

to paper

electrophoresis

it traveled

at the

lamp

where

rate

same

was

as pup,

into

uridine-3’-phosphate-5’-pyrophosphate

again

recovered

indicate

the presence

point

of the polyuridylic

tiation

Similar rivative

results

acid been

acid

polynucleotide aspect

after

The side

chain

reaction reaction

since

positions

philic

may come from enzyme-nucleoside of the molecule

of polyuridylic

the chain

an enzymically

group;

3’-OH

of 14C - polyadenylic the absence product

of the

to be a fundamental

not involve

initiation

and elongation attack

of chain

polymer initiation,

the aqueous reaction monophosphate nucleoside diphosphate

451

medium complex.

into

For would

is

chain

on elong-

be the nucleo-

however, resulting

yield

the

both group

the hydroxyl in an

Subsequent monophosphate with

would

It

reactions

of a hydroxyl

diphosphate.

of the growing

of the bound

nucleo-

1966).

(Godefroy,

catalyzed

in the case

a free

of radioactivity

acid

of the nucleoside

of nucleoside

been

of (2- 14c> uridine -5’ -mono containing unlabeled uridine di-

t,erminal

not lead

the 3’ hydroxyl

a un-

has

Thus,

appears does

to incorporation

a -phosphate

yielded

the addition medium

a.tion

de-

was virtually

,196Q.

does

the

which

hydrolysis

to a reaction

that

at the inia purine

5’-diphosphate

phosphate possible

could

results

group

employing

acid

phosphate

involve

These

mechanism.

initiation

monophosphate

of radioactivity

in the polymerization

phosphorylase

experiments of ribonuclease

of (p- 32 P)ADP

alkaline

group

of the enzymic

Similar

where

chains.

and Grunberg-Manago

of a 5’-pyrophosphate

pH 3.5)

which

obtained

Polymerization

identified (Williams

and sublected

citrate,

the incubation.

high molecular weight polyadenylic 14 C-adenosine-3’, labeled. Further, tentatively

The

migrates. eluted

of a monophosphate

have

as substrate.

ppUp

in no incorporation from

-

which material

pup.

ppUp

the added

of thechro

in the presence

and unlabeled be quantitatively

resu1te.d

in n-pro-

but no detectable was

as authentic

was

a compound

M sodium

polymerized

After

mixture

Examination

authentic

to

overnight

revealed pup,

(0.05

COMMUNICATIONS

substrate.

reaction

and developed

identified

( 14 C> UDP

in which

a polymeric entire

authentic

in the region tentatively

not be susceptible

(50 : 10 : 35, v/v/v>.

an ultraviolet

compound,

would the

HR paper

: water

under

mltgrated was

2 hours,

on Whatman

pa-no1 : ammonia

RESEARCH

requiring

at 37’ for

streaked

BIOPHYSICAL

therefore,

by a phosphatase

incubation

AND

pNpN

reaction a second

which

could

-

Vol.

23,

No.

then

4, 1966

BIOCHEMICAL

act as a nucleus

for

AND

BIOPHYSICAL

further

chain

RESEARCH

COMMUNICATIONS

elongation.

R.Harvey is a N.I.H. feHoG.This work was supported by the Centre National de la Recherche Screntifique,N. I.H.(grant CA 04580),D.G. R. S. T .(grant no61 FR-087, L .N.F.C .C. (Comrte de la Seine), F. R.M. F. and CEA

REFERENCES T. Godefroy,

unpublished

M. Grunberg-Manago, Acta, M c Grunberg-Manago

, Ann.

U. Maitra,A.Novogrodsky, Biophys A.Michelson,

observations

P . J . Ortiz 20 ,269 (1956) Rev.

Biochem.

3,66

Biochim ,s,

91,

M. Grunberg-Manago (1964)

Biochem.

1 (1964)

, Biochim.

452

aBiophys

301 (1962)

D. Baltimore, J. Hurwitz, . Res . Communs . ,g,BOI (1965)

Biochim.Biophys.Acta,

F . R . Williams,

(1966)

, S . Ochoa,

Biophys

. Acta,

.