Studies on the breakdown of messenger RNA

Vol. 11, No. 6, 1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

STUDIES ON THE BREAKDOWN OF MESSENGER RNA C. WOESE, S. NAONO, R. SOFFER and F. GROS Service

de Biochimie

Institut

Received

April

23,

Pasteur

(Jacob,and

readily

verified

incorporated

properties

1961).

It

exists,

if

between

nomycin

D to prevent

into sis

that acid

is

soluble

specific

in vivo, this

rate

E. coli

and that not

et al,

1961

what

sort

of correlation

synthesis.

Using et al,

during

to actinomycin

is

this

acti-

1962)

have

decomposed

protein

synthe-

D but we shall

and proflavine, in

most

RNA fraction

functioning

of DNA transcription

with

this

(Levinthal

dinitrophenol

exist

that

of B. subtilis

sensitive

substances,

inhibitors

and protein

its

indeed

which

(Brenner

and to establish

RNA synthesis,

is

did

carrier

RNA fraction

Experiments

RNA fraction

to be shown

breakdown

labeled

fragments

on two other

however

RNA originally

turnover.

a particular

at a rapid

completely

the rapidly

catalytic.

report like

any,

rapid

of an information

remained

breakdown

messenger

was its

that

precursors

undergoes

shown

1961)

the prediction

of the expected

also

(France)

of bacterial

Monod,

labelled

Gros et al,

- Paris

1963

One of the characteristics postulated

Cellulaire

which

behave

organism.

Experiments Addition centration a uracil radioactive resuspending addition

of DNP to an exponentially

of 5 x 10 label

into

-3 M leads cold

internal

of DNP. Figure

rapid

5 % TCA insoluble

pool

the cells

to the

growing

is

also

subject

cessation material. to this

in a medium containing 1 illustrates

the 435

culture

C time

12

at a final

con-

of incorporation Assimilation

inhibition uracil

dependent

of from

a

as shown by prior

to the

disappearance

of

;

Vol. 11, No. 6, 1963

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 0

YIYUTES AFTER ONP AOOITIOI

Figure1 labelled TCA insoluble material after various durations.

14 DNP terminated C uracil

pulses of

It can be sunnnarized as follows :

1 - The decay is approximately first

order and a half life

period

of about 8 minutes can be derived from the data for the one minute pulse. 2 - There is a residual stable fraction

of 20 % which is probably a

minimal value since it can also be observed after

pulses as short as 10

seconds. 3 - The percentage of stable material

increases with increasing

pulse length reaching 85 % for a pulse of one generation time (60 minutes). Characterization

of the labile

The following

RNAfraction.

observations suggest that the RNAdisappearing after

treatment with DNPincludes what is generally

recognized as messenger RNA.

1 - The 14 S peak which can be demonstrated in a sucrose gradient after

a short exposure to a labeled RNAprecursor (Gros et al, 1961) is

markedly reduced if the cells are incubated with DNPprior

to extraction.

Furthermore there is no corresponding net increase of radioactivity remainder of the profile. .?L

in the

Vol. 11, No. 6, 1963

BIOCHEMICAL

2 - The ability incorporation

of extracted

diminishes

trophenol

AND BIOPHYSICAL RESEARCH COMMUNICATIONS ENA to stimulate

dramatically

as shown in Table

after

exposure

amino

of the cells

0’

15'

20'

30'

60'

100

35

26

17

11

% remaining incorporating activity

loo

12

16

9

not detectable

incubated

in the presence

very

from

low endogenous

stimulated from

non-DNP

treated

T2 DNA primed

amino

In vivo

protein Figure

valine

(10

protein

cells. acid

synthesis

-2 M) into

using

detectable

If

increase

though

the

decreases

rate

in enzyme.

the

tein

which

that

3 % of the

synthesis.

that

the

obtained

However,

if

is

added

for

initially

three

at least

lower

14

same data

simultaneously

DNP is

can be demonstrated

the

as an index

added

of C

as a function

to DNP. Essentially

are

support

1962).

diminishes

of D-galactosidase

synthesis

1961)

of incorporation

material

are

than

of speciffc there

is no

minutes

after

10 minutes the control

aland

time.

&wing

culated

that

DNP and inducer

of this

with

of DNP.

presence

formation

but

such extracts

in the

TCA insoluble

enzyme synthesis

dialysed,

(Wood and Berg,

of the culture

the

synthesis.

inducer,

hot

if

a

in vitro

and Matthaei,

incorporation

fact

of DNP have

aminoacids

In addition

the

of exposure

obtained

to incorporate

U or by ENA (Nirenberg

2 illustrates

of the time are

cells

capacity

by Poly

to dini-

I

% remaining TCA insoluble Cl4

3 - Extracts

acid

1. TABLE

Time after DNP addition

in vitro

it

specific

activity the amount

constitutes, total

a triplet This

indicates

of valine,

ENA is

that

of ENA broken

messenger

of bases

(Gros

must be used there

the percentage

is 437

et al, about

probably

down,

pro-

and assuming

1961 b), six

of bulk

times

no obligatory

it

can be cal-

in protein destruction

Vol. 11, No. 6, 1963

BIOCHEMICAL

AND BIOPHYSICAL

I I I s IO IS YINUTES AFTER DNP ADDITION

RESEARCH COMMUNICATIONS

I 20

Figure 2 of a molecule cule

of messenger

of protein.

That

breakdown

of the

fact

chloramphenicol

that

of RNA after

In vivo

there

label

however,

reduces

3 shows the

the synthesis

a relationship

by a factor

uracil

effect

of 70 a/ml

Cl4 uracil.

thmic

and the half-life

which

can be systematically

decay

with

of a mole-

between

of protein,

is

of three

the rate

the rate

implied

of

by the

of decay

pulse.

of action et al. inhibits

2 provides

of adding

is

thus

of cold

(1962)

growing

acid

5.4 minutes.

There

insoluble is

at a final

culture

45 seconds

a stable

from

corrected,

of 3.5 minutes.

the data

is

fraction

to yield

logariof 22 %

an exponential

and proflavine.

had already

the activity

sulphate

material

subtracted

of dinitrophenol

additional

proflavine

to an exponentially

Disappearance

a half-life

Hurwitz

Table

specifies

RNA and the synthesis

after

centrations

it

of proflavine.

concentration

Mechanism

is,

a DNP terminated

effect Figure

RNA after

shown that

of the DNA primed

evidence

that

proflavine

proflavine

at high

RNA synthesis acts

at the

con-

in vitro. level

of

Vol. 11, No. 6, 1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

LOSS Or 5%TCA IW5OL”GLL MATLRIAL tOLLOWING TLRWIN*TIOY OF A 45SECOND WL5L or C~UR*CIL B”*DGITION OF PROFlA”INE

La C.P. 300 250

\

corrected

t %-3.5min

.

I

4 6 12 minur~rafteraddition

16

h

I

I

I

I

,

20 24 25 of Pmflavine Sulphatr

1

32

Figure3

the transcription

of information

case for dinitrophenol.

from DNAto RNAand that this is also the

Thus each agent can be seen to inhibit

ted amino acid incorporation

DNAdirec-

in a cell free system at concentrations,

which

if anything are stimulatory

for the poly U primed synthesis of polyphenyl-

alanine by the sameextract.

These data suggest that the observed effects

on RNAand protein synthesis in vivo,

are a consequence of the cessation

of DNAprimed RNAsynthesis. TABLEJ II Effect

of DNPand Proflavine on DNAand Poly U Dependent Amino Acid Incorporation T2 DNAdirected Threonine incorporation

Complete system Minus T2 DNA Minus Poly U Plus DNP-Final Concentration 4 1, Plus DNP " 1.6 'I Plus DNP " 0.8 Plus Proflavine sulphate final 40 &ml

6578 166 -3 x 10-3 M x 10-3 M x 10 M concentration

Poly U directed Phenylalanine incorporation 1088

3231 4320 6515

160 l480 1513 1186

108

1651

Vol. 11, No. 6, 1963

BIOCHEMICAL

The extract DNP treated

was an extensively

culture

The complete tion

contained

pH 7.8,

reaction

mixture

the following

ethanol,

1.5

kinase,

1.5

; 1-threonine,

The complete

for

threonine

; poly

0.5

0.025

preparation

DNA directed

from a

; KCl,

mixture

for

acids,

activity

phenylalanine

0.05

identical

did

pH 7.8,

; GTP, ; S-mercapto-

pg. not

include

6 ; MgS04,

(specific to that

buffer

; pyruvate

; T2 DNA, 43.5

0.023

: 45 minutes

each

10 PC/~)

in um : Tris

was otherwise

Incubation

; CTP, 0.2

incorporation

1 ; 1-phenylalanine,

U, 20 pg, but

incorporation.

l-amino

incorpora-

of 0.5 ml : Tris

18 ; UTP, 0.2

(specific

contained

threonine

volume

200 ~1 (ca 4.6 mg protein)

; P-mercaptoethanol,

*c/p

alumina

in ua~ in a total

mso4, UTP, CTP, or T2 DNA. It

10.7

for

; PEP, 5 ; 19 unlabelled

15 ng ; extract

GTP, 0.1

dialysed

of ML 308.

21 ; MgSO4, 6 ; MnSO4,

; ATP,

0.35

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

21 ;

activity described

at 37'.

REFERENCES Brenner, S., Jacob, F., and Messelson, M., Nature, 190, 576 (1961). Gros, F., Hiatt, H., Gilbert, W., Kurland, C., Risebrough, R., and Watson, J.D., Nature, 190, 581 (1961 a). Gros, F., Gilbert, W., Hiatt, H.H., Attardi, G., Spahr, P.F., and Watson, Cold Spr. Har. Quant. Biol., a, 111 (1961 b). Hurwitz, J., Furth, J.J., Malamy, MJ, and Alexander, M., Proc. Nat. Acad. 48, 1222 (1962). Jacob, F., and Monod, J., J. Mol. Biol., 2, 318 (1961). Levinthal, C., Keynan, A., and Higa, A., Proc. Nat. Acad. Sci., 48, 1631 (1962). Nierenberg, M., and Matthaei, J., Proc: Nat. Acad. Sci., 47, 1388 (1961). Wood, W.B., and Berg, P., Proc. Nat. Acad. Sci., 48, 94 (1962).

J.D. Sci..