Effects of intercalating agents on the structure of the ribosome

Vol. 65, No. 2, 1 9 7 5

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EFFECTS OF INTERCALATING AGENTS ON THE STRUCTURE OF THE RIBOSOME T. Suryanarayana*

and D. P. Burma

M o l e c u l a r Biology Unit, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University Varanasi 221005, U.P., India Received

June 3,1975

SUMMARY R i b o s o m e s t r e a t e d w i t h an i n t e r c a l a t i n g dye (ethidium bromide or acridine orange) are degraded m o r e r a p i d l y b y RNase I t h a n u n t r e a t e d ribosomes. Spermidine or spermine counteracts to great extent the effect produced by the intercalating dye. It appears that the intercalation leads to the 'loosening' of the structure of the ribosome which is also reflected in the decreased inhibitory capacity of the treated r i b o s o m e s t o w a r d s RNase I . Intercalation appears to decrease the affinity o f rRNA towards proteins causing the structure of the r i b o s o m e t o become u n f o l d e d .

INTRODUCTION The intercalating

dyes like ethldium bromide and

acridine orange have been extensively used to study the structure

of polynueleotides

to RNA in yeast ribosomes

(1,2).

Acridine

(3); ethldlum bromide binds to

free rRNA as well as to rRNA in ribosomes laboratory with

the mechanism of artificial

30S s u b u n i t

(5)

is being

Because the hydrolytic

attack

d e p e n d s on t h e c o n f o r m a t i o n tion

can be utilised

ribosomes.

to

(4).

investigated

of the the

In this

association in detail

o f RNase I on t h e

study

In the present

orange binds

ribosome structural

communication the

o f RNase I (6).

ribosome

(7) this

associa-

topography effects

of

* Senior Research Fellow under a project financed by the Indian Council of Medical Research.

Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.

708

of

VoI. 65, No. 2, 1975

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Intercalating monitored

agents

on t h e

b y RNase I w i l l

structure

of the

ribosomes

as

be described.

MATERIALS AND METHODS P o l y ( A ) was t h e p r o d u c t o f M i l e s L a b o r a t o r i e s , U.S.A. S p e r m i d i n e , s p e r m i n e , b o v i n e s e r u m a l b u m i n and e t h i d i u m bromide were purchased from Sigma Chemical Co., U.S.A. Acridine o r a n g e was t h e p r o d u c t o f B r i t i s h D r u g H o u s e , E n g l a n d . E . e o l i r i b o s o m e s a n d S . t y p h i m u r i u m RNase I w e r e prepared a~ribed earlier (~). Ribosomes were treated with ethidium bromide or acridine orange according to the m e t h o d o f S t e v e n s and P a s c o e ( 4 ) . The degradation of ethidium bromide-or acridine orange-treated and untreated r i b o s o m e s b y RNase I was f o l l o w e d i n Z e i s s PMQ I I s p e c t r o p h o t o m e t e r . The r e a c t i o n was c a r r i e d o u t i n t h e c u v e t t e a t 25°C i n a t o t a l v o l u m e o f 1 ml c o n t a i n i n g 0 . 1 M T r i s - H C 1 pH 7 , 1 A 2 6 0 . u n i t o f t r e a t e d or untreated r i b o s o m e , 4 u n i t s o f RNase I t 6 ) and d e s i r e d a m o u n t o f Mg ++. A b l a n k m i x t u r e w i t h o u t t h e e n z y m e was u s e d t o a d j u s t t h e initial absorhancy of the reaction mixture to zero. The i n h i b i t i o n o f RNase I - e a t a l y s e d hydrolysis of poly(A) by treated and u n t r e a t e d r i b o s o m e s was m e a s u r e d a c c o r d i n g t o t h e m e t h o d d e s c r i b e d b y D a t t a and Burma ( 6 ) . RESULTS AND DISCUSSION Degradation . of ethidium ribosomes

b~ RNase I :

produce a large (Fig.lA).

bromide-and

amount of distortion

are completely

protected

in the

untreated

ribosomes

Similar trations.

degraded

differences Even a t

treated

ribosomes

Similar

results

orange

(Fig.lB).

at

ribosomal

against

treated ribosomes are degraded at ++ When Mg concentration is lowered to are

seems t o structure

o f 1 . 3 5 mM t h e u n t r e a t e d

the

ribosomes

orange-treated

Ethtdtum bromide treatment

At a Mg ++ c o n c e n t r a t i o n

ribosomes

acridine

a much f a s t e r

RNase I ,

whereas

a reasonably

fast

0 . 3 5 mM t h e

treated

rate

whereas

rate.

the

are degraded

s l o w l y and i n c o m p l e t e l y . ++ a r e a l s o o b s e r v e d a t o t h e r Mg concert÷÷ a v e r y h i g h Mg concentration (5 mM) t h e

are not completely

were

obtained

These

results

protected

following suggest

709

against

treatment that

with

RNase I . acridine

the distortion

Vol. 65, No. 2, 1975

I

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

|

I

|

I0

15

A ]

I

I

I

5

I0

15

50

O 40

~A

_Z 30

~ zo

io

5

20

20

MINUTES

Fig.l.Degradation,,of ethidium bromide (A) and acridine orange (B) treated ,rlbosomes by RNase I at different Mg ++ concentrations. The treatment of ribosomes with ethidlum bromide an~cridine o r a n g e and t h e a s s a y w e r e c a r r i e d o u t a s described under 'Materials and M e t h o d s ' Closed symbols and o p e n s y m b o l s r e p r e s e n t u n t r e a t e d a,nd t r e a t e d r i b o s o m e s respectively. Mg ++ c o n c e n t r a t i o n s (mM) a r e A . e O 0 . 3 5 ! A A 0.7|m ~1,355[]~2.35|m ® 5.0 B.e o 0.355A/k 0.7~Br~l.0; m~l.35;B ~ 2.35.

produced by the the

overall

the

attack

structure

of polycationtc

of the

degradation amines:

on t h e

leads

to the unfolding

r i b o s o m e and m a k e s i t

of

amenable to

It

is well

spermidine

and s p e r m i n e

of treate d ribosome in presence

Polycationtc

structure

(4,7-10).

amines have profound

and f u n c t i o n

known t h a t cause the

ribosome to become tightened Spermidine

agent

o f RNase I .

RNase I c a t a l y s e d

influence

intercalating

of the

polycationic overall

ribosome amines such as

structure

of the

and t h u s mimick t h e e f f e c t

o r s p e r m i n e was f o u n d t o d e c r e a s e

710

the

rates

o f Mg of

++

Vol. 65, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

degradation of both ethidium bromide-treated and untreated ribosomes (Flg.2).

Similar results were obtained with

ribosomes treated with acridine orange (data not shown). the

presence

produced

in the

Inhibition a~ents:

of a polycationic ribosome

agent

by the

ability

of

a polynucleottde

as catalysed

is

dependent

concentration

capacity

on t h e

which

is

dependent

T - - T - -

the distortion

intercalating

of RNase I by ribosomes

Ribosomes have the

reverses

treated to

agent. with

inhibit

the

intercalatin~

the hydrolysis

by RNase I and this o f Mg + + ( 6 ) .

on t h e

overall

A

Thus

inhibition

This

inhibitory

structure

of the

I

T

f

5

I0

15

B

50 o


"

_z ,,<, :50 Z

20

-

I0

5

I0

15

MINUTES

Fig.2.De~radation .of e t h i d i u m b r o m i d e t r e a t e d ribosomes In p r e s e n c e o f s p e r m i d i n e (A) a n d s p e r m t n e ( B ) . The assay was c a r r i e d out as described under tMaterials and Methods I excepting that indicated amount of spermtdine or spermine was p r e s e n t w h e r e m e n t i o n e d . The concentration o f Mg ++ during assay was invariably 0 . 3 5 raM. C l o s e d s y m b o l s a n d open symbols represent untreated and treated ribosomes respectively. Spermidine concentrations (raM) a r e A.eOntl~A/\0.05~ID 0.251U ~ 0.5. B.• 0 nil|A/\0.01! ID0.05! !1~ 0.125.

711

Vol. 65, No. 2, 1 9 7 5

ribosome,

is

also

intercalating tion

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

profoundly

dyes

(Fig.3).

o f 0 . 8 mM t h e

ribosomes

are

inhibition, observed

untreated

ribosomes

inhibition

ribosomes

with

0.02 I I

0

0

0.04 I

the

orange-treated considerable

of ribosomes, ++ A t 5 mM Mg the

capacities

produced by the

treated

acridine

ribosomes.

becomes smaller

carried

the

concentration

inhibitory

Studies

with

a t a Mg ++ c o n c e n t r a -

o f RNase I w h e r e a s

on t h e

the untreated

in the

less

bromide-or

poor inhibitors

difference

by treatment

For example,

ethidium

dependent with

affected

out

of the

hut

treated

in this

thiol

there

treated is

is

and

definitely

ribosomes. laboratory

reacting

with

reagents

the

like

N-ethyl-

AMOUNT OF RIBOSOME(A26O) .-o-. 0"02 0.04 0.06 0.06 I AI

0"06

0,08

o;2

003

oo4

~

0"01

0'02

0-03 0"04 AMOUNT OF RIBOSOME(Az6O)

Ftg.3.Inhtbitton of RNase I by ribosomes treated with ethtdium b r o m i d e (A) a n d a 0 r i d i u . e o r a n g e ( B ) . The treatment of ribosomes with ethldium bromide and acridine orange and Inhibition assay have been described in the text. Closed s y m b o l s and o p e n s y m b o l s r e p r e s e n t untreated and treated rlbosome respectively.@ O a s s a y e d a t 0 . 8 mM Mg ++ A~assayed a t 5 . 0 mM Mg ÷+.

712

Vol. 65, No. 2, 1 9 7 5

maleimide,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

parachloromercuribenzoate,

benzoic acid),

indicate

the ribosome.

Apparently the distortion

structure

of either

similar

5,5t-dithiobis(2-nitro-

unfolding

rRNAs o r p r o t e i n s

and t h u s t h e

of the ribosome. monitoring

~loosening'

in t h e r i b o s o m e

between nucleic

of the overall

RNase I a p p e a r s t o be a u s e f u l

such c o n f o r m a t i o n a l

of

produced in the

present

leads to the weakening of the interaction and p r o t e i n s

of the structure

acids

structure

tool for

change.

REFERENCES /l/

(3)

Waring, J . , N a t u r e , 219, 1 3 2 0 ( 1 9 6 8 ) . Carlberg-Bacq~C.M., in Fundamentals of Biochemical Pharmacology (Z.M.Bacq,R.Capek,R.Paoletti, and J . R e n s o n , E d s . ) Pergamon P r e s s , p . 4 7 6 ( 1 9 7 1 ) . M o r g a n , R . S . , and D . G . R h o a d s , B i o c h i m . B i o p h y s . A c t a , 102, 311 ( 1 9 6 5 ) . Neu,H.C. and L . A . H e p p e l , ~ , 1267 ( 1 9 6 4 ) .

Proc.Nat.Acad:Sci.,

U.S.A.,

Suryanarayana,T., Ph.D.Dissertation submit~e'd t o B a n a r a s Hindu U n i v e r s i t y , V a r a n a s i , I n d i a ( 1 9 7 5 ) . (8) B r e t t h a u e r , R . K . , L.Marcus,J.Chaloupka,H.0:IIalvorson, and R.M.Bock, B i o c h e m i s t r y , 2, 1079 ( 1 9 6 3 ) . 9) T a k e d a , Y . , B i o e h i m . B i o l D h y s . A c t a , 182, 2 5 ~ ( 1 9 6 9 ) . 10) S p i r i n , A . S . , in ' M o l e c u l a r mechanism o f a n t i b i o t i c a c t i o n on p r o t e i n s y n t h e s i s and membranes' (E.Munoz, F.Garcia-Ferrandiz, and D . ~ a z q u e z , Eds. ) E l s e v i e r Scientific P u b l i s h i n g C o . , Amsterdam, p . l l ( 1 9 7 2 ) .

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