Ribosome degradation in escherichia coli induced by colicin E2

BIOCHEMICAL

Vol. 54, No. 3, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RIBOSOME DEGRADATION IN ESCHERICHIA COLI INDUCED BY COLICIN E2

Akira

Yanai*,

Teruhiko

Beppu and Kei Arima

Laboratory of Microbiology and Fermentation Department of Agricultural Chemistry University of Tokyo Tokyo, Japan * Present address:Basic Research Toray Industries, Inc., 1,111 Kamakura 248 Japan Received

August

27,

Laboratory Tebiro,

1973 SUMMARY

The mode of action of colicin E2 on ribosomes in Escherichia coli cells was investigated by zonal centrifugation analysis. Ribosome particles, both 50s and 3OS, were degraded to smaller contents with the lapse of time by the action of colicin E2. Gradual reduction of S values of each particles could not be observed and degradative intermediates of possible RNA-protein complex were detected only at the position between 30s and 4s in the zonal centrifugation profile, which indicated the destruction 50s ribosome fraction of ribosome in burst-out attitude. influenced by colicin E2 contained both 23s and half-sized RNA. From these data, the mode of action of colicin E2 on ribosomes in E. coli was discussed.

INTRODUCTION Colicin

E2, one of the

Enterobacteriaceae 60,000) genus

(11, (2).

inhibition

t-RNA

division

DNA (2,4,5)

degradation in Escherichia

of

protein

by certain (molecular

strains

biochemical

action

induction

weight

of the is

(4),

colicin

23s and 16s ribosomal 413,

a defective

of degradation

1185

E2 - challenge RNA, but mutant

same

the

and RNA (4,6).

coli

Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

its

produced

to certain

(3) and the

previously of

a simple only

The main feature

As we reported induces

is

and bactericidal

of cell

of cellular

strains,

bacteriocins

for

not of

Vol. 54, No. 3,1973

ribonuclease

BIOCHEMICAL

I.

degradative

This

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

suggests

enzyme for

that

RNaseI,

RNA in E. coli

which

cells,

is the main

does not participate

in the r-RNA degradation. We investigated alteration

further

of ribosome

and followings degraded

constitution

action

reduction

of

only

ribosomes

could

at the

split

retain

contents

position

halves

both

not be

30s and 4s in

the ribosome

in 50s ribosomes,

before

particle

suggested

structure

of

RNA-protein

indicated

E2, which

their

could

between

Second,

into

were

were decreased.

of possible

which

by colicin

yet

cells

action,

particles

each particles

attitude.

23s RNA was first induced

of

profile,

in burst-out

destruction

E2, and the

sensitive

and the

the colicin

ribosome

intermediates

centrifugation

destruction

First,

S values

were increased

the zonal

their

in the

and degradative

complex

during

of colicin

50s and 305 particles

observed

r-RNA degradation

were suggested.

by the

Gradual

the

with

that

50s

the damaged RNA.

MATERIALS AND METHODS Colicin

E2 was prepared

was cultured (Difco)

with

supplemented

(0.5pCi/ml.) of

(10,000xg, washed cells

At

Sakaguchi

with

at

intervals,

(10,000xg, dryice-acetone

to the

E. coli

1% Penassay or

cell

Q13

broth

[3Hllysine

concentration

and washed by centrifugation

in 70ml.

broth

of

at 37“C.

1% Penassay

After

broth

the

resumed

E2 was added at 6x108 cells/ml.

50). 20ml.

20min.)

flask

37OC, colicin

multiplicity

of

(7).

(0.2pCi/ml.)

1% Penassay

resuspended

growing

(lOug/ml.,

[ 14Cluracil

and collected

15min.)

before

at 37OC in 70ml.

with

in 500ml

5.5xlOS/ml.,

normal

shaking

as reported

aliquots

of the

and the pellets

and stored

overnight

1186

culture

were rapidly at

-75'C.

were centrifuged freezed

by

The collected

BIOCHEMICAL

Vol. 54, No. 3,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig.1 Effect of colicin E2 on zonal centrifugation profile of ribosomes in E. coli Q13 labelled with [l4C]uracil. A: Omin., B: 30min., C: 70min. Detailes are shown in Materials and Methods. Fig.2 Effect of colicin E2 on zonal centrifugation profile of ribosomes in E. coli Q13 labelled with [3H]lysine. A: Omin., B: 30min., C: 70min. cells

were suspended

buffer

(pH 7.4)

sonically

for

a needle

at

profiles

were obtained

of each fraction

The diluted

COONa, pH5.1)

(pH5.1)

SDS at and zonally (1.5

37'C,

of

and zonal

O.lM

for

centrifuged

RPS40 rotor.

1187

37,000 by

centrifugation of O.lml.

NaCl-0.05M

scintillation

- 15% sucrose

in a Hitachi

at

the radioactivities

0.4ml.

by liquid

0.2ml.

linear

was performed

505 and 30s peak fractions

0.3% recrystallized

density-gradients

with

on 4.8ml.

RPS40 rotor

of tubes

by measuring

20min.,

in 1OmM Tris-HCl-O.lmM

Fractionation

the bottom

diluted

buffer

(8),

were charged

and

condition

at lO,OOOxg,

and CentrifugedinHitachi

piercing

O.lmM MgC12

disintegration

(S-2O%w/w)

at 4OC.

Gesteland

constant

fractions

170min.,

Na-acetate

1OmMTris-HCl

centrifugation

density-gradients

MgC12 (pH 7.4)

of

30mM NH4Cl-6mM 2-mercaptoethanol

under

After

supernatant

sucrose

rpm,

containing

oscillated

(35W, -/min.). of the

in 10ml.

counting.

were incubated

30min., through in O.lM

according linear NaCl-0.05M

with

to R.F. sucrose CH3-

23s and 16s peaks of

BIOCHEMICAL

Vol. 54, No. 3,1973

RNA were detected scintillation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by measuring

[ 14C)radioactivities

by liquid

counting.

RESULTS AND DISCUSSION In zonal phenol

centrifugation

method from E. coli

appeared

three

We had already

induced

gradual

ribosomal

RNAs except

particles

during

investigated

reported

here inzonal

sucrose

possessing

In the profile

be observed hand,

with

we could

profile

These data

of colicin

labelled

after

the

suggested

degradative suggested degraded gradually

of RNA-protein intermediates that

ribosome

abruptly decreasing

into

smaller size

of

On the other

(Fig.2),

increased

in

there

also

same position

substances

of ribosome

Furthermore,

the

found between

fragments intermediates. 1188

not

E2 challenge.

especially

particles,

could

density-gradient

30s and 4S, the

intermediates

were not

heights

of radioactivity

[3H]lysine

these

complex.

(Fig.l-b,

decreased

In sucrose

colicin

that

cells

(S-value)

increase

with

between

size

as

particles.

E2 challenge.

the gradual

30s and 4s were degradative consisted

E. coli

in their

30s and 4s peaks.

c,

of E. coli

showed the profile

decrease

a peak increased

as in Fig.l-b,

E2 was

fraction

SOS and 305 with

of ribosomes

appeared

supernatant

lapse

two

profile.

E2-challenged

recognize

the peak between

by colicin

peaks of

the

E2

change in ribosome

density-gradient

of colicin

but gradual

of colicin

and amounts of the

Structural

(4).

there

16s r-RNA and 4s

50s and 305 peaks of ribosome

I two radioactive

appeared

S-values

by SDS-

[14C]uracil,

the addition

centrifugation

of the

through

in Fig.l-a

that

of both

t-RNA

with

23s r-RNA,

such RNA degradation

Centrifugation sonicate

peaks,

decrease

of RNAs extracted

Q13 labelled

radioactive

t-RNA.

cl

profile

particles fact

that

50s and 30s

50s particles,

without

between

producing

BIOCHEMICAL

Vol. 54, No. 3,1973

a.

8-

b

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c

165

3-

235

b

a

C

0

r z “2.

Fig.3 Zonal centrifugation profile RNA in 50s ribosomes treated with colicin A: Omin., B: 30min., C: 70min.

of E2.

Fig.4 Zonal centrifugation profile RNA in 30s ribosomes treated with colicin A: Omin., B: 30min., C: 70min.

of E2.

Possibility particles

could

not

in the colicin-treated

extracting

operations

artificial

products

In order process

cells

further

was found treated

consisted

shoulder

S-values,

contained

but

In contrast,

reported

might

Q13 at

RNA of of the

in 50s

about

30s peaks in The

come from the contamination

activity

1189

of

of Fig.l-c.

storage

O'C for

16s

colicin-

as is shown in Fig.4.

of

purified

ribosome

a few weeks resulted

23s and 16S, to the

and endonuclease

RNAs respecRNAs or not.

each RNAs from

in 1969 that

both

intact

smaller

in the profile

from E. coli

from the

their

50s particles

of one 16s peak,

of r-RNA,

were

one peak of 23s RNA was found

cells(a),

intermediates

W. Szer

cleavage

only

shown in Fig.4-c

degradative

particles

c).

extracted

they

235 RNA in the

cells(b,

to the

on the degradation

50s and 30s particles

whether

the control

besides

ribosome

intermediates"

information

were isolated,

As shown in Fig.3, from

the

became unstable

"degradative

particles,

and analyzed

ribosome

that

by sonication.

of ribosome

tively,

however,

cells

and the

to obtain

colicin-treated

Fig.1

be excluded

fragments

of RNase IV type

with

in a half

in split

BIOCHEMICAL

Vol. 54, No. 3, 1973

protein

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was assumed to be involved

in this

limited

degradation

(9). It dation

seems possible in E. coli

follows.

to account

413 cells

Adsorption

influences change,

slight

of RNase IV type a nick

or nicks

at first

into

in the pieces

Structural when this

entities

limited

But ribosomes

loosened

and made sensitive

loss

of

particle

fragments. give

In 50s ribosome,

about

degradation

Then further

half

with

Investigation

breakdown

unmasked and makes 23s RNA is

of r-RNA results

Dr.

into

2. 3. 4. 5. 6. 7. 8. 9.

intermediate

structure. Takahisa

Ohta

Herschman, H. R., and Helinski, D. R. (1963) J. Biol. Chem. 242, 5360-5368. Nomura, M. (1967) Ann. Rev. Microbial. 21, 257-284. Beppu, T., and Arima, K. (1972) J. Bacterial. 110, 485-493. Yanai, A., Beppu, T., and Arima, K. (1970) Agr. Biol. Chem. 34, 149-151. Beppu, T., and Arima, K. (1971) J. Biochem.(Tokyo) 7J, 263-271. Nose, K., Mizuno, D., and Ozeki, H. (1966) Biochem. Biophys. Acta. 9, 636-638. Beppu, T., and Arima, K. (1967) J. Bacterial. 93, 80-85. Gesteland, R. F. (1966) J. Mol. Biol. 18, 356-371. Szer, W. (1969) Biochem. Biophys. Res. Commun. 35, 653-658.

1190

even

from in

small

REFERENCES 1.

split

has

RNase distinct

degradative

on the ribosome to thank

RNase activity

can be retained

and degradation the

structural

damaged r-RNA are

to cytoplasmic

about

Acknowledgement: We wish for his useful advices.

induce

RNA component

a limitedly

enzymatic

somehow

S-value.

of their

conformation

some information

gets

of the ribosomes

occurred.

RNase I.

protein(s)

degra-

E2 as

surface

in the particles.

r-RNA.

with

of colicin

one by one to

unfolding, split

mode of ribosome

E2 to cell

in the cells

in the

the

by the action

of colicin

ribosomes e.g.,

for

may