Ribonucleic acid synthesis in streptomyces antibioticus: Stable ribonucleic acid species synthesized by young and old cells

Vol. 63, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RIBONUCLEIC ACID SYNTHESIS IN Streptomyces antibioticus: STABLE RIBONUCLEIC ACID SPECIES SYNTHESIZED BY YOUNG AND OLD CELLS George H. Jones Department of Zoology University of Michigan Ann Arbor, Michigan 48104 Received

December

9,1974

SUMMARY : In studies of RNA synthesis by intact cells and cell-free extracts of Streptomyces antibioticus, it has been found that 48 hr cells (producing actinomycin) and cell-free extracts are less efficient than 12 hr cells (not producing actinomycin) and extracts in the synthesis of RNA. Analysis of the products of "in viva" and "in vitro" RNA synthesis by sucrose gradient cent trifugation reveals that both 12 and 48 hr cultures and cell-free extracts synthesize ribosomal RNA as well as RNA species of higher and lower molecular weights. However, 50-60% of the 3H-uridine labelled RNA synthesized by intact cells sediments as rRNA as compared with only S-10% of the cellfree product. The addition of 2 x 10 -5 M actinomycin D to incubation mixtures for cell-free RNA synthesis does not significantly alter the relative amounts of the various RNA species synthesized by 12 or 48 hr extracts.

Streptomyces

antibioticus

the antibiotic,

actinomycin

that

in the

a decrease

the production

are maintained pite

synthesis

antibiotic. the

metabolite It

that

was somewhat which

secondary

results

the cells

have

during

this

surprising

period

in

intact

cells studies

strain

5. antibioticus, described actose

(5). glutamic

After acid

Copyright o 19 75 by Academic Press, Inc. All rights of reproduction reserved.

continued

cells.,

this

to syn-

and RNA synthesis phase,

des-

in secondary

RNA synthesis

in an

RNA synthesis

basis

certain

and cell-free are presented

48 hr in medium

begin

stationary

the molecular

3720,

cells

is utilized

of DNA-depandent

MATERIALS

accompanies

(3,4).

to observe

an inhibitor

in 2. antibioticus

of these

reached

shown

In particular,

of protein

energy

produces

has been

synthesis as the

low levels

which

it

(2-4).

dramatically

To examine

metabolite.

RNA synthesis

macromolecular

of the cellular

produces

of RNA synthesis the

after

studies,

by 2. antibioticus decline

most

production

organism

for

Nevertheless, long

fact

In previous

capacity

of actinomycin

RNA and protein thesize

is one member of the genus (1).

for

the persistence

characteristics

extracts

as a of

has been examined,

and

below.

AND METHODS was grown medium,

containing

in NZ-amine the

cells

0.1% glucose 469

medium as previously

were (5).

transferred It

is

in the

to gallat-

Vol. 63, No. 2, 1975

ter

medium

BIOCHEMICAL

that

the

200 ml cultures, medium were

cells

grown

labelled

produce

for

washed

of 1.4

1 hr with

M NaCl,

and sonicated

pH 5.0.

from

and was analyzed cates

were

collected

cribed

in detail

exogenous bations

performed

Ci/mmol),

1.25 about

are

tures

is

extracts (in

were

under

the growth

was extracted

from

was determined

will

0.1 M Na acetate, SB-283

rotor

were

as sources

the legends

to Figs.

dient

were

tubes

the bottom and its

found

G-

of RNA polymeri-

pH 7.5,

0.1 nmole; Incubations

the medium

conditions

employed

mixtures

without

MgC12,

analyzed IEC B-60

0.5 nmole; umole carried

determined

fibre

filters.

The RNA samples

prior

to liquid

scintillation

were for

Each fraction

were

the

was diluted Thereafter,

counting. 470

cul-

observation). (3).

gradients

RNA Protein

prepared

centrifuged times

in

indicated

were

with

in

an in

the gra-

collected

from

0.5 ml Hz0

0.5 mg of total

were added to each RNA was collected

dissolved

30

concentration

centrifugations,

of 10 drops

at 260 nm.

This

described

sucrose

At the end of the

RNA and 0.5 ml of 50% trichloroacetic acid After standing 10 min at 0", the precipitated

for

(6).

Gradients

ultracentrifuge, and fractions

extract out

D, at a concen-

(unpublished

on 12 ml 5-20%

1 and 2.

bovine each; 3H-UTP,

of 48 hr 2. antibioticus

et al.

pH 5.0.

10 pmoles;

and crude

were

as previously

of Lowry,

of each gradient.

absorbance

Sephadex

proceded

ATP, GTP and CTP, 0.15

in

punctured

Briefly,

40 umoles;

phosphate,

dithiothreitol,

0.1 M NaCl,

of the

as cpm

be des-

through

extracts

pH 7.8,

potassium

by the method

RNA samples

were

expressed

DNase and RNase. Cell-free incu3 of 1 ml using H-UTP as a precursor. In-

Tris-HCl,

reaction

(3).

of the soni-

preparation).

filtered

in these

2.5 mg protein.

that

Total

extract.

endogenous DNA as template. Actinomycin -5 of 2 x 10 M was present in some incubations.

tration

g.

and the precipitates

min at 37" using of actinomycin

0.1

described

Aliquots Results

purification

in volumes

nmole;

at a setting

as previously

of cell-free

0.5 mg; unlabelled

(12 or 48 hr),

unit

fil-

to both

contained:

EDTA, 0.1 pmole;

serum albumin, (19.8

sonic

At

by suction

power

acid

publication

further

acid-glucose

10 min at 20,000

and counted.

in the

studies,

(49 Ci/mmol).

centrifugation.

3H-UTP incorporation

mixtures

viva"

of 0.1 M Na acetate,

g supernatants

the preparation

used without

were

potassium

for

trichloroacetic

DNA and was sensitive

cubation

centrifuged

of 12 and 48 hr cultures

activity.

"in

harvested

volumes

filters

in a subsequent

extracts

25 and were zing

for

nere

an MSE ultrasonic

gradient

with fibre

cells

in three

2a,QaQ

per mg protein

Procedures sonic

were

the

precipitated

incorporated

with

by sucrose

on glass

the

suspension

Sonicates

RNA was extracted

For the

50 nCi of 3H-uridine

period,

after

amperes,

actinomycin.

RESEARCH COMMUNICATIONS

11 or 47 hr in galactose-glutamic

for

the end of the labelling tration,

AND BIOPHYSICAL

yeast

fraction. on glass

in 1 ml of 95% Protosol

Vol. 63, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION Incorporation

of labelled

-S. antibioticus to synthesize sors.

For

cells these

after

innoculation) synthesis.

was found

at levels

and cell-free

RNA, using

mycin it

RNA precursors

studies,

24,870

extracts

and 9,433

(48 hr after

cpmfmg extract

protein,

the decreased

ability

of actinomycin

with

younger

cells

thesis.

An interesting,

greater

inhibited extent

decreased

in

(in

Sucrose

analysis

with

RNA species

(unessential

sis

12 hr cells

the observed the

intact

to the

an increased

possibilities,

rate

"in

be explained

48 hr cell)

those

labelled protein

that

of 2 x 10 -5

with

the for

levels

this

in a subsequent viva"

if

of

difpubli-

was analyzed in Fig.

1.

from

whose of all

the nature

by sucrose

The data

16s and 23s ribosomal uridine (4)

into

ribosomal

and RNA synthesis

of certain

types

RNA, despite (see preceding

471

from

accomthese by

12 and 48 hr 12 and 48 hr

with

the results

12 and 48 hr cells

48 hr cells their

the

synthe-

synthesized

labelled

section).

rein

to examine

centrifugation

and that

for

of RNA might

RNA

both

might

persists

capacity

of the RNA's

that

by RNA

inhibited.

In order

indicate

RNA's,

synthesis

synthesis

of 3H-uridine

gradient

clearly

the

in cellular

in 2. antibioticus.

sonicates

vitroV

to synthesize

of RNA by 48 hr extracts

of degradation)

and to determine

and "in

was preferentially

species

a decrease

production

The RNA extracted shown

syn-

to a much

The basis

cell-free extracts, the labelled -S. antibioticus cells and extracts has been analyzed.

contain

RNA as

by 12 and 48 hr extracts

of 48 hr cells

synthesis

of only

Alternatively,

actinomycin

cultures

verify

antibiotic

the presence

be reported

ability could

cell-free

formation

cell. (or

in

of RNA synthesized

- The decreased

S. antibioticus

Then,

results

to synthesize

as compared

will

12 of

was the observation

of the antibiotic.

sensitivity

Similarly,

in 12 hr extracts Thus,

RNA

preparation).

gradient

present

finding,

of actino-

RNA at levels

commenced

of 3H-UMP incorporation

the absence

as compared

pany

levels

in actinomycin

cation

unexpected

(12 hr

into

These

cells

yet

by 71% and 3%, respectively,

incorporation ference

have not

in 48 hr extracts.

D, the

'H-uridine

respectively.

.'H-UMP incorporation

than

M actinomycin were

but

before

was measured,

respectively. 3H-UMP into

producing

which

been used

the onset

incorporated

incorporated

Intact

as RNA precur-

net RNA synthesis

cpm/mg protein

extracts

-

have

cultures

innoculation)

in which

and 18,431

from

vitro"

cells

harvested

were

compared actinomycin

of these

cells

12 hr and 48 hr cells

of 86,715

and "in

and 3H-UTP respectively,

and after

hr and 48 hr cell-free

viva"

3H-uridine

In experiments that

"in

do incorporate

reduced Fig.

capacity 1 also

for shows

BIOCHEMICAL

Vol. 63, No. 2, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2

'8

23,

16,

t

t

10

5 Tube

E :

7";

15

number

Fig. 1 - RNA's extractd from sonicates of 3H-uridine labelled 12 hr (top panel) and 48 hr (bottom panel) cultures of 2. antibioticus were analyzed by sucrose gradient centrifugation. See Materials and Methods for details of the analysis. Gradients were centrifuged for 13 hr at 36,000 rpm and 10 drop fractions were collected from the bottom of each gradient and processed as described in Materials and Methods. The positions of 16s and 23s rRNA's were determined using marker RNA's prepared from E. coli cells and run on parallel gradients. The direction of sedimentation is from right to left.

that

both

than

16s and more rapidly

12 and 48 hr cells

hr cell-free It

extracts

can be seen that

rRNA's patterns

were

The data and their

when

D were

analyzed

I give

labelled

extracts.

It

material

sedimenting

RNA species in the

and lower (data

not

some information

of Fig.

the synthesis weights.

synthesized

in

2. of

Similar the presence

shown). on the relative

RNA species

synthesized

can be seen

that

472

by 12 and 48

profiles

support

molecular

the RNA species

more slowly

synthesized

gradient

12 and 48 hr extracts of higher

obtained

of Table

of the various

23s.

are displayed both

and RNA species

of actinomycin

contain than

there

proportions

by 12 and 48 hr cells is

no great

difference

in

BIOCHEMICAL

Vol. 63, No. 2,1975

AND BIOPHYSICAL

Tube

Fig. 2 - Sucrose gradients and 48 hr (bottom panel) 36,000 rpm and processed

the

relative

cultures that

amounts during

net

reduced

the

RNA from

RNA sedimenting

more

of stable to those portion

observed

with

slowly

labelled

by cell-free

35-40%

under

(6%).

despite is

I show that

by cell-free

significantly Labelled

16s RNA, 35-40%

Eighty-five "in

extracts

by 12 hr extracts

to ninety

sediments

per

amounts

are not identical A smaller pro-

conditions. by cell-free

synthesized

faster

the relative

extracts

labelling

two

the fact

section).

20% 23s RNA, 35-40%

RNA synthesized

of the RNA labelled

I),

by the

16 s and Z-3% RNA sedimenting

vivo"

extracts

(Table

(see preceding

of Table

synthesized "in

synthesized

by 48 hr cells

about

than

and more rRNA is

by 48 hr extracts thesized

contains

RNA species

&lo%)

period

3H-uridine

the data

Further,

of the

somal

RNA species

1 hr labelling of

cultures

23 s.

containing RNA's synthesized by 12 hr (top panel) cell-free extracts were centrifuged for 12 hr at as described in Materials and Methods.

to the 12 hr culture

both

than

“umber

of the various

incorporation relative

RESEARCH COMMUNICATIONS

cent

is

ribo-

(11%)

than

of the RNA syn-

at less than 16s as compared A slight increase in the proviva."

473

Vol. 63, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Relative

proportions of 3H-labelled RNA observed lysis of 12 and 48 hr -S. antibioticus

RNA synthesized in vivo by 12 hr

ana-

of label 23s

in RNA'ssedimenting 16s

at ~16s

cells

1.5

20.2

36.7

41.6

48 hr cells

2.6

19.9

42.6

34.8

12 hr extracts

1.6

4.0

7.1

87.3

48 hr extracts

3.0

1.5

4.4

90.1

in vitro

by

12 hr extracts

+ Act.

D

4.0

3.8

8.6

83.6

48 hr extracts

+ Act.

D

5.4

2.2

3.9

88.5

An estimate rose gradients representing 16s; (4)more of the total data presented

portion

3 RNA species in the sucof the relative proportions of H-labelled of Figs. 1 and 2 was obtained by summing the counts per minute RNA's sedimenting: (1)more rapidly than 23s; (2)at 23s; (3)at Each sum was then expressed as the percentage slowly than 16s. The radioactivity recovered from the gradient in question. represent the average of two experiments.

of the labelled

in the nally, sized

as compared

Table

that

I shows cell-free

to those

Patterns

of Fig. amounts

Between

by a factor to incorporate fested

1 indicate

of three.

species inhibition the cells

that

there

the amount

in the cells

into decreased (Table

is

decrease

RNA species

First,

Fi-

syntheD are

the decrease

in

RNA ascompared

I).

These

of all data

in the

indicate

stable

ability

474

of 48 hr

the major

cells

decreases

of 48 hr cells

12 hr cells

RNA species

rela-

12 hr cells.

the cells

that

pat-

in the

with

the ability with

conclusions

decrease

as compared

of 16 + 23 s RNA in

production

general

the absorbance

a significant

of the synthesis of specific begin to make actinomycin.

The observed

products.

of actinomycin

- Several

above.

in 48 hr cells

Second,

23s was observed

absence.

presented

?I-uridine

as a generally

of stable

in S. antibioticus

of rRNA present

than

12 hr cell-free

in the presence

in its

the data

12 and 48 hr,

the

the proportions

observed

from

at greater

with

conditions

of RNA synthesis

can be drawn terns

RNA sedimenting

48 hr profile under

similar

tive

Percentage >23s

on sucrose gradient total RNA

is manistable

RNA

no selective occurs

as

to incorporate

Vol. 63, No. 2, 1975

3

I-I-uridine

as compared

in the size in

the overall

rate

(Fig.

between

changes

bination

in

This pool

of the

two)

in 48 hr cells.

size

to support

levels

obtained

extracts

reveal

hibition

of the

extracts,

12 hr extracts.

This

thesis

by 48 hr cell-free

the antibiotic hr cells

is not

to synthesize

synthesized

under

are

crude

represent

RNA.

S. A.,

(Interscience),

some com-

capacity

to syntheribosomes

have been

is

responsible

for

should

produce

of all

would

observed

observation rather

suggest

may not

reflect

extracts.

RNA syn-

insensitive that

in

to

the presence ability

of 48

the presumed the

of

rRNA

initiation

of

The enzyme systems

to preexisting,

by these nascent

by grant number 5ROl-CA-12752-03 U. S. Public Health Service.

Formation

present

that

the decreased that

in-

by cell-free

the species

be noted

Actinomycin-Nature,

antibio-

a selective

rRNA synthesized

of nucleotides

New York,

extracts

may

rRNA chains.

from

the

and Activities.

Wiley

1968.

Eats, Jones,

E. and Weissbach,

G. H. and Weissbach,

4.

Collett,

M. S. and Jones,

5.

Gallo,

M. and Katz,

6.

Lowry,

0. H., Biol.

extracts

and the observed

3.

J.

the

It

2.

(1951),

with

conditions

the addition

Waksman,

coupled

in theoall-free

ones,

whe-

in RNA synthe-

RNA species

level

chains

This research was supported National Cancer Institute,

1.

stable

the

cell-free

new polynucleotide used

itself

(or

of RNA by 2.

does not

above),

(see

by an in-

sufficient

which

synthesis

of particular decreases

by actinomycin

48

been determined

to supply

actinomycin

finding,

be explained

the decrease

synthesis

of RNA deintact

of rRNA occurs

or degradation

on the cell-free

generally

catalyzed

has not

decline

(4).

but

inhibition

not

it

causing

of protein

synthesis

rate

degradation

increase

(2)a

from

rRNA and the residual

cultures that

increased

could

However,

may be necessary

antibioticus

The data ticus

observation

The residual

the low 2.

(3)an

extensive

the factors

rRNA in 48 hr cells

in aging

that

(l>an

in 48 hr cells;

of RNA synthesis

are

by:

of the RNA obtained

size.

the rates

pool or

profile

1) does suggest

in precursor

may be caused

precursor

of RNA synthesis;

12 and 48 hr.

crease

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

12 hr cells

The absorbance

hr cells

sis

with

of the nucleotide

gradation.

ther

BIOCHEMICAL

H.

(1963), H.

G. H.(1974),

E. (1972),

Rosebrough, Chem. 193,

J.

(1970),

N. J.,

Biol. Arch.

265-275.

475

666-675.

Biochem.

Biophys.

.I. Ultrastructure

Res.

J. Bacterial. Farr,

Chem. 238,

109,

137, 46,

659-667.

A. L. and Randall,

R. .I.

558-575. 452-465.