T1 ribonuclease inhibition of polyuridylic acid-stimulated polyphenylalanine synthesis

BIOCHEMICAL

Vol. 11, No. 4, 1963

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Tl RIBONUCLEASE INRIBITION

OF POLYURIDYLIC ACID-STIMULATED

lQLY-

PHENYLALANINE SYNTHESIS David

W. Allen

and Paul

C. Zamecnik

The John Collins Warren Laboratories of the Huntington Hospital of Harvard University at the Massachusetts Hospital, Boston

Memorial General

Received April 10, 1963 Polyuridylic phenylalanine cell-free

(phe) system

1962;

Arlinghaus

study

both

(Arnstein

on a multiple

1963).

Poly

aggregated active

5 ribosomes

phe synthesis

RNase which

RNA depolymerize (Arnstein,

the

linking

the ribosomes

RNA (Warner

al -et -.'

To study reticulocyte produced lutions

to combine

in the 1963;

this

and inhibit

protein

of both

pancreatic

and RNase T1 CT1) were

ration

both

with

and without

amounts

hydrolysis

un-

of of

synthesis

to hydrolyze

the RNA

the messenger

1963). further

a cell-free

in which 14

-phe

used to inhibit poly

U. 294

rabbit

the addition

incorporation.

RNase (Worthington

lized)

with

polysomes

polysome , presumably

in C

contain-

Gierer,

Small

gross

Gierer,

cell-free

preferentially

1963).

to

synthesis

1963;

negligible

phenomenon

increase

phe)

and to produce

system was employed a 5-fold

--et al.,

(Gierer,

polysome

is possible

the polysome,

The RNase is considered

1961).

it

and Schechter,

Hemoglobin

(Warner

produce

reticulocyte

(poly

structure,

ribosomes

to stimulate

Weinstein

Thus

conditions.

ribosomal

reticulocyte

pancreatic

rabbit

1962;

1962).

U has been found

in poly

in the

and polyphenylalanine

comparable

approximately

U) has been found

et - al -.'

and Schweet,

under

occurs

(poly

incorporation

hemoglobin

synthesis

ing

acid

Poly

Serial

3 times 14 C -phe U is

of poly

digested

di-

crystalincorpoby pan-

U

Vol. 11, No. 4, 1963 creatic hemoglobin

BIOCHEMICAL

RNase but

not T1,

messenger

whereas

RNA should

Pancreatic

-7

LoG

AND BIOPHYSICAL RESEARCH COMMUNICATIONS a mixed

polynucleotide

be hydrolyzed

by both

-5

-3

such as enzymes.

RNase

-6

-4

ENZYME RIBOSOMAL

-2

RNA

Fig. 1. The results of several experiments compare the inhibition of phe incorporation with or without poly U by either pancreatic RNase or T1. Rabbit reticulocyte ribosomes were prepared by the method of Schweet et al. (1958) and stored as the pellet at -15'C under The ribosomes were suspended in 0.01 r2YM sucrose. M magnesium acetate, 0.06 M KCl, and 0.01 M tris HCl A complete cell-free system was used buffer, pH 7.8. similar to that previously described (Allen and Zamecnik, 19621, except for the alteration in the medium noted above, reduction of the final volume to 0.25 ml, and the addition of 0.25 mg/ml yeast soluble RNA, an amount which yielded maximal incorporation under these To alternate tubes 20 micrograms of poly conditions. Serial dilutions of pancreatic RNase, U were added. or T were added as well as 0.08 micromoles of Cl4 DLphe t 1.82 x lo6 cpm/micromole), the tubes incubated 45 minutes at 37OC, and the protein precipitated and counted as previously described (Allen and Zamecnik, 1962). Results are expressed by plotting the percent of the proper uninhibited control (e.g. 1223 cpm/mg poly U> against with poly U, or 257 cpm/mg without the logarithm of the ratio of the weight of the enzyme to the weight of the ribosomal RNA. Aliquots inhibited by T1 are represented as 0 with poly U, and inhibited by pancreatic u without poly U. Aliquots RNase are represented by @ with poly U and l without PolY u. 295

Vol. 11, No. 4, 1963

BIOCHEMICAL

Fig.

1 shows that

tion

there

hemoglobin poration

the

synthesis

and poly

poly

synthesis.

only

of action

taken

if of Tl,

since

with

of poly

phe

synthesis

RNA is

the poly

as a

incorporation

as a measure

the messenger

incor-

is taken

of polyphenylalanine

be predicted

site

of both

by squares)

is

of dilu-

Cl4 -phe

and C 14-phe

by circles)

range

inhibition

phe synthesis.

synthesis

The inhibition

not

a large

same percentage

IJ (indicated

of hemoglobin

U (indicated

would

each enzyme over

is nearly

without

measure poly

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

considered

U messenger

the

RNA should

be immune to T1 action. This

finding

the Tl which

acts

is not on the

ments was prepared using

a modification

and has been involving

tions

duced

with

no change

activity

duced lowing

that

in poly

T1 inhibits

poly

U messenger,

polysome

technique were

a similarly

Bergquist

and Sober

(1962)

phosphodiester

protein

bona

test,

poly

synthesis

hydrolyzed

U

95%.

poly

U, Tl

by chromatography,

phe synthesis

pro-

nor

--et al.

and labeled poly

without

in

gradient (1963)

at 37OC with tube

depolymerized

296

U in-

analysis, (Fig.

minus

fol-

2).

by a 15 minute

U and C14-phe.

control

hyarolyz-

of T1 on the poly

by sucrose

15 minutes

has largely

experi-

RNase or by Tl at concentra-

the effect

with

in these

As a further

poly

produced

in

phe incorporation.

of Warner

for

the

U detectable

incubated Tl

for

impurity

Peter

of Rushizky

inhibited

was studied

incubation

2 shows that

method

RNase completely

at 37OC of ribosomes

with

by Dr.

pancreatic

in stimulatLng

The polysomes

further

takadiastase

either

Since

the

The T1 used

3' phosphate.

pancreatic

ing the

U.

shown to be specific

of the enzymes

Whereas

poly

of the

guanosine

was treated

its

from

due to an enzymatic

incubation

The effect T1 is the

the poly

of a

compared

enzyme. U induced

Fig.

Vol. 11, No. 4, 1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2400 k 2 2000 P f$ 1600 Q 2 I200 3 2

600 400

Fig. 2. Depolymerization of poly U induced polysomes by T1 as studied by the sucrose gradient technique. Two samples of 8 mg of ribosomes containing 3.6 mg of RNA were incubated with the complete cell-free system as described in Fig. 1, 0.2 mg poly U, 0.4 micromoles DL phe (1.82 x 106 cpm/micromole) in a final volume of 1.2 ml for 15 minutes at 370C. Then 8 micrograms of Tl were added to one tube and both samples incubated 15 minutes longer. One ml of the incubation mixture was layered on 30 ml of a linear 5-20% sucrose gradient in 0.0015 M MgC12, 0.01 M tris HCl, pH 7.8, and 0.01 M KC1 and centrifuged 25,000 rpm at 5OC for 3 hours in the Spinco SW25 swinging bucket centrifuge. Ten drop fractions were collected from the bottom of the tube and the absorbancy at 260 rnp (- 0 -) and radioactivity (--0 --) determined, following the technique of Warner --et al. (1963).

polysomes

in tubes

component

which

nearly

component

around

tube

remains

attached demonstrated

ized

the polysomes. at a slower

are

compared

with

shows the majority

resulting coincides

25 (the

in a more slowly with

the major

80 s monomer).

to the disaggregated

ments

but

2-18,

that

pancreatic

Incubation

rate.

For this

an incubated of radioactive

260 rnp absorbing The C14-poly

ribosomes. RNase similarly

alone

produced

reason, control.

sedimenting

the Tl

Other

297

experi-

depolymerdepolymerization, treated

An unincubated

labeling

phe

in the heavier

polysomes control polysome

BIOCHEMICAL

Vol. 11, No. 4, 1963 fractions

(around

tube

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14) and only

a small

percent

of the

counts

in the 80 s fraction. Thus Tl by addition

of poly

may be concluded of the polysome. treated

with

compared that

Tl

poly

U is

depolymerizes

Tl,

U but

to act

then

with

but

of ribosomes

on the

conclusion,

show inhibited

by the protective

poly

U (Table

binding

action

U.

Tl

for

then

formation

phe synthesis

controls.

ribosomal

produced

ribosomes

poly

washed

the

poly

RNA required

this

similarly

on RNA at or near

with

act

on a ribosomal

washed,

demonstrated

of ribosomes

does not

Consistent

to untreated acts

an aggregate

Evidence site

for

of preincubation

I). TABLE I

EFFECT OF PRBINCUBATION WITH POLY U ON Tl &+VAL

v%g

C14phe Control

2. 3.

Preincubated with poly U Poly after

U added preincubation

No poly

OF

AND C14PBE INCORPORATION

No.

1.

INHIBITION

U added

Tl

C14val Control

Tl

1174

523

164

13

1237

309

192

15

192

28

270

16

All samples contained 1.6 mg of ribosomes (0.8 mg ribosomal RNA) and the complete cell-free system described in Fig. 1, and were preincubated for 10 minutes at 37'C prior to addition of the radioactive label, one third of the samples (No. 1) containing in addition at this time 20 micrograms of poly U per tube. After chilling the tubes to O°C the same amount of poly U was added to a second third of the tubes (No. 2) and the final third received no poly U (No. 3). Where stated, the tubes received 1.6 micrograms of Tl RNase (Tl) or no enzyme (control). Either 0.08 micromoles of DL or 0.04 micromoles L C14phe (1.82 x lo6 cpm/micromole) Cl4val (4.8 x lo6 cpm/micromole) was used as a radioactive label. The samples were incubated for 45 minutes at 37OC, then the protein precipitated and the radioactivity determined as previously described. The results are averages of duplicate tubes which agreed within 10%. 298

BIOCHEMICAL

Vol. 11, No. 4, 1963 Preincubation C14phe

of the

incorporation

-vs 309 cpm/mg), corporation spared

with

effect

preincubation

with

preincubation Tl

protein

synthesis

of this

T 1 sensitive

rather

than

question

poly of poly

polypeptide

to the messenger;

of the

poly

The first for

together dition

and it

ribosomal peptide the

binding

and messenger,

nascent

Three

soluble

is the

the

the

remains

with

1963)

on the

ribosomal

binding

and the

protective

simplicity,

to nascent

poly-

the single (Fig.

299

2).

ad-

the

by the observation

bility

by its

by the

of T1 action,

RNA linked

polysome

favored

polysome

an unnecessarily

site

of the

T1 acts

is no evi-

of ribosomes

soluble

site

together.

RNA holding

possible

the

binding

there

(Gierer,

RNA

the RNA in

since

polymerization

this

is hydrogen

ribosomes

messenger

functions

RNA to which

ribosomal

seems remote

of the

(1) (2)

that

for

possible

and which

T1 depolymerization that

and

evidence

suggested:

is made unlikely

polypeptide

provides

together;

the

The second site

this

without

RNA may be essential

it

messenger

concept.

I (including

was 26:3%

attached

holding

makes the

of artificial

complicated

for

is

than

U

reproducible.

deviation)

polysome

possibility

an RNA other

poly

tube

formation.

or (3)

U messenger

with

of the control

RNA are

site

bonded

dence

messenger

the

chain

in Table

phe synthesis

ribosomal U holds

system

U was 4625%.

and polysome

poly

in-

similarly

is quite

shown

with

is the binding

growing

of the

percentage

than

(C14va1)

is not

incorporation

U (2 standard

RNA other

cpm/mgm

U (13 cpm/mgm ~9 15 cpm/mg>.

poly

inhibition

a ribosomal

synthesis)

of the experiment the average

(523

of C14valine

poly

of Cl'phe

protects

T1 action

of preincubation

T1 inhibition

experiment),

U partially

inhibition

hemoglobin

by preincubation

In 6 replications

poly

subsequent

Tl

(measuring

against

with

against whereas

The protective

with

system

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ribosome The third site effect

for

that following possi-

poly

U is

of preincu-

Vol. 11, No. 4, 1963 bation

with

BIOCHEMICAL poly

somal binding tivity, ternal

site

lication

for

I>.

poly

This

guanylic

acid work

Society

The authors

wish

McCully

helpful

for

considering

its

for Tl

a ribosensi-

one or more in-

residues.

was supported

of the

evidence

RNA containing

by Grant

and by ABC contract

#1113

provides

U which,

may be a single-stranded

This Cancer

U (Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cancer

to thank

discussions

#AT(30-11-2643.

Commission

Drs.

#E63 from

Marvin

of Harvard R. Lamborg

the American This

is pub-

University. and Kilmer

S.

and advice. RBF'BRENCES

Allen,

D. W. and Zamecnik, P. C., Biochim. Biophys. Acta 2, 865 (1962). Arlinghaus, R. and Schweet, R., Biochem. Biophys. Res. Comm. 2, 482 (1962). Arnstein, H. R. V., Biochem. J. 8l-, 24P (1961). Arnstein, H. R. V., Cox, R. A. and Hunt, J. A., Nature -194, 1042 (1962). 2, 148 (1963). Gierer, A., J. Mol. Biol. Rushizky, G. W. and Sober, H. A., J. Biol. Chem. 237, 834 (1962). Schweet, R., Lamfrom, l-l. and Allen, E., Proc. NatrAcad. Sci. ", 1029 (1958). Warner, J. R., Knopf, P. M. and Rich, A., Proc. Natl. Acad. Sci. 49, 122 (1963). Weinstein, I. B. and Schechter, A. N., Proc. Natl. Acad. Sci. 48, 1686 (1962).

300