Maturation of chloroplast rRNA in Euglena gracilis

Vol. 56, No. 1,1974

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATURATION OF CHLOROPLAST RRNA IN Philippe Section

Received

November

EUGLENA

GRACILIS

HEIZMANN

de B i o l o g i c g ~ n ~ r a l e et a p p l i q u ~ e Laboratoire associ~ au C N R S U n i v e r s i t @ de L y o n I 69621 VILLEURBANNE France

8,

1973

SUMMARY phosphorus into RNA of greening Eu16 S and 23 S chloroplast rRNA (0.55 and i.I × 106 dalton), and two minor "p 16" and "p 23" chloroplast species, slightly larger than 16 S and 23 S RNA (0.62 and 1.2 × 106 dalton). Their role of precursors for 16 S and 23 S RNA has been demonstrated by quantitative conversion of "p 16" to 16 S RNA. The incorporation

of radioactive

glena labels 2 1 S and 25 S cytoplasmic rRNA,

INTRODUCTION In e u c a r y o t e s lar w e i g h t

as in p r o c a r y o t e s ,

RNAs s e e m to r e s u l t f r o m

ge p r e c u r s o r m o l e c u l e . T h i s ces of e a c h

of the rRNA,

In m a m m a l i a n cleus the

cells

rRNAs

are t h e n

and

In b a c t e r i a , "p 16"

precursor

for b o t h

"p 23"

rRNAs

active

and

Haselkorn

phosphorus

and m a t u r a t i o n in Nicotiana, i.I

× 106 and

precursors,

2.7

and H a r t l e y 0.56

rRNA

× 106 d a l t o n

level,

could

In

by d i f f e r e n t (4)

without however,

sequen-

in

the nu-

cleaved

steps.

in

Mature

(7)

of a c o m m o n

authors

observation Munsche

of p r e c u r s o r

rRNA

indicated possess

of r e s p e c t i v e l y

spinach,

and

of r a d i o -

and W o l l g i e h n

in s p i n a c h ,

be a p o s s i b l e

(2)(3).

in Chlamydomonas,

incorporation

chloroplast

112 Copyright ©1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

is

documented

showed

weights

× 106 d a l t o n .

× 106 d a l t o n

proteins,

and W i l s o n

and E l l i s

molecular

appears

(i). The o c c u r e n c e

suggested

Recently,

the

RNA.

16 S and 23 S RNA a p p e a r as s l i g h t -

in Euglena

in m a t u r e

steps.

with

and 0 . 6 5 - 0 . 7 0

was

with

besides

lar-

cytoplasm.

species

Chiang

(5)

to

which

high molecu-

of a s i n g l e

excess

by w e l l

to the

precursors

and

In c h l o r o p l a s t s , Brown

associated

nucleoplasm

transported

ly l a r g e r

contains,

nonconserved

the p r e c u r s o r ,

as a 45 S m o l e c u l e nucleolus

ribosomal

the p r o c e s s i n g

precursor

some

both

common

that

larger

1.2-1.3

a species

(6)

× 106

migrating

precursor.

at

Vol. 56, No. 1,1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

In this p a p e r

we show that

from the m a t u r a t i o n 1.2

× 106 d a l t o n

of larger

precursors

RNA molecules.

the chloroplasts. 0.62

chloroplast

moving

These

as

0.62

precursors

A precursor-product

x 106 d a l t o n RNA and the 0.55

in Euglena result

RNAs

are

relationship

x 106 d a l t o n

× 106 and located

between

in the

RNA has been d e m o n s -

trated. As this work was Eisenstadt

in p r e p a r a t i o n

tro, newly s y n t h e s i z e d ture

rRNA as

molecules

Carritt

incubated

slightly

larger

and

in vi-

than ma-

RNAs.

MATERIALS Euglena gracilis, phosphate

limited

stationary tions KCI

for p u b l i c a t i o n ,

in Euglena c h l o r o p l a s t s

(8) d e s c r i b e d

under

vation

fluorescent H332p04

at a c o n c e n t r a t i o n ed in a SS-I

purchased

of 30-50 mCi/l.

Sorvall

centrifuge,

After

star-

illuminated

level

under

of the cultures).

from CEA,

Aliquots

condi-

in a 3 × 10 -6 M

8 x 106 cells/ml. they were

on a of the

in sterile

and r e s u s p e n d e d

of

(3000 lux at the free,

in the dark,

centrifuged

1 to 5 days,

light

(carrier

grown

(99 . At the b e g i n n i n g

dim illumination

for

METHODS

were

were

at a c o n c e n t r a t i o n

in the dark

white

Z,

[PC m e d i u m

the c u l t u r e s

green

solution

strain

medium

phase,

AND

were

and the p e l l e t s

France)

was added

rapidly

sediment-

frozen in a c e t o n e -

dry ice. RNA was e x t r a c t e d an aqueous MgCI2 lene

phase

5.10 -3 M

containing

; sodium

sulfonate

Worthington)

1%.

contact

RNA species effect

scans were

for 2 hours

scanned

Regulix

in UV

sheets.

method

0.i M, pH 7.3

0.05

M

at O°C.

a

with

;NaCI O.i M

DNase

(RNase

The RNA was % gels

Joyce

Loebl

Autoradiographs

HS films

these

with ;

; triisopropylnaphta-

on 2.4-2.8

with

(i0),

were

dry gels,

free,

analyzed (ii).

Chromoscan, obtained

by

and scanned

the Chromoscan. Quantitative

were

thioglycolate

on glass p a p e r

of Kodak

Tris-HCl

gel e l e c t r o p h o r e s i s

The gels were

with

:

phenol-m-cresol

The p r o d u c t was t r e a t e d w i t h

at 50 pg/ml

by p o l y a c r y l a m i d e

and dried

by the

were

estimations

obtained

in a P a c k a r d

normalized expressed obtained

3375

with

with

respect

a D-MAC

sliced

Scintillation to the

in cm 2 (i cm2A/

with

of r a d i o a c t i v i t y

1.50D

pencil

gels

counted

by C e r e n k o v

Spectrometer

; the counts

16 S a b s o r b a n c e units).

follower

113

of the d i f f e r e n t

peaks

on UV

Surface e s t i m a t i o n s

and a ~ I

500 computer.

Vol. 56, No. 1, 1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

RESULTS Fig.

1 describes

the

results

of an e x p e r i m e n t

in w h i c h

Eu-

1b-15min.

/i

!I

ii

:t t

:

i ! i i

i i i

11

+

+ i i i "+ i

,

;b

51

I I

I

t t i +

I I

i

i

i i

t

r

I

I

I

i

•

'

|

J

i

M,M&Iff5 55 ~

85

|

,

111213.5

P, it

lc-30min.

ld-60

it

min.

t,

II II II II

1

I I Ii

I

I I

Jl I!

I,

I | 1

I t

,,.

/\

t ,/jki

5.5 ~ .

I !

A

',/

,

-

8.5

i

I I

I~| I I1~1 I I

'

~ i

MW,,IO'5

A I| lil

I~

I I

I

I l

~.;,.____-'z==-~

I

I' I

il

i

,.,

i

M.W.l©

1112 13.5

Fig. I - 2,8 % polyacrylamide gel electrophoresis of total RNA from Euglena. Etiolated cells were starved in the dark for 48 hours after harvestin~ then illuminated for 5 hours ; they received 32p (50 ~Ci/ml) for 5, 15, 30 or 60 minutes. ------ UV scan of the gel ; densitometric scan of the autoradiograph obtained with the same dry gel.

glena

were

illuminated

Radioactive

phosphate

sis of c h l o r o p l a s t mination)

(12).

after was

rRNA

two

added

days during

(3-20 h o u r s

Chloroplast

rRNAis

of

starvation

the p h a s e

after

in the dark.

of r a p i d

the b e g i n n i n g

then rapidly

syntheof

illu-

and p r e f e r e n t i a l -

ly l a b e l e d . In the first,

16 S r e g i o n

without

of b a c t e r i a

lag

(i) and

(fig.

of the gel, 1 a),

slightly

the

radioactivity

in a s p e c i e s

larger

114

(0.62

similar

appears

to the

× 106 dalton)

"p 16"

than

the

VoI. 56, No. 1, 1974

mature

16 S

constant after

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

(0.55

level

some

experiment

× 106

of

lag,

dalton).

labeling.

This

longer

than

5 minutes

1 b).

This

stable

(fig.

species

The m a t u r e

rapidly

16 S

species

in the

product

reaches

conditions

accumulates

a

is l a b e l e d of t h i s

radioacti-

vity. In the mature nutes

23 S R N A

to the

and m o v e s The

"p 23"

with

an a p p a r e n t

membranes, RNAs. can be

free

chloroplast satellite

large

detected

isolated

This methods

and the

No v e r y

molecular

RNA,

on the

a lag s h o r t e r heavy

weight

green

were

radioactivity

side

and

of

1.2

cells

than

provides

remains

amounts,

× 106

dalton. RNAs

DNA and

in

with32p04

altered

ribosomal

on

chlo-

material

to

plastidial

of t h e s e

chloroplast

c o m m o n p r e c u r s o r tothe two

gel b e t w e e n

mipeak,

by f l o t t a t i o n

attached

the

dalton

greatly

are p a r t

5

of t h i s

furnished

and purified

species

possible

1.2

x 106

of c y t o p l a s m i c

RNA

enters

It is p r e s e n t in s m a l l

was showed with

(13).

: only

with on the

x 106

chloroplasts

2)

the

of 0.62

r o p l a s t s , but e s s e n t i a l l y (fig.

dalton)

appears

of b a c t e r i a .

localization

% sucrose

of the gel,

× 106

shoulder

chloroplast

Their 50

(i.i

; a small

similar

the

23 S r e g i o n

the

1.2

rRNAs,

× 106 d a l t o n

peak.

DNA \

h4X~&lO-5

5.5 6.2

,

11 12

F{g. 2 - 2,4 % polyacrylamide gel electrophoresis of RNA from purified chloroplasts. Green cells (48 hours starvation in the dark plus 48 hours ~lumination) received 32p (50 ~Ci/ml) for 30 minutes. The chloroplasts were purified by the flottation method of Brawerman (13), and their RNA extracted as described in Method8 ; the label in the DNA peak disappeared after DNase treatment . . . . UV scan of the gel ; densitometric scan of the correspondant autoradiograph.

115

Vol. 56, No. l, 1 9 7 4

The

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

stoichiometric

into 0.55

× 106 dalton

transformation

RNA has been d e m o n s t r a t e d

incorporation

of r a d i o a c t i v e

after

labeling,

a pulse

precursor

Brown

added

and al.

(14)

We tried

With

siderably

slower

by

the

blocking

species

transformation

that t r a n s c r i p t i o n

to use this

of

Despite

(fig.

during

inhibitor

its pe-

labeling

of

for 4 days on KCI me-

the m a t u r a t i o n

conditions

the drug

to allow

to stop the

starved

used

in the

these precautions,

the course

of c h l o r o p l a s t

in Euglena, p r o v i d e d

starvation,

than w i t h

3 a).

of t r a n s c r i p t i o n

rate was

con-

first experi-

complete

of the e x p e r i m e n t

inhibition

could

not be

3 b).

Chase w i t h

cold phosphate,

tion of the r a d i o a c t i v e of the r a d i o a c t i v i t y tant

RNA

Precursor

the

of d i m e t h y l s u l f o x i d e ,

such p r o l o n g e d

(fig.

obtained

showed

RNA in Euglena c u l t u r e s

chloroplast

ment

in the

following

by r i f a m p i c i n

in the p r e s e n c e

netration.

dium.

phosphate

and by

x 106 d a l t o n

to product.

RNA could be inhibited was

of 0.62

appearance

however,

tracer

after

in the 0.62

be measured.

This

of the larger

species

precursor-product

clearly

some

× 106

of r a d i o a c t i v i t y

into the smaller (fig.

lag

extensive

dilu-

and d i s a p p e a r a n c e

dalton

in 0.55

demonstrated

relationship

caused

RNA w i t h

concomit-

× 106 dalton

RNA could

stoichiometric

conversion

one,

characteristic

of

a

3 c).

DISCUSSION Our results

for

16 S RNA is easily

23 S RNA seems more te. No e v i d e n c e has b e e n

found

after

transient

exponential

starvation

growth.

maturation

In b o t h cases

while

steps

the pre-

the p r e c u r s o r

for

of its rapid m a t u r a t i o n p r e c u r s o r for both heavy

of labeling

state

It b e c o m e s

without

strongly

roplast

rRNA accumulation

of the cultures.

or if the cells

affecting

slower

either

as

are

rates.

116

It

rRNAs

synthesis

de-

is rapid

illuminated

starvation

chlorophyll

ra-

in vivo.

rate of 16 S c h l o r o p l a s t RNA is e x t r e m e l y

on the p h y s i o l o g i c a l

a short

because

our c o n d i t i o n s

between

rRNA.

detected,

of a p o s s i b l e common under

The m a t u r a t i o n pendent

analogy

and Euglena c h l o r o p l a s t

of b a c t e r i a l cursor

show a p e r f e c t

during

increases, or chlo-

Vol. 56, No. 1, 1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

15 >

~J

}1c

/

Z

1

Hours 3b 15

O

/

E L

/

ii

"~--__pl~

/

"-II,

//

5

// f"

I

I

I

I

I

I

I

2

3

4

5

6

I

7

I

I

8

9

Hours

3c

o - - - - rn16+___0.89_.~p16 - IC

/F"', /

E z

l,

7",,

/ I

F~.

2

3

"'"--.... .... 2. 4

5

6

7

8

9

Hours

3 - Kinetics of labeling "p 16" and "m 16" RNA. E t i o l a t e d cells starved for 96 hours in the dark after h a r v e s t i n g and i l l u m i n a t e d for 5 hours, r e c e i v e d 50 ~Ci/ml 32po 4 . The amounts of r a d i o a c t i v i t y were normalized w i t h r e s p e c t to the 16 S a b s o r b a n c e peak (see Methods). "m 16" ; w - - - - p 16" ; "m 16" + 0,89 "p 16" (89 % of "p 16" is conserved), a. Control ; b. Cultures added with r i f a m p i c i n (10 pg/ml) and d i m e t h y l s u l f o x i d e (i %)45 m i n u t e s after the addition of 32p ; c. C u l t u r e s added w i t h cold p h o s p h a t e (0,3 mM) 45 minutes after the addition of 32p.

117

Vol. 56, No. 1, 1974

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

Preliminary dicate protein

that

results

chloroplast

with

rRNA

protein synthesis

synthesis is

inhibitors

strictly

in-

dependent

on

synthesis.

ACKNOWLEDGMENTS I am grateful to Drs H. Duranton, F. Gros, R. Monier, V. Nigon and J.P. Weil for their kind criticism. This work was partly supported by a grant from the Commissariat ~ l'Energie Atomique (CEA).

BIBLIOGRAPHY I.

Adesnik, M. and Levinthal, C.

2.

Miller Jr, O.L. and Hamkalo, B.A.

J. Mol. Biol., 46, 281-303 (1969).

3.

Kossman, C.R., Stamato, T.D. and Pettijohn, D.E. Nature 234, 102-104 (1971).

4.

Wilson, R. and Chiang, K.S.

5.

Brown, R.D. and Haselkorn, R.

J. Mol. Biol., 59, 491-503 (1971).

Intern. Rev. Cytol., 33, 1-25 (1972). (London) New Biol.

J. Cell. Biol., 55, 285 a (1972).

6.

Munsche, D. and Wollgiehn, R.

Biochim. Biophys. Acta, 294, 106-117 (1973).

7.

Hartley, M.R. and Ellis, R.J.

Biochem. J., 134, 249-262 (1973).

8.

Carritt, B. and Eisenstadt, J.M.

9.

FEBS Letters, 36, 116-120 (1973).

Freyssinet, G., Heizmann, P., Verdier, G.,

Trabuchet,

G.

and

Nigon, V.

Physiol. V~g~t., i0, 421-442 (1972). iO. Parish, J.H. and Kirby, K.S. Bioahim. Biophys. Aeta, 129, 554-562 ii. Loening, U.E.L. 12. Heizmann,

P.

13. Brawerman, G.

(1966).

Biochem J., 102, 251-257 (1967). Biochim. Biophys. Acta, 224, 144-154 (197o). Biochim. Biophys. Acta, 72, 317-331 (1963).

14. Brown, R.D., Bastia, D. and Haselkorn, R. (1970). In RNA Polymerase and Transcription (Silvestre, L. ed), pp. 309-328, North-Holland, Amsterdam.

118