Interaction of the radioactive translation initiation factor IF3 with ribosomes

BIOCHIMIE, 1972, 54, 829-835.

Interaction of the radioactive translation initiation factor IF3 with ribosomes. Jean THIBAULT,A n n i c k CI:IESTIER, D o m i n i q u e VIDAL et F r a n c o i s G n o s . S e r v i c e de B i o c h i m i e cellulaire. Institut Pasteur. (25/6/1972).

S u m m a r y . - - This work describes a p r e p a r a t i v e procedure to o b t a i n IFs, one of the m a i n t r a n s l a t i o n i n i t i a t i o n factors, in a 35S labelled form. 3.~S IF~ t h u s p r e p a r e d was 80 p. cent pure. The m a i n f e a t u r e s of 35S IF~ b i n d i n g to E. colt ribosomes h a v e been specified by m e a n s of the sucrose density s e d i m e n t a t i o n technique. At 5.10-3 M Mg ÷*, :~5S IF,~ readily f o r m s a stable complex w i t h 30S s u b u n i t s - - p r e v i o u s l y 'washed free of t h e i r endogeneous factors - - b u t not w i t h 50S s u b n n i t s . W h e n the Mg ÷÷ c o n c e n t r a t i o n is lowered to 2.10 4 M, appreciable b i n d i n g to the 50S s u b u n i t s is observed. After m i x i n g 35S IF~ w i t h 70S ribosomes, one can detect the a p p e a r a n c e of 30S-35S IF3 complexes as a r e s u l t of the dissociating effect of IF~. The s t a e c h i o m e t r y of this b i n d i n g corresponds to one molecule of factor per 30S s u b u n i t , -~S-IFz is released f r o m 30S s u b u n i t s a f t e r or d u r i n g f o r m a t i o n of an i n i t i a t i o n complex. In the presence of s t r e p t o m y c i n or n e o m y c i n at doses i n h i b i t i n g s p o n t a n e o u s 70S dissociation, f o r m a t i o n of 30S-35S-IFz complex c a n n o t be detected. These antibiotics as well as k a s u g a m y c i n or colicin F~ do not i n h i b i t 35S-IF:, b i n d i n g to pre-dissociated 30S at low Mg ÷÷.

It is g e n e r a l l y a d m i t t e d t h a t i n i t i a t i o n o f p r o tein synthesis, namely the messenger directed bind i n g of i n i t i a t o r t R N A t o r i b o s o m e s , is a c o m p l e x p h e n o m e n o n i n v o l v i n g n o less t h a n t h r e e d i s t i n c t p r o t e i n f a c t o r s El, 2, 3]. L i t t l e is k n o w n a b o u t tile m o d e of a c t i o n of t h e s e t r a n s l a t i o n f a c t o r s w i t h r e s p e c t to t h e i r d i r e c t i n d i v i d u a l e f f e c t o n t h e v a r i o u s c o m p o n e n t s of t h e i n i t i a t i o n r e a c t i o n . O n e w a y to t a c k l e t h i s p r o b l e m c o n s i s t s i n a n a l y z i n g i n d i v i d u a l s t e p s of t h i s r e a c t i o n s u c h , f o r i n s t a n c e , as t h e f o r m a t i o n of s p e c i f i c c o m p l e x e s b e t ween defined radioactive factors and ribosomes.

S i n c e IF:~ h a s b e e n s h o w n to c a u s e r i b o s o m a l d i s s o c i a t i o n [7] o r to p r e v e n t r e a s s o c i a t i o n of ribosomal subunits even in the absence of any c o m p l e m e n t a r y e l e m e n t of t h e i n i t i a t i o n r e a c t i o n [8], o n e c o u l d e x p e c t it to b i n d e f f i c e n t l y w h e n a d d e d a l o n e to r i b o s o m e s . W o r k b y S a b o l a n d O c h o a [9] h a s s h o w n t h i s to b e t h e c a s e . IF:~ d i r e c t e d 70S d i s s o c i a t i o n w a s c o n c o m i t a n t w i t h t h e f o r m a t i o n of a r a d i o a c t i v e /F:~-30S c o m p l e x . As i n t h e c a s e of I F p r e c o n s t i t u t i o n of a 70S d i m e r following the primary initiation step apparently c a u s e d I F a r e l e a s e [9].

It w a s a l r e a d y f o u n d t h a t r a d i o a c t i v e I F I c a n n o t f o r m a n y s t a b l e c o m p l e x w i t h t h e 30S s u b u n i t , as shown by zonal sedimentation unless the other e f f e c t o r s of i n i t i a t i o n : IF2, G T P , a n A U G t r i p l e t a n d fMet t R N A w e r e also p r e s e n t . R e f o r m a t i o n of a d i m e r u p o n a d d i t i o n of a 50S s u b u n i t w a s s h o w n to c a u s e r e l e a s e of I F I f r o m r i b o s o m e s [4].

We had independently carried out some studies w i t h :sss l a b e l l e d IF:~. R e s u l t s p r e s e n t l y d e s c r i b e d support Sabol and Ochoa's findings. They also provide information concerning the comparative e f f e c t s of c e r t a i n a m i n o g l y c o s i d i c a n t i b i o t i c s o n d i s s o c i a t i o n a n d r i b o s o m a l b i n d i n g of r a d i o a c t i v e I F 3.

Similarly, radioactive IF 2 has been prepared [5, 61 a n d i t w a s o b s e r v e d t h a t it b i n d s to t h e s m a l l r i b o s o m a l s u b u n i t , o n l y i n t h e p r e s e n c e of I F 3 + I F I [5], [6j. T h u s it s e e m s t h a t I F I a n d I F , e i t h e r i n t e r a c t w i t h r i b o s o m e s o n l y if t h e y a r e i n t h e f o r m of a d e f i n e d c o m p l e x w i t h o t h e r t r a n s l a t i o n f a c t o r s o r a s s o c i a t i v e b i n d i n g r e q u i r e s suit a b l e c o n f o r m a t i o n a l c h a n g e s of t h e r i b o s o m e s , these changes being produced by the accompanying factors plus or minus the other effectors.

While this manuscript was in preparation, data f r o m P o n et al. [10] b a s e d u p o n t h e u s e o f 14CH~IF~ h a v e also b e e n p u b l i s h e d . M o s t of t h e i r c o n clusions are in good agreement with those from S a b o l a n d O c h o a [9] a n d f r o m o u r s e l v e s . MATERIA~LS A N D M E T H O D S . STRAINS AND MISCELLANEOUS.

E. colt M R E - 6 0 0 , R N A a s e I less w a s t i s e d t h r o u g h o u t ; N - f o r m y l - m e t h i o n y ! - t R N A (fMet-

830

Jean Thibault, A n n i c k Chestier, Dominique Vidal el Francois Gros.

tRNA) was p r e p a r e d a c c o r d i n g to [ H I . GTP and GMPPCP w e r e p r o d u c t s f r o m Miles. Kasugainycin, S t r e p t o m y c i n an d N e o m y c i n - B w e r e gifts f r o m Roussel-UCLAF. Colicin E.~ was g e n e r o u s l y given by Dr. Boon. C r u d e i n i t i a t i o n factors (IF) an d NH4C1 w a s h e d r i b o s o m e s w e r e p r e p a r e d as already d e s c r i b e d (I). PREPARATION OF 3 5 S - I F 3.

i) Bacterial labelling.

it) (s~S-IFT)purifieation

(fig. 1).

a) 35S-IF T w a s c h a r g e d on a 4 × 15 cm DEAES e p h a d e x As0 column. After w a s h i n g the dialysis buffer .(see p r e v i o u s section), elution was p e r f o r m ed with a l i n e a r g r a d i e n t of 0 to 0.4 M NH4CI, in <>. F r a c t i o n s eluted b e t w e e n 0.05 an d 0.2 M w e r e pooled. This gave rise to about 2,0,0 ml of c r u d e IF 3 (15 rag). At this stage IF 3 a c t i v i t y was d e t e r m i n e d a c c o r d i n g to Sabol and O e h o a [7].

E. coli MRE-600 w a s g r o w n b a t c h w i s e on l o w sulfate m e d i u m of the f o l l o w i n g c o m p o s i t i o n (in one liter) : Tris, 12 g ; NaC1, 0,5 g ; N H4CI, 1.1 g ; Mg Acetate, 0.5 g ; KH2PO ~, 0.1 g ; F e r r i c Citrate, 0.0.05 g ; v i t a m i n B1, 0.005 g ; glucose, 5 g ; (NH4)~S04, 10 mg. Medium was adjusted to pH 7.3. W h e n the d e n s i t y at 420 nm w a s 0.2, 50 millicuries of c a r r i e r - f r e e :~5S04H~ (Centre de l ' E n e r g i e Atomique, Saclay) w e r e a d d e d to each flask and c u l t i v a t i o n was c o n t i n u e d u n d e r m e c h a n i c a l shak i n g until and O.D. of 4.4 ( d i v i s i o n time, 50 rain was r e a c h e d . At this stage, about 75 p. cent of 3,~S exogeneous r a d i o a c t i v i t y w a s i n c o r p o r a t e d . Labelled cultures w e r e p o u r e d r a p i d l y onto c r u s h e d ice. About 2.5 g (wet w e i g h t of cells) w e r e collected by c e n t r i f u g a t i o n in the cold. T h e y w e r e w a s h e d t w i c e w i t h 250 ml (buffer I : Tris-HC1 p H 7.4, 5.10 -2 M ; Mg Acetate, 10~ M ; DTT, 10-a M) ; and kept frozen at - - 2 0 ° C . In o r d e r to p r o c e s s b a c t e r i a for IFa e x t r a c ti o n , 35S labelled cells w e r e first s u s p e n d e d in buffer I, in the p r e s e n c e of 48 g of <> u n l a b e l l e d b a c t e r i a g r o w n u n d e r separate conditions. The s u s p e n s i o n was s o n i c a t e d t h r e e times (1 rain p e r i o d each) in a MIlL s o n i c a t o r at m a x i m a l frequency. After r e m o v i n g the cell debris, r i b o s o m e s w e r e co l l ect ed by two cycles of l o w (30,0,0,0 g) a n d h i g h s p eed (150,000 g) c e n t r i f u g a t i o n . To e x t r a c t the m i x t u r e , of i n i t i a t i o n factors r i b o s o m e s w e r e s u s p e n d e d in t h e f o l l o w i n g b u f f e r : Tris-HC1, p H 7.4 ; Mg Acetate, 2.10 -u M ; NH4CI, 1 M ; D.T.T., 10-3 M ; g l y c e r o l 10 p. cent. After 8 h o u r s at 0°C, the r i b o s o m e s u s p e n s i o n was c e n t r i f u g e d 30 rain at 30,000 g and the s u p e r n a t a n t l a y e r e d onto 20 p. cent g l y c e r o l c u s h i o n s (5 ml) in the same buffer. After c e n t r i f u g i n g at 1,00,000 g o v e r n i g h t , the u p p e r 9/10 of this r e s u l t i n g s u p e r n a t a n t was w i t h d r a w n . It c o n s t i t u t e d the 3~S labelled c r u d e I F f r a c t i o n (IFT). ~5S labelled IFT w a s d i a l y z e d against Tris-HC1, 2.1,0~3 M, p H 7.4 ; EDTA, 10-3 M ; D.T.T., 10 -3 M ; glycerol, 10 p. cent. A slight p r e c i pitate was d i s c a r d e d and about 100 ml of 3~.S I F (5 m g / m l ) w i t h a Specific r a d i o a c t i v i t y of 12.106 c p m / m g w a s o b t a i n e d (35S-IF~).

BIOCHIMIE, 119,72, 54, n ° 7.

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FIG. 1. - - The three main steps involved in the purification of .~5S labelled IF~ are described. (see Material and Techniques for details) : A. DEAE'-Sephadex (A-50) elution profile; B. Phosphocellulose-elution profile; C. Gel-filtration on a G-75 Sephadex eolum, n, in the presence of 6 M-urea. At. these three steps, radioactivity was measured by counting aliquots of the various subfraction in 5 ml Bray's solution. The solid lines represent the radioactivity profiles. The dashed areas indicate the distribution of the dissociating (IFsmediated) activity. Ribosomal dissociating activity was determined by assaying various IFs snbfractions on 1 M NH~C1 washed 70 S ribosomes according to Sabol and Ochoa [4].

b) 20,0 ml <> w e r e adsorbed on a 1.4 )< 7 cm P h o s p h o c e l l u l o s e c o l u m n (Mannex). P h o s p h o c e l l u l o s e w a s p r e p a r e d in buffer C of T h a c h [3]. After w a s h i n g w i t h 0.4 M NH4C1 , elution was c a r r i e d out w i t h a NHxCI l i n e a r g r a d i e n t .(0.4 to 0.8 M). A f r a c t i o n e x h i b i t i n g strong I F 3 a c t i v i t y was eluted at 0.55 M NH4Cl. The f r a c t i o n thus obtained a p p e a r s to give a symet r i c a l peak as d e t e r m i n e d b o t h by the biological assay and the r a d i o a c t i v i t y . At this stage, the P h o s p h o c e i l u l o s e - p u r i f i e d 35S IF~ (PC=35S~IFa)

Radioactive factor IF~ and ribosomes. g a v e t w o m a j o r b a n d s o n a c r y l a n f i d e gel. It w a s d i a l y s e d a g a i n s t 0.05 a m m o n i u m b i c a r b o n a t e a n d lyophylised.

t h e a m o u n t o f 30 S p l u s 50 S s u b u n i t s o r i g i n a t i n g f r o m t h e 70 S i n c r e a s e s i n p r o p o r t i o n to t h e a m o u n t of f a c t o r a d d e d . O n e ~g o f p u r i f i e d f a c t o r is r e q u i r e d f o r c o m p l e t e d i s s o c i a t i o n ( a f t e r a 15 r a i n

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Fro. 2. - - Binding of .~5S labelled IF~ to 30 S s u b u n i l s in the course of ribosomal dissociation. One A~_,,ounit of IM NH, C1 w a s h e d E. colt (MRE-600) 70 S ribosomes w a s incubated w i t h o u t (A) or w i t h 0.5 (B) or 1 ug (C) of :35S-IF~ u n d e r the conditions described in Material and Methods. At the end of this incubation period, each m i x t u r e was cooled down to zero 0°C., layered on sucrose gradients and centrifuged for 90 m i n at 48,000 r.p.m, in a SW-50-1 Beckman rotor at 4°C ; 23 subfractions were collected in scintillating vials, the O.D. profile being recorded in a Beckman DB spectrophotometer. Radioactivity was measured as usual. O.D. at 260 nm. O ......O 3~S radioactivity.

c) T h e l y o p h y l i s e d p o w d e r w a s d i s s o l v e d i n 1 ml, 6 M u r e a . A f t e r f i l t r a t i o n o n G-75, t h e f r a c tion eluted after the dead volume was dialysed a g a i n s t T r i s - H C l p H 7.4, 10 r a M ; NH4C1, 10 -1 M, D.T.T., 10 3 M. A b o u t 6 m l o f a s o l u t i o n c o n t a i n i n g 0.025 m g p r o t e i n s / m l w e r e o b t a i n e d w i t h a s p e c i fic r a d i o a c t i v i t y o f 5,000 cpmh.~g. A t w o f o l d c o n centration was achieved by partial lyophylisation. At t h i s p u r i f i c a t i o n s t e p a c r y l a m i d e gel e l e c t r o phoresis, in the p r e s e n c e of urea, r e v e a l e d o n e m a j o r b a n d a m o u n t i n g to a b o u t 80 p. c e n t of t h e s t a i n i n g i n t e n s i t y a n d r a d i o a c t i v i t y w i t h an estim a t e d M.W. of 21,500. 1 ~g of p u r i f i e d asS-IF:~ w a s s h o w n to d i s s o c i a t e 216,0 n m O.D. u n i t (25 ixmoles) o f 70S r i b o s o m e s .

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FIG. 3. - - A. Binding of ssS-IF~ to << deriued >> 30 S subunils. 2 ~g z5S-IF:~ were incubated for 15 m i n at 37°C with : 0.5,1 and 1.5 A~, units of 30 S obtained by low Mg+~ dissociation (2.10-4 M) of 1 M NH,C1 washed 70 S ribosomes. Mixture were layered on sucrose gradients in the same conditions as those described tlgure 2. Centrifugation was for 150 rain. O.D. 260 nm. 0 0 asS radioactivity. Top of the gradient on the left. B. The a m o u n t of r i b o s o m e - b o u n d 35S-IF,, (calculated f r o m tim e x p e r i m e n t of panel A) is plotted against the q u a n t i t y of 30 S subunits p r e s e n t in the mixture. . . . . . .

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A f i x e d a m o u n t of ¢ NH4CI w a s h e d ~> 70 S r i b o s o m e s w a s i n c u b a t e d w i t h i n c r e a s i n g q u a n t i t i e s of ~5S-IF:~ u n d e r t b e c o n d i t i o n s d e s c r i b e d b y O c b o a [9]. At t h e e n d of t h e i n c u b a t i o n p e r i o d , t h e m i x ture was layered on a sucrose gradient and the e x t e n t of 70 S d i s s o c i a t i o n as w e l l as t h e a m o u n t of b o u n d r ' ~ d i o a c t i v i t y w e r e d e t e r m i n e d . As s h o w n in figure 2 i n c o n f i r m a t i o n o f p r e v i o u s r e s u l t s [91,

BIOCHIMIE, 1'972, 54, n ° 7.

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FIG. 4 . - - - Binding of ass-It::, to ribosomes at different Mg ++ concentrations. I n c u b a t i o n and gradient analysis were p e r f o r m e d in the conditions described in the legend of figure 1. OD 260 nm. O ...... O 35S radioactivity.

i n c u b a t i o n p e r i o d at 37°C) of o n e 2 6 , 0 0 . D . u n i t e q u i v a l e n t of 70 S. C a l c u l a t i o n i n d i c a t e s t h a t a 2/1 r a t i o o f f a c t o r to r i b o s o m e , w o u l d b e r e q u i r e d f o r

Jean Thibault, Annick Chestier, Dominique Vidal et Fran(ois Gros.

832

complete dissociation. Such an unexpected figure m i g h t b e r e l a t e d to t h e r e l a t i v e l y s l o w 70 S d i s s o c i a t i o n e q u i l i b r i u m w h e n p u r i f i e d IFa (i.e. d e v o i d e d of I F I) is u s e d [12].

When a increasing increasing the small was again

f i x e d a m o u n t of 35S-IF 3 w a s m i x e d w i t h a n m u n t s of p u r i f i e d 30 S s u b u n i t s , a n q u a n t i t y of l a b e l c o s e d i m e n t i e d w i t h s u b u n i t (fig. 3), a n d a I : I s t o i c h i o m e t r y found. TABLE

I.

Effect of ssS-IF s on fMet Puromycin formation. pmoles ~1t lMet Puromyein --

Messenger . . . . . . --

AUG

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1R,sRNA _~_ IF;;.. ~2

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FIG. 5. A p p a r e n t release of 35S-IF~ a c c o m p a n y i n g f o r m a t i o n of a 70 S i n i t i a t i o n complex. The reac*tion m i x t u r e c o n t a i n e d in 0.1 m l the following : Tris-HC1, pH 7.4, 50' mM~ ; NH,C1, 6,0, m M ; M,a gnesium acetate, 5 mM ; ~ m e r c a p t o - e t h a n o l , 7 mM ; GTP 1 mM ; 70 S ribosomes, 1.5 260 OD u n i t ; poly AUG, 0 . 2 0 . D . u n i t : 3H-fMet-tRNA (600 c p m / p m o l e ) , 20 pmoles ; IF1 (IF~ free), 20 ,~g; :35S-IFs, 2 j~g e i t h e r in the absence of IF2 (upper figure) or plus 50 ,~tg IFe lower figure. Samples "were i n c u b a t e d f o r 15 m i n at 37°C and centrifuged on sucrose g r a d i e n t s for 60 m i n at 4°C in the SW-65 Titane Beckman rotor, at 60,000 rpm. Profiles 'were recorded a n d r a d i o a c t i v i t y d e t e r m i n e d as described i n figure 2. O.D. 260 nm. © ...... O ~5S radioactivity. +--+ 3H-fMet-tRNA radioactivity. -

-

T h e m o r e 70 S d i s s o c i a t e d , t h e m o r e a~S-IF 3 w a s f o u n d a t t a c h e d to t h e 30 S s u b u n i t (fig. 2). At t h e M g +÷ c o n c e n t r a t i o n u s e d f o r t h e d i s s o c i a t i o n r e a c t i o n , n o s t a b l e c o m p l e x w i t h t h e 50 S s u b u n i t o r w i t h t h e r e m a i n i n g 70 S w a s v i s i b l e . T h e r e w a s a b o u t 1 m o l e c u l e of I F 3 b o u n d p e r 30 S r i b o s o m e .

BIOCHIMIE, 1972, 54, n ° 7.

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4 34 48

The reaction m i x t u r e c o n t a i n e d in 50 Ixl, the following : Tris-HC1 pH 7.4,5.10-2 M, Magnesium acetate : 8.10~3 M, ~ M e r e a p t o - e t h a n o l 7.10-~ M, GTP : 10-3 M, 3HfMet tRNA : 20 p Moles (600 e.p.m/pmole), pAUG. 0.2 A~,~,m unit. For n a t u r a l messengers, T:~ mRNA or R~rRNA : 2 A~.m units, IF~ p a r t i a l l y purified : 20 p,g, IF= p a r t i a l l y purified : 50 'Bxg, IF~ : 1 ~g.

As a l r e a d y s t a t e d , w h e n 35S-IF 3 is i n c u b a t e d w i t h 70 S r i b o s o m e s , o r w i t h d e r i v e d 5,0, S s u b u n i t s at t h e Mg +~ c o n c e n t r a t i o n a p p r o p r i a t e to t h e d i s s o c i a t i o n s t u d i e s (5.10 -3 M) n o a p p r e a c i a b l e b i n d i n g t o 50 S is n o t i c e a b l e . B y c o n t r a s t , w h e n t h e a s S - I F 3 p r e p a r a t i o n ~vas m i x e d w i t h 70 S r i b o somes which had been artificially dissociated at l o w Mg ++ (10 -4 M), a p p r e c i a b l e b i n d i n g of r a d i o a c t i v i t y to t h e 50 S s u b u n i t s c o u l d b e d e t e c t e d a f t e r s e d i m e n t i n g o n s u c r o s e g r a d i e n t (fig. 4). S i n c e o u r f a c t o r p r e p a r a t i o n is at l e a s t 80 p. c e n t p u r e a n d t h e p e r c e n t of b o u n d r a d i o a c t i v i t y c l e a r l y e x c e e d s 20 p. c e n t of t h e a m o u n t i n i t i a l l y i n t r o d u c e d , a n a p p r e c i a b l e f r a c t i o n of t h e 50 S b o u n d r a d i o a c t i v e m a t e r i a l m u s t b e e q u i v a l e n t to I F 3.

ssS-IF s release during initiation. I n o r d e r to s t u d y t h e f a t e of 30 S b o u n d I F 3 i n t h e c o u r s e of i n i t i a t i o n t h e f o l l o w i n g e x p e r i m e n t w a s d o n e (fig. 5) : 7,0 S r i b o s o m e s w e r e i n c u b a t e d w i t h a n a m o u n t of 35S-IF 3 s u f f i c i e n t to c a u s e 8,0 p. c e n t d i s s o c i a t i o n ; p u r i f i e d u n l a b e l l e d IF1, 1F 2 p l u s G T P , nH-fMet-tR.NA a n d m e s s e n g e r R,NA ( p o l y AUG, T 4 o r R17 m e s s e n g e r h a v e b e e n u s e d ) were then added to the predissociated mixture. T h a t t h i s r e s u l t e d i n t h e f o r m a t i o n of a n i n i t i a tion complex was demonstrated in two ways:

R a d i o a c t i v e f a c t o r IFs a n d r i b o s o m e s . F i r s t , aH-fMet-tRNA w a s s h o w n to r e a c t r e a d i l y w i t h p u r o m y c i n to f o r m a r a d i o a c t i v e ZH-fMetp u r o m y c i n p r o d u c t (table I). M o r e o v e r , r a d i o a c t i v i t y f r o m ~H-fMet-tRNA w a s f o u n d at t h e p o s i t i o n of t h e 70 S p e a k o n a s u c r o s e g r a d i e n t . In c o n t r a s t , no a~S r a d i o a c t i v i t y c o u l d be d e t e c t e d at t h e 70 S l o c a t i o n b u t c o u l d be r e c o v e r e d as f r e e (i.e. s l o w l y s e d i m e n t i n g ) z~S-IF s. A c o n t r o l s a m p l e in w h i c h '~sS-I,F,~ w a s i n c u b a t e d w i t h 70 S r i b o -

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Fro. 6. - Effect of neomgcin, Streptomycin and Kasugamgcin on 70 S (IFs mediated) dissociation and ssS-IF~ binding. One A 260 nm unit of 70 S 1 M NH4CI 'washed ribosomes was mixed on the usual dissociation condition with 0.1 mM Kasugamycin, Streptomycin or Neomycin, as described in the text. After incubating 2 rain at 4°C, 1 ~g 35S-IF.~ was added and incubation proceded for 15 min at 37°C. Samples were analysed as usual..

s o m e s w i t h o u t f u r t h e r a d d i t i o n of the o t h e r p r o t e i n f a c t o r s a n d i n i t i a t i o n e f f e c t o r s but m i n u s I F 2 w a s also s e d i n l e n t e d . An a p p r e c i a b l e a m o u n t of a~s label w a s f o u n d , as a l r e a d y d e s c r i b e d , at t h e p o s i t i o n of d i s s o c i a t e d 30 S s u b u n i t s . C a l c u l a t i o n i n d i c a t e d t h a t m o r e :~S r a d i o a c t i v i t y w a s f o u n d in the s u p e r n a t a n t a f t e r i n i t i a t i o n t h e n in the c e n t r a l s a m p l e in w h i c h I F e w a s l a c k i n g , s u g g e s t i n g o n c e a g a i n t h a t IF:~ is rel e a s e d f r o m the 30 S in the c o u r s e of its reassoc i a t i o n w i t h t h e 50 S s u b u n i t ,

833

p l e t e l y i n t e r f e r w i t h t h e IF~ c a t a l y s e d ¢ dissoc i a t i o n >> of 70 S r i b o s o m e s , t h e i n h i b i t i o n r e a c h i n g 70 a n d 100 p. c e n t r e s p e c t i v e l y . A n a l y s i s of tile .~.~,S-IF3 b o u n d to t h e 3,0 S s u b u n i t in tlle c o u r s e of <> i n d i c a t e d t h a t t h e a m o u n t of :~sS-IFs-30 S c o m p l e x is r e d u c e d in t h e s a m e p r o p o r t i o n as t h e e x t e n t of d i s s o c i a tion. W i t h n e o m y c i n , in p a r t i c u l a r , t h e w h o l e :~5S l a b e l is f o u n d in t h e s u p e r n a t a n t p o r t i o n of the g r a d i e n t . It is i n t e r e s t i n g , h o w e v e r t h a t n e i t h e r s t r e p t o m y c i n n o r n e o m y c i n i n h i b i t 35S-IF:~ b i n d i n g to <> 30 S s u b u n i t s . T h i s s i t u a t i o n w i l l be f u r t h e r c o m m e n t e d u p o n i n t h e <> s e c t i o n . K a s u g a m y c i n , e v e n at doses i n h i b i t i n g f a c t o r - d e p e n d e n t b i n d i n g of f M e t - t R N A to a r i b o s o m e - A U G c o m p l e x , affects n e i t h e r t h e <~dissoc i a t i o n >> n o r t h e b i n d i n g of '~sS-IF 3 to t h e r e s u l t i n g 30 S s u b u n i t . F i n a l l y , t h e effect of c o l i e i n E.z w h i c h is k n o w n to s e v e r e l y i n h i b i t in vitro t r a n s l a t i o n of n a t u r a l m e s s e n g e r R N A ' s , w i t h o u t a p p r e ciably interfering with poly U-directed polyphen y l a n i n e s y n t h e s i s [16] w a s also i n v e s t i g a t e d . At doses w h i c h c a u s e d a m o r e t h a n t w o - f o l d r e d u c t i o n in t h e e x t e n t of A U G - d e p e n d e n t i n i t i a t o r t R N A b i n d i n g , c o l i c i n E~ e x h i b i t e d no a p p r e c i a b l e effect on t h e I F z - p r o m o t e d ¢ d i s s o c i a t i o n ~ o r on 3~S-IF:~ b i n d i n g to t h e 30 S s u b u n i t .

DISCUSSION.

Effect of certain initiation reaction inhibitors on s~S-1F s binding to ribosomes. S i n c e the r e s u l t s so far d e s c r i b e d s l m w t h a t IFa is able to b i n d t h e 30 S s u b u n i t , a n d to c a u s e t h e d i s s o c i a t i o n of a 70 S d i m e r o r p r e v e n t its r e f o r m a t i o n f r o m t h e s u b u n i t s , t h e effect of cert a i n i n h i b i t o r s of t h e i n i t i a t i o n r e a c t i o n w a s investigated.

I F l [4] o r I F u [5, 61 w h e n a d d e d s i n g l y to 70 S o r 30 S c a n n o t c o m p l e x t h e s e r i b o s o m e s in a f o r m w h i c h can s u s t a i n s u c r o s e g r a d i e n t s e d i m e n t a t i o n w i t h o u t the p r e s e n c e of o t h e r c o m p o n e n t s of the i n i t i a t i o n r e a c t i o n . I n c o n t r a s t , IF:~ c a n b i n d by itself t h e 30 S m o i e t y , w h e t h e r t h i s s u b u n i t is a r t i f i c i a l l y <~d e r i v e d >> or p r o d u c e d d u r i n g f a c t o r n l e d i a t e d 70 S <>. T h e a m o u n t of r a d i o a c t i v e IF:~ b o u n d p e r 30 S c o r r e s p o n d s to a I:l ratio taking into account the specific radioa c t i v i t y of t h e f a c t o r a n d a s s u m i n g a m o l e c u l a r w e i g h t of a b o u t 21,5,00. T h i s r e s u l t is in g o o d a c c o r d a n c e w i t h p r e v i o u s l y p u b l i s h e d d a t a of Sabol a n d O c h o a [9]. T h a t IF:~ c a n d i r e c t l y b i n d to the 30 S s u b u n i t w h i l e t h e o t h e r f a c t o r s o n l y do so i n d i r e c t l y suggests that f o r m a t i o n of a<< 30 SIF:~ c o m p l e x >> is a p r e r e q u i s i t e for t h e I F 2 catal y s e d f o r l n a t i o n of an i n i t i a t i o n c o m p l e x .

Anmng these inhibitors some anfinoglycosidic a n t i b i o t i c s k n o w n to i n t e r f e r e w i t h the f o r m a t i o n , o r t h e stability of the i n i t i a t i o n c o m p l e x s u c h as s t r e p t o m y c i n [13, 141, n e o m y c i n a n d k a s u g a m y c i n [15] w e r e first e x a m i n e d . As s h o w n figure 6, strept o m y c i n and n e o m y c i n at 10 -a M s t r o n g l y , or c o m -

A l t h o u g h no a p p r e c i a b l e b i n d i n g of r a d i o a c h v e IF.~ to t h e 50 S s u b u n i t a p p e a r s to t a k e p l a c e u p o n r i b o s o m a l d i s s o c i a t i o n , f o r m a t i o n of a 50 S-IF:~ c o m p l e x c l e a r l y o c c u r s at l o w Mg +÷ (10 -4 M). S i n c e lF:i is a b a s i c p r o t e i n , this r e s u l t p o s s i b l y suggests a w e a k i n t e r a c t i o n b e t w e e n IF:; a n d the 50 S t h a t

BIOCHIMIE, 1972, 54, n ° 7.

834

Jean Thibault, A n n i c k Chestier, D o m i n i q u e Vidal et F r a n f o i s Gros.

w o u l d be c o m p e t e d by d i v a l e n t cations. W h e t h e r this 50 S-IF 3 is of i m p o r t a n c e d u r i n g "the ribosomal d i s s o c i a t i o n step is u n k n o w n . The present work touches upon another important aspect of the IF~-ribosome i n t e r a c t i o n since it is o b s e r v e d that the d i s s o c i a t i o n step, 30 Sb o u n d I F 3 is s u b s e q u e n t l y released f r o m the complex that r e f o r m s u p o n the c o m b i n e d a d d i t i o n of a 50 S s u b u n i t plus the r e q u i s i t e c o m p o n e n t s of the i n i t i a t i o n action. It is not yet c le a r h o w e v e r if the t r i g g e r f or this release is the 3,0 S-50 S j u n c t i o n per se or some o th e r e v e n t a c c o m p a n y i n g the b u i l d i n g up of the i n i t i a t i o n c o m p l e x . The p o s s i b i l i t y could also exist that d u r i n g the posit i o n i n g of fMet-tRNA on the m e s s e n g e r - r i b o s o m e c o m p l e x , IF3-30 S i n t e r a c t i o n is o n l y w e a k e n e d , release b e i n g actually caused by the h y d r o s t a t i c p r e s s u r e i m p o s e d d u r i n g zonal c e n t r i f u g a t i o n . It must be r e c a l l e d that I F 1 a n d IF.~ are also released f r o m r i b o s o m e s at some stage d u r i n g the fMettRNA a t t a c h m e n t to r i b o s o m e s [4, 5, 6]. As far as the m e c h a n i s m of the I F 3 - p r o m o t e d 7:0 S <5 is c o n c e r n e d , our results and those f r o m p u b l i s h e d w o r k [9] i n d i c a t e that dissoc iat i o n is cl ear l y c o n c o m i t a n t w i t h the f o r m a t i o n of a stable c o m p l e x b e t w e e n IF a a n d the 30 S sub u n i t an d not m e r e l y caused by some c a t a l y t i c i n t e r a c t i o n b e t w e e n I F 3 a n d the 70 S d i m e r : for each d i s s o ci at ed 70 S p a r t i c l e one finds a p p r o x i m a t e l y one I F 3 molecu-le a t t a c h e d to the 30 s subunit. Moreover, agents s u c h as s t r e p t o m y c i n inhib i t 70 S d i s s o c i a t i o n a n d the a p p e a r a n c e of a 35S-IF~ c o m p l e x to the same extent. K a s u g a m y c i n w h i c h has no effect on d i s s o c i a t i o n has no effect on IFa b i n d i n g . A p o i n t that deserves c o m m e n t is the fact that s t r e p t o m y c i n and n e o m y c i n s t r o n g l y i n h i b i t the IF:~ cat al y s ed d i s s o c i a t i o n but h a v e v e r y little effect w h e n 3~S-IFa is m i x e d w i t h p r e e x i s t i n g 30 S. T h i s result could be e x p l a i n e d by a s s u m i n g that I F s c a n n o t p r i m a r i l y i n t e r a c t w i t h the r i b o s o m a l target as long as the 30 S subunit is c o m b i n e d w i t h the 50 S. The s t i m u l a t o r y IF a effect on <> w o u l d r e s u l t f r o m s w i t c h i n g the equil i b r i u m b e t w e e n the d i m e r and its subunits I8], r a t h e r than i n d u c i n g a c o n f o r m a t i o n a l change of the dimer. Since n e o m y c i n , for i n s t a n c e is precisely k n o w n to i n c r e a s e the stability of 7.0 S ribosomes at Mg +÷ c o n c e n t r a t i o n w h i c h o t h e r w i s e f a v o u r the p r e d o m i n a n c e of 50 S and 30 S over 70 S I17], it w o u l d m e r e l y p r e v e n t the <5 a p p e a r a n c e of 30 S subunits w h i c h r e p r e s e n t the t r u e target for IF~ b i n d i n g . If one c o n s i d e r s the opposite m o d e l [1.8] n a m e l y that I F 3 i n d u c e s d i s s o c i a t i o n by d i r e c t l y i n t e r a c t i n g

BIOCHIMIE, 19'72, 54, n ° 7.

w i t h <5 70 S one is led to a m i n o g l y c o s i d i c a n t i b i o t i c s do not same alteration on the 30 S w h e n this in its <> as w h e n it is

t h i n k that cause the subunit is <5.

Effects of a m i n o g l y c o s i d i c a n t i b i o t i c s on dissoci at i o n and 35S-IF~ b i n d i n g could be i n t e r p r e t e d in t w o w a y s : the a n t i b i o t i c s could p r e v e n t IF a access to the 3 0 S m o i e t y of the diiner or else they could p r e v e n t s p o n t a n e o u s dissociation of 70 S ribosomes [17] and thus i n h i b i t the a p p e a r a n c e of free 30 S subunits w h i c h w o u l d constitute the only r i b o s o m a l target w i t h w h i c h IF 8 interacts. It is i n t e r e s t i n g to n o t i c e in this r e s p e c t that n e i t h e r s t r e p t o m y c i n n o r n e o m y c i n caused in our h a n d s any i n h i b i t i o n of s5S-IF:~ b i n d i n g to p r e e x i s t i n g 30 S ribosomes. This could be i n t e r p r e t e d to m e a n that IF:: c a n n o t i n t e r a c t w i t h its r i b o s o m a l target as l o n g as 30 S r i b o so m es are b o u n d to 59 S subunits. It must be noted that in t h ei r r e c e n t paper, Pon

et al. [10] have o b t a i n e d data c o n f l i c t i n g w i t h our o w n since they o b s e r v e i n h i b i t i o n by streptomycin or n e o m y c i n of 14CH3-IF:~, b i n d i n g to 30 S ribosomes. Although the d i s c r e p a n c y is not yet fully u n d e r s t o o d , it m i g h t be related to the fact that in e x p e r i m e n t s by P o n et al. [10] r i b o s o m e s w e r e prei n c u b a t e d w i t h the antibiotics for 10 nfin at 30°,C b e f o r e the r i b o so m es w e r e m i x e d at the same temp e r a t u r e w i t h r a d i o a c t i v e IFa w h i l e in our case the antibiotics w e r e i n t r o d u c e d only 2 rain before r a d i o a c t i v e IF;~ at 4°,C and the b i n d i n g m i x t u r e w as f u r t h e r i n c u b a t e d at 37°C for 15 rain before zonal centrifugation. W i t h r e s p e c t to the p a r t of the 3.0 S r i b o s o m e to w h i c h IF 3 does b i n d still little is known. Prel i m i n a r y results i n d i c a t e that at 5.10-~ M. Mg ÷+, 35S-IF 3 r e a d i l y b i n d s p u r i f i e d 1,6 S RNA ~qth a s t o i c h i o m e t r y close to one m o l e c u l e p er RNA c h a i n [6] but since I F 3 also tightly b i n d s messenger RNA, the significance of this is difficult to assess. Use of a n t i b o d i e s against s p e c i f i c ribosomal p r o t e i n s m i g h t shed some light on the problem. W o r k along these lines is actually in progress.

A cknorwledgements. We thank Dr. P. Cornuet for his generous gift of R17 RNA. This work was supported by grants from the Fonds de D~velop,pement de la Recherche Scientifique et Technique, the Cmnmissariat h l'Energie AtOmique, the Centre National de la Recherche Seientifique, the Ligue Nalionale Fran~aise contre Ie Cancer, and the Fondation pour la Recherche M~dicale Fran~aise.

Radioactive

factor IFz and ribosomes.

RI~SUM~. Le p r 6 s e n t t r a v a i l dderit la p r S p a r a t i o n d ' u n des f a c t e u r s d ' i n i t i a t i o n de la s y n t h 6 s e p r o t S i q n e , le fact e u r IF:~ h l'6tat de c o m p o s 6 m a r q u 6 a u 35S. iF:~-35S a 6t6 o b t e n u p u r h 80 p. cent. Les m o d a l i t d s qui p r d s i d e n t h l ' a t t a c h e m e n t d'IF~-35S a u x r i b o s o m e s d'E. coli s o n t prScisdes grfice h des dtudes de sg,d i m e n t a t i o n e n g r a d i e n t de s a c c h a r o s e . IF~-35S f o r m e u n c o m p l e x e s t a b l e avec les s o u s - u n i t d s 30 S p r S a l a b l e m e n t d 6 b a r r a s s 6 e s de l e u r s f a c t e u r s endog~nes. L o r s q u e la c o n c e n t r a t i o n e n i o n s Mg ++ c o r r e s p o n d h celle raise en oeuvre p o u r la f o r m a t i o n du c o m p l e x e d ' i n i t i a t i o n , soil 5.10 ~ M, on n ' o b s e r v e a u c u n a t t a c h e m e n t a u x s o u s - u n i t 6 s 50 S. P o u r de f a i b l e s c o n c e n t r a t i o n s , 2.10-4 M, on ddc61e p a r c o n t r e u n e f i x a t i o n n o t a b l e . Apr6s m d l a n g e d'IFa-35S et de r i b o s o m e s 70 S, on o b s e r v e u n e fixation a u x s o u s - u n i t 6 s 30 S r d s u l t a n t de l'effet d i s s o c i a n t du f a c t e u r . II se fixe e n v i r o n 1 m o l 6 c u l e d'IFa-3.~S p a r r i b o s o m e 30 S. Apr6s f o r m a t i o n d ' u n c o m p l e x e d ' i n i t i a t i o n s u r les r i b o s o m e s 70 S, le f a c t e u r IFa se t r o u v e 6jectd. En p r 6 s e n e e de s t r e p t o m,vcine ou de n ~ o m y c i n e q u i i u h i b e n t la d i s s o c i a t i o n des r i b o s o m e s 70 S, on n ' o b s e r v e p a s la f o r m a t i o n de c o m p l e x e s 30 S - IF:~-:a5S. Ces m ~ m e s a n t i b i o t i q u e s , p a s p l u s d ' a i l l e u r s que la k a s u g a m y c i n e ou la coliciue E.~ n ' i n h i b e n t la tixation d'IF~, r a d i o a c t i f s u r les s o u s u n i t 5 30 S p r d a l a b l e m e n t isoldes. ZUSAMMENFASSUNG.

Die v o r l i e g e n d e Arheit b e s e h r e i b t die D a r s t e l l u n g eines d e r I n i t i a t i o n s f a k t o r e n d e r P r o t e i n s y n t h e s e , des F a k t o r s IF~,, als eine m i t a s s m a r k i e r t e n V e r b i n d u n g . 35S-IF~ ist 80 p. cent r e i n e r h a l t e n w o r d e n . Die Modalitiiten der B i n d u n g y o n 35S-IF:~ a n die R i b o s o m e n yon E. coli w e r d e n m i t t e l s U n t e r s u c h u n g e n d u r c b S e d i m e n t a t i o n i m S a c c h a r n s e g r a d i e n t n~iher b e s t i m m L 35S-1F:~ bildet e i n e n s t a b i l e n K o m p l e x m it den 30 S - U n t c r c i n heiten, w e l c h e z u e r s t v o n ihcen c n d o g e n e n F a k t o r c n g e t r e n n t w u r d e n . W e n n die t ( o n z e n t r a t i o n a n Mg +~I o n e n d e r j e n i g e n e n t s p r i e h t , w e l c h e ftir (tie B i l d u n g des I n i t i a t i o n s k o m p l e x e s v e r w e n d e t w i r d , d.h. 5.1(}-'3 M, so b e o b a e h t e t m a n :keine B i n d u n g a n die 50 S - U n t e r e i n h e i t e n . Fiir s c b w a c h e K o n z e n t r a t i o n e n , 2.10 4 M, w e i s t m a n h i n g e g e n eine h e d e u t e n d e B i n d n n g naclL Nach M i s c h u n g von 35S-IF:, m i t 7{) S - R i b o s o m e n

BIOCHIM1E, 1972, 54, 11" 7.

835

b e o b a c h t e t m a n eine B i n d u n g a n die 30 S - U n t e r e i n h e i t e n , ~velcbe d u r c h die d i s s o z i i e r e n d e W i r k u n g des F a k t o r s v e r u r s a c h t w i r d . Es b i n d e r sich u n g e f S h r ein Molekiil 35S-IF~ p r o 30 S - R i b o s o m . N a c h d e r B i l d u n g eines l n i t i a t i o n s k o m p l e x e s a u f die 70 S - R i b o s o m e n w i r d d e r F a k t o r IF.~ a u s g e w o r f e n . I n G e g e n w a r t y o n S t r e p t o m y c i n oder N e o m y c i n , w e l c h e die D i s s o z i a t i o n der 70 S - R i b o s o m e n h e m l n e n , b e o b a c h t e t m a n k e i n e B i l d u n g v o n 30 S-35S-IF~-Komlplexen. D i e s e l b e n Antib i o t i k a , wie a u c h das K a r u g a m y c i n oder d a s E~-Colicin, i n h i b i e r e n die B i n d u n g des r a d i o a k t i v e n IF:, a n die v o r h e r i s o l i e r t e n 30 S - E i n h e i t e n nicht. REFERENCES. 1. Revel, M., Lelong, J. C., B r a w e r m a n n , G. & Gros, F. (1968) Nature, 219, 1016. 2. I w a s a k i , K., Sabol, S., W a h b a , A. J. & Ochoa, S. (1968) Arch. Biochem. Biophys., 125, 542. 3. H e r s h e y , S. W. B., R e n i o l d O ' D o n n e l , E., K o l a k o f sky, D., Dewey, K. F. & T h a e h , R. E. (1971) Methods in E n z y m o l o g y , S. P. Colowick a n d N. O. K a p l a n Eds, 20, 235. 4. H e r s h e y , S. W. B., Dewey, K. F. & T h a e h , R. E., (1969) Nature, 222, 944. 5. T r a u t , P e r s o n a l c o m m u n i c a t i o n . 6. Thil)ault, J. & Gros, F. U n p u b l i s h e d w o r k . 7. Sabol, S., Sillero, M. A. G., I w a s a k i , K. & Ochoa, S. (1970) Nature, 2'.28, 12'69. 8. K a e m p f e r , R. (1970) Nature, 534. 9. Sabol, S., & Ochoa, S. (1971) Nature N e w Biology, 234. 10. P o n , C. L., F r i e d m a n , S. M. & G u a l e r z i , C. (1972) M.G.G., 116, 192. 11. Marker, K. ,6 S a n g e r , F. (1964) J. Mol. Biol., 8, 835. 12. Noll, H. P e r s o n a l c o m m u n i c a t i o n . 13. Modolell, J. & Davies, B. D. (1970) Proc. U. S. Nat. Acad. Sci., 67, 1148. 14. Lelong, J. C., Cousin, M., G r u n b e r g - M a n a g o , M. Gros, F. (1971) Biochem. and Biophys. Res. Commun., 42, 530. " 15. O k u y a m a , A., M a c h i y a m a , N., K i n o s h i t a , T. &T a n a k a , N. (1971) Biochem. and Biophys. Res. Corn., 43, 196. 16. Boon, T. (1971) Proc. U. S. Nat. Acad. Sci., 68, 2421. 17. S p i r i u , A. S. a L i n e v s k a y a , E. B. (1971) F.E.B.S. Letters, 14, 114. 18. S u b r a m a n i a n , A. R., Davies, B. 1). & Beller, R. J. (19(19) (:old. Spr. Harb. Syrup. Quanl. Biol., 34, 223.