An altered pattern of ribosome synthesis in a mutant of E. coli

Vol. 25, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AN ALTERED PATTERN OF RIBOSOMESYNTHESIS IN A MUTANT OF E. m Leon J. Lewandowski and Barbara (The Wistar

Received

Institute

November

of Anatomy and Biology,

ribonucleoprotein 1965; Sypherd,

either

inhibitors

direction,

growth

to the trans-

exists

growing

cells

that the products

occurring

the study of ribosome

nucleoproteins synthesis

that mutants might exist

growth conditions,

in amounts large

of

1966; Hosokawa and

seem analogous

we considered

(Nakada,

1965) or by addition

and the normally

Approaching

under balanced

would accumulate

ribo-

enough to make possible

their

dependence and resistance

to

and characterization.

Recalling streptomycin

that

sensitivity,

(SM) resides

Speyer et al.,

19621, it

on the ribosomes seemed possible

the presence of a suppressor

bacterium

of unbalanced

19621, the possibility

of

whose metabolism

found in exponentially

manipulations

somal precursors

for

coli

and Horowitz,

precursors

are not identical.

isolation

by conditions

(Hills

(McCarthy et al.,

from another

in Escherichia

While such particles

ribosomal

of metabolic

Pa.)

to study the formation

1965; Nakada and Marquisee,

Nomura, 1965).

which,

become fashionable

particles

has been altered

sitory

Philadelphia,

9, 1966

It has recently

metabolic

L. Brownstein

to SM might display

that

(Flaks

et al,

that mutants selected

changed the response

a change in the physical

554

1962; of the

properties

BIOCHEMICAL

Vol. 25, No. 5, 1966

of the the

ribosomes

case.

or in

This

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the kinetics

paper

of synthesis.

describes

tracts

of one of our mutants,

growth

conditions,

the

particles

MATERIALS We have

this

isolated

the strain

properties

of this

(Brownstein Cultures

and Graham, glucose.

cell.

of the

in

mentation Beckman

L-2

fractions

sucrose

collected

extracts

Tris

buffer

in

fully

else-

cultures

(Paranchych and 0.2%

in early

were prepared (0.01

log at 4OC

M, pH 7.4)

contain-

at 15OcOpsi

in a French

pressure

and debris

and unbroken

cells

dodecyl

gradients

on filter

from

(Sagik

were analyzed

by sedi-

in an SW 39 rotor

were punctured paper

with

2 ml of PPO-POPOP-toluene

a Packard

4000

Tri-Carb

liquid

in a

and three-drop

in vials.

covered

555

such ex-

sulfate-phenol

fractions

Tubes

directly

and

medium

RNA was isolated

ultracentrifuge.

were dried,

and counted

5-20s

with

Cell

sodium

from

(K-106).

more

20 pg methionine/ml

RNA and ribosome

through

reported

with

by centrifugation.

1962).

selected

characterization is

and,

in preparation).

was added,

with

have

minimal

in

of

mutant,

Tris-maleate

and disrupting

by extraction

et al.,

rate

strain

to SM-independence

were performed

washed cells

mM MgC12,

(19601,

mutant

supplemented

DNase (5 pg/ml)

tracts

a sedimentation

a SM-dependent

isolation,

particular

studies

were removed

ters

K-100)

and Lewandowski,

1962)

by suspending 0.1

exponential

SM-sensitive

revertant

were grown

All

a wild-type

(5-40 x 107 cells/ml).

phase

with

of Hashimoto

a spontaneous description

where

under

of ex-

AND METHODS

number

techniques

A detailed

ing

from

K12 (laboratory

utilizing

which,

analyses

43s.

of approximately

&. &

sedimentation

K-106,

accumulates

Such has proven

scintillation

These

fil-

solution counter.

Vol. 25, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS Figure of extracts

almost

after aside

equal

30 minutes

shows the

from

the

labeling

has markedly

areas

are more

by 60 minutes increased

clearly

the in

in

amount

proportion

centrifugation

sampled

It

of the

of isotope

gradient

cultures

of H3-uridine.

distribution these

of sucrose

growing

addition

and 30s peaks; peaks

results

of exponentially

60 minutes minutes,

la

AND CONCLUSIONS

is

clear

4s component, three

main

at 15, 30 and

there areas.

differentiated of label

at 15

that

is By

into in the

an

50,

43

50 and 30s

to the 43s material.

pattern of K-106 ribosomes. (la) At Figure 1. Sedimentation time 0, 150 PC HJ-uridine (1 mc/mg) was added to 100 ml of an Twenty-ml samples were exponentially growing culture of K-106. harvested and cell extracts obtained as described in Methods. Centrifugation was for 190 minutes at 38,000 rpm. Thirty fractions were collected from each gradient. Arrows from left to right mark the positions of 50, 43 and 30s respectively. ,-B-m Fifteen minute samples WW,~WW~; 30 minute ; 60 minute . ... ....... ... .

556

Vol. 25, No. 5, 1966

In

the

120 minutes for

label

experiment

a culture

with

H3-uridine,

divided,

Sucrose

unchased

of label

of the

total

possibility

label

is

suggested

label

after

chase

43s component by the

sedimentation in

Further

gradient

50 and 43s peaks

each of the total

in the the

presence

the

addition

is

patterns respective

(lb) K-106 incubated for 120 minutes the culture was analyzed immediately--.-culture was incubated for an additional uridine as described in text are omitted from figures; conditions

of the of

in Figure

lb,

unlabeled

chased

The dis37%

the

50s peak.

a precursor

of the

lb

of Figure

with

the

(dashed

gradients.

and

approximately

that

in Figure

unlabeled of the

patterns

is under

observation

557

of excess

shows that

for

was incubated

incorporation

sedimentation

the

was labeled

and one-half

shown superimposed

after

label that

in the

of the

are

of K-106

the

immediately

gradient

samples

tribution

in

as a chase.

RNA ceased

uridine.

particle

90 minutes

(100 pg/ml) into

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

next

an additional

uridine

for

BIOCHEMICAL

The 50s

sum of the line) la is

and also

A detailed

37% kinetic

H3-uridine one-half ; the re;ainder of 90 minutes with excess . For convenience,, points are the same as above.

BIOCHEMICAL

Vol. 25, No. 5, 1966

evaluation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is now in progress

from these results incorporated

into

-- that

the material

which accumulate

to the l'core particle"

resulting

somes (Hosokawa et al,

1966).

in the 43s region

is later

in K-106 are not identical

from CsCl treatment When a mixture

are exposed to CsCl, the majority

is converted

what seems probable

50s ribosomes.

The 43s particles

particles

to corroborate

to 43s particles

while

the initial

of 50s ribo-

of 50s and 43s

of the 50s material 43s particles

sediment at 37s. To determine K-106, perties,

whether

and the wild-type,

the 50 and 30s ribosomes K-100, have similar

we analyzed an extract

prepared

of the mutant,

sedimentation

from a mixture

proof C'4-

FRACTION

Figure 2. Comparison of ribosomal sedimentation pattern of K-106 and wild-type, K-100. A sample of K-106 labeled with H3-uridine for 120 minutes was mixed with a sample of K-100 labeled with Cl4 -uridine for 120 minutes. Cells were lysed and extracts edimented for 190 minutes at 38000 rpm. UU..U.."" , cl e .

558

H3;

BIOCHEMICAL

Vol. 25, No. 5, 1966

labeled for

K-100 and H3-labeled

120 minutes.

the accumulation not seem to be

K-106, each exposed to labeled

The 30 and 50s peaks coincide;

lacks any distinguishable the generation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

accumulation

however,

of 43s material.

K-100 Although

time of K-106 is 1.5 times that of the wild of 43s material merely

a

in one and not the other

function

uridine

of the slower growth rate.

type, does No

FRACTION

Figure 3. Comparison of RNA extracted from K-106 and wild-type. An aliquot of t e mixture of K-106 labeled with H3, and K-100 labeled with C1b described in Figure 2, was treated with phenolSDS and sedimentid for 6.5 hrs. at 390 0 rpm. Arrows mark the E3 position of 23 and 16s RNA. c14. Insert: RNA extracted from purified 43s parti%s~~;O6 and from crude extract of K-100 .,-...LU=II .

559

BIOCHEMICAL

Vol. 25, No. 5, 1966

comparable

43s peak

5 and 120 minutes

second

sample

mentation the

of

in

the

exactly

RNA extracted (Insert,

from

Figure It

between

with

al.

that

(19621,

are

now analyzing gel

proteins

any visible

RNA of K-106.

than

sediments

The

mutant

90% of the

with

23s RNA

step(s)

in

the

of the

from

assembly

a large

those the

of

production

of

the

43s particle

completed

or

particle.

43 and 50s components

to determine the

in

to the

of an otherwise proteins

than

a step

of proteins

electrophoresis

are missing

transitory

represents

addition

the

we may be observing

less

which

rearrangement

acrylamide

in K-106

The final

may be the

a molecular

show that

cause

More

control.

43s particles

particles,

50s ribosomes.

ribosome

The sedi-

material

for

a

3).

of neosome-like et --

between

from

23 and 16s RNAs of the

of the

purified

2.

does not pattern

the

that

seems therefore

McCarthy

resulting

43s particle

sedimentation

of label

coincides

of

the

overall

distribution

pool

in

was prepared

in Figure

3) of the

point

uridine.

extract

analyzed

(Figure

at any time

of labeled

a phenol

the material

profiles

in K-100

addition

control

RNA contained

change

accumulates

after

As a further

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

whether

We by

specific

43s particle.

ACKNOWLEDGMENTS This Health

investigation

Service

tute

of Allergy

from

the

from

the

Research

Grant

and Infectious

National

U. S. Public

was supported

Health

National

Science Service Institute

in

AI 02454-09 Diseases

Foundation. Research of General

560

part from

by U. S. Public the

and Research L.J.L.

Training Medical

is

National Grant

a trainee

Grant

InstiGB-4410 under

GM 00142-08

Sciences.

Vol. 25, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Flaks,

J.G., Cox, E.C., and White, J.R. Biochem. Biophys. Res. Comm. 2, 385, 1962. Genetics s, 49, 1960. Hashimoto, K. Hills, D. C., and Horowitz, J. Biochemistry 2, 1625, 1966. Hosokawa, K., and Nomura, M. J. Mol. Biol. l2, 225, 1965. Hosokawa, K., Fujimura, R. and Nomura, M. Proc. Natl. Acad. Sci. E, 198, 1966. McCarthy, B.J., Britten, R.J., and Roberts, R. B. Biophys. J. 2,

57, 1962.

Nakada, D. J. Mol. Biol. l2, 695, 1965. Nakada, D., and Marquisee, M. J. J. Mol. Biol. I.& 351, 1965. Paranchych, W., and Graham, A. F. J. Cell. Comp. Physiol. 60, 199. 1962. Sagik, B..P:, Greeb, M. H., Hayashi, M., and Spiegelman, S. Biophys. J. 2, 409, 1962. Speyer, J. F., Len el, P., and Basilio, C. Proc. Natl. Acad. Sci. 48, 6 @I , 1962. Sypherd, P. S. J. Bact. 90, 403, 1965.

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