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The binding of Escherichia coli ribosomes to matrix bound messenger RNA
BIOCHIMIE, 1'972, 54, 823-827.
The binding of Escherichia coli ribosomes to matrix bound messenger RNA Jean PETriE, Alex BOLLEN, P i e r r e NOKIN an d H e n r i GROSJEAN.
D e p a r t m e n t o[ Molecular Biology. University of Brussels, Rue des Chevaux, 67-B-1640 Rhode Saint Genbse, Belgium. (17-6-1972).
Summary. - - The formation of a hydrazone derivative between periodate oxidized RNA and a linear polymer of acrylic hydrazide entrapped in an agar gel affords a convenient method to prepare a resin bearing chemically eoupled synthetic messenger RNA. The use of such an RNA derivative instead of the free messenger rrmkes it possible to isolate 30S or 70S ribosomes involved in the initiation complex programmed by poly(A,G,U) as messenger, and of 70S ribosomes bound to a poly(U) template. The complex isolated is specific and, as judged by the puromycin reaction, it retains a high activity to transfer bound N-blocked anainoaeyl-transfer RNA. Therefore, this method appears an unique way to prepare a population of active ribosomes presumably homogeDeous with respect to their functional state. INTRODUCTION. A c o n s i d e r a b l e a m o u n t of w o r k has been devoted for m a n y years to the k n o w l e d g e of the successive steps of p o l y p e p t i d e b i o s y n t h e s is , and the different c o m p l e x e s i n v o l v e d in the i n i ti a t io n , elongation an d t e r m i n a t i o n m o d e s of the ribosomes have been d e s c r i b e d by m a n y w o r k e r s . Until now, the c h a r a c t e r i z a t i o n of these com.plexes and the d e m o n s t r a t i o n of t h e i r f u n c t i o n w a s a c h i e v e d e i t h e r by d e n s i t y g r a d i e n t u l t r a c e n t r i f u g a t i o n or by n i t r o c e l l u l o s e f i l t e r b i n d i n g assay. These t wo methods, in spite of t h e i r w i d e use and r e l i a b i l i t y , suffer f r o m some m a j o r limit a t i o n s ; a l t h o u g h the f o r m e r allows s o m e t i m e s the isolation of c o m p l e x e s for f u r t h e r p r o c e s s i n g , the latter i r r e v e r s i b l y destroys them. F u r t h e r more, none of these m e t h o d s enables the separation of the active c o m p l e x , w h i c h usually i n v o l v e s only a few p e r c e n t s of the r i b o s o m e s engaged, f r o m the bulk of p a r t i c l e s that did not p a r t i c i pate to the r e a c t i o n assayed. C o n s i d e r i n g these facts, one of ns (H.G.) propos ed a n e w m e t h o d of i n v e s t i g a t i o n based on the p r o p e r t i e s of m e s s e n g e r RNA c h e m i c a l l y c ou p l ed to an insoluble support.
Abbreviations : EDTA, ethylenediamine tetra-acetic acid. poly(U), polvuridylic acid. poly(A,G,U),'poly adenylic, guanylie, uridylie aeid. Met-tRNA, methionyl-transfer RNA. Phe-tRNA, phenylalanyl-transfer RNA. fMet-tRNA, N-formylmethionyl-transfer RNA. N-aeetylPhe-tRNA, N-aeetylphenylalanyl-tran sfev RNA. GTP, guanosine triphosphate.
The basic p r o c e d u r e , d e s c r i b e d by K n o r r e and c o w o r k e r s [1~, i n v o l v e s the f o r m a t i o n of a h y d r a zone d e r i v a t i v e b e t ~ ' e e n p e r i o d a t e o x i d i z e d RNA and a l i n e a r p o l y m e r of a c r y l i c h y d r a z i d e e n t r a p p ed in an agar gel. This c h e m i c a l c o u p l i n g features a v e r y high stability in d i f f er en t pH, i o n i c strcnth and t e m p e r a t u r e c o n d i t i o n s [2] and the RNA d e r i v a t i v e r e m a i n s c o m p l e t e l y u n d a m a ged after i n c u b a t i o n in the s t a n d a r d buffers used for assays in p o l y p e p t i d e s b i o sy n t h esi s. The m e t h o d has been a p p l i e d before to the p a r t i a l p u r i f i c a t i o n of an a m i n o a c y l - t R N A ligase by affinity c h r o m a t o g r a p h y on a c o l u m n c h a r g e d w i t h a speeific t r a n s f e r RNA [3]. A c o l u m n charged w i t h polyIU) w a s also used to d e m o n s t r a t e the specific r e t a r d a t i o n of Phe-tRNA by codona n t i e o d o n p a i r i n g [4]. In this p a p e r , we s h o w that this resin c h a r g e d w i t h s y n t h e t i c m e s s e n g e r RNA m a y actually be used i n st ead of the free template to p r o g r a m the a c t i v i t y of ribosomes. Since in this ease, the only r i b o s o m e s that are b o u n d to the linked m e s s e n g e r are r e t a i n e d on the resin, this p r o c e d u r e p r o v i d e s a v e r y efficient w a y to isolate active c o m p l e x e s i n v o l v i n g m e s s e n g e r RNA, 30S or 70S r i b o s o m e s and o t h e r ligands.
MATERIALS AND METHODS.
Escherichia colt (strain B) r i b o s o m e s and initiation factors from strain MRE 600 w e r e p r e p a red a c c o r d i n g to N o m u r a et al. [5J ; labelled ribosomes w e r e o b t a i n e d f r o m cells g r o w n on m i n i -
824
Jean Petre, A l e x Bollen, Pierre N o k i n a n d H e n r i Grosjean.
mal m e d i u m s u p p l e m e n t e d w i t h labelled a m i n o acids (14C or all). Ribosomal s u b u n i t s w e r e o b t a i n e d by g r a d i e n t c e n t r i f u g a t i o n in low Mg2÷ buffer, p r e c i p i t a t e d w i t h e t h a n o l [6] a n d dialyzed against buffer 1. They were r o u t i n e l y reactivated in the p r e s e n c e of a m m o n i u m chloride before use [7].
E. colt B t r a n s f e r RNAs (Schwarz Bioresearch) were r o u t i n e l y acylated w i t h m e t h i o n i n e (14C) or p h e n y l a l a n i n e (14C) ; fMet-tRNA (Met-14C) was o b t a i n e d b y e n z y m i c f o r m y l a t i o n of Met-(14C)tRNA [8], a n d the u n f o r m y l a t e d p r o d u c t s were deacyluted by t r e a t m e n t w i t h 1 mM CuSO 4 [9]. The final p r o d u c t s were purified by DEAE-celhdose c h r o m a t o g r a p h y and ethanol p r e c i p i t a t i o n . Na c e t y l P h e 4 R N A (Phe-SH) was k i n d l y supplied by Dr. J.-C. L.elong. Adenosine was oxidized by s o d i m n m e t a p e r i o date a n d passed t h r o u g h an a n i o n exchange resin to remove the excess reagents. Poly(U) and poly(A,G,U) (Boehringer M a n n h e i m ) were oxidized by the method of K n o r r e el al. [1], p r e c i p i t a ted w i t h e t h a n o l a n d stored in 10 mM s o d i u m acetate buffer (pH 4.8). The synthesis of p o l y a c r y l i c h y d r a z i d e and the i n c l u s i o n of the p o l y m e r into an agar gel was p e r f o r m e d as d e s c r i b e d by K n o r r e el al. [1?, but the final p o l y m e r c o n c e n t r a t i o n was b r o u g h t to 1 p. cent ( w / w ) . The r e s i n was sieved twice t h r o u g h a 60 mesh screen a n d stored in cold water. It w a s washed for at least 14 hours before use, until no free h y d r a z i n e could be detected i n the eluate. The c o u p l i n g reaction w i t h oxidized RNA was c a r r i e d out in the following c o n d i t i o n s : 0.2-0.35 g (wet weight) bed was dispersed inta a final volume of 1 ml c o n t a i n i n g 0.2 M sodium acetate buffer (pH 4.8), 20 mM m a g n e s i u m acetate and 1214 a b s o r b a n c y u n i t s (258 nm) of the oxidized RNA. The m i x t u r e was vigorously stirred at 4 ° for at least 3 h o u r s and p r e f e r a b l y overnight ; 8090 p. cent of the RNA was u s u a l l y coupled to the resin u n d e r these conditions. In o r d e r to block the excess of h y d r a z i d e groups, the resin was then s t i r r e d in 1 ml of the above buffer c o n t a i n i n g 250 a b s o r b a n c y u n i t s of oxidized a d e n o s i n e ; it was finally w a s h e d a n d e q u i l i b r a t e d before use in buffer 1 or 4. The f o r m a t i o n of the i n i t i a t i o n complex on resin coupled poly(A,G,U) a n d the b i n d i n g of 70S ribosomes to the poly(U) derivative were performed as described in the text ; following incubation, the sample was chilled, t r a n s f e r r e d into a small c o l u m n and washed w i t h 10-15 ml of buffer 1 or 4. The complex was r o u t i n e l y eluted
BIOCH1MIE, 1972, 54, n ° 7.
by buffer 2 (15 mM EDTA) at 45 m l / h flow rate, or by buffer 3 (0.1 mM Mg2+) ; in the latter case, the flow rate was r e d u c e d to 4.5 m l / h to allow complete recovery of the ribosomes from the column. 0.6 ml fractions were c o u n t e d in T r i t o n X-100 based scintillation fluid.
Buffers. Buffer 1 : Tris-C1 10 mM (pH 7.8), NH4C1 30 mM, Mg acetate 10 mM, m e r c a p t o e t h a n o l 6 mM. Buffer 2 : Tris-C1 10 mM (pH 7.8), NH4C1 30 mM, EDTA 15 raM, m e r c a p t o e t h a n o l 6 raM. Buffer 3 : Tris-C1 10 mM (pH 7.8), NH4C1 30 raM, Mg acetate 0.1 mM, m e r c a p t o e t h a n o l 6 mM. Buffer 4 : T r i s - C 1 10 mM (pH 7.8), NH4C1 160 mM, Mg acetate 10 raM, m e r c a p t o e t h a n o l 6 raM. RESULTS AND DISCUSSION.
1. Formalion and specificity of the iniliulion complex on resin bound poly (A, G, U). The f o r m a t i o n of the i n i t i a t i o n c o m p l e x requires the p r e s e n c e of p r o t e i n factors a n d involves a specific i n t e r a c t i o n b e t w e e n messenger RNA a n d 30S subparticles, followed by the b i n d i n g of i n i t i a t o r tRNA a n d GTP. The large subparticle then joins this complex a n d makes it competent to accept an aminoacyl-tRNA a n d to achieve the s y n t h e s i s of the first p e p t i d e b o n d [for a review, see ref. 10]. The first step i n v o l v i n g the i n t e r a c t i o n between 30S ribosomes and messenger RNA, it is expected that all the i n t e r m e d i a t e s o c c u r r i n g in the above process should be isolable as resin b o u n d products b y the bias of messenger RNA. In o r d e r to check this point, the a b i l i t y of resin b o u n d poly (A, G, U) to p r o g r a m the formation of the messenger RNA : 30S : GTP : fMet-tRNA complex i n the presence of crude i n i t i a t i o n factors w a s assayed. After w a s h i n g any u n b o u n d material, the complex was recovered by elution with a low m a g n e s i u m buffer (fig. la) or b y EDTA t r e a t m e n t (fig. l b ) ; the latter method, w h i c h is expected to p r o d u c e some more drastic effects on the breakage of tile complex, did not release more r a d i o a c t i v i t y t h a n the low m a g n e s i u m b u f f e r , although a n o t i c e a b l e a m o u n t of r a d i o a c t i v e material r e m a i n e d b o u n d after the successive elution steps. F i g u r e l b clearly shows that the residual r a d i o a c t i v i t y is released only after r i b o n u c l e a s e t r e a t m e n t ; however, since it is o b t a i n e d in irrep r o d u c i b l e amounts even i n columns l a c k i n g messenger RNA (fig. lc), we suspect that it corresp o n d s to unspecific aggregation product~ of degraded or n o n r i b o s o m a l c o n t a m i n a t i n g material.
B i n d i n g of r i b o s o m e s to m a t r i x b o u n d m R N A . I n o r d e r to p r o v e t h a t t h e c o m p l e x f o r m e d o n t h e r e s i n b o u n d m e s s e n g e r R N A c o r r e s p o n d s to a true initiation intermediate, we checked several of i t s c h a r a c t e r i s t i c s , n a m e l y t h e r e q u i r e m e n t f o r initiation factors, initiator tRNA and AUG codon.
825
Since ribosomes used in this experiment were not salt washed, they do not exhibit a strong requirement for added initiation factors for bind i n g f M e t - t R N A . H o w e v e r , m u c h m o r e 30S p a r t i c l e s b i n d to t h e m e s s e n g e r i n t h e a b s e n c e of i n i tiation factors than in the complete system, wher e a s t h e l e v e l of t R N A b i n d i n g is d e c r e a s e d u n d e r these conditions. Both observations suggest that t h e i n h i b i t i o n of n o n - i n i t i a t o r t R N A b i n d i n g i n d u c e d b y i n i t i a t i o n f a c t o r s [11] m i g h t o r i g i n a t e i n t h e s e l e c t i v e i n h i b i t i o n of r i b o s o m e a t t a c h e m e n t to n o n - i n i t i a t o r c o d o n s . To ascertain whether the isolated complex r e t a i n s i t s f u l l a c t i v i t y , t h e a b i l i t y of b o u n d fMett R N A to r e a c t w i t h p u r o m y c i n i n t h e p r e s e n c e of G T P a n d 50 S p a r t i c l e s w a s a s s a y e d (fig. 2). T h e
0 r ~< L,
a
Z
ft.. 0
TABLE I . o
Specificity of the initiation complex formed on resin bound poly(A,G,U). Eluted (picomoles) !1
0
0
w
20
r¢~i 40
FRACTIONS
FIG. 1.
--
Isolation of the initiation complex on resin bound poly(A,G,U).
The i n i t i a t i o n complex w a s p e r f o r m e d i n a polya l l o m e r tube b y 10 m i n i n c u b a t i o n at 37 ° in a final volume of 0.5 m l c o n t a i n i n g 10 mM Tris-C1 buffer (pH 7.8), 8 mM Mg acetate, 30 mM NH~C1, 2 mM GTP, 6 mM m e r c a p t o e t h a n o l , 350 ilxg crude i n i t i a t i o n factors, 250-300 I~g fMet-tRNA ( M e t - 1 4 C ) , 600 'ltg 30S ribgsomes ( u n l a b e l l e d or 3H) a n d 400 l~g poly(A,G,U) supplied as resin coupled derivative. After chilling, the sample was t r a n s f e r r e d into a s m a l l column, washed at 4 ° w i t h buffer 1, t h e n eluted w i t h buffer 2, buffer 3 or buffer 2 c o n t a i n i n g p a n c r e a t i c r i b o n u c l e a s e (10 u g / m l ) as indicated i n the figures. Fig. l a a n d l b : complete s y s t e m ; fig. l c : poly(A,G,U) o m i t t e d (resin s a t u r a t e d w i t h oxidized adenosine) ; - - - 30S (3H) net counts, fMet-tRNA (Met-14C) counts. Arrows indicate buffer changes.
M o r e o v e r , w e t e s t e d w h e t h e r P h e - t R N A o r MettRNA (unformylated) could replace fMet-tRNA in the assay. These operations were all made with t r i t i u m l a b e l l e d r i b o s o m e s a n d a m i n o a c y l (14C)tRNA. The data are summarized in table I ; they show clearly that we are dealing with a true initiation c o m p l e x . I n d e e d , it a p p e a r s t h a t m e s s e n g e r R N A is n e e d e d to a l l o w a s i g n i f i c a n t b i n d i n g of r i b o somes and tRNA ; furthermore, the'initiator tRNA engages in the complex with a stoichiometry close to 1 : 1, a n d it c a n n o t b e r e p l a c e d b y P h e - o r MettRNA.
BIOCHIM1E, 1972, 54, n ° 7.
System 30S
aa-tRNA
complete (fMet-tRNA) . . . . . . . . . . - - fMet-tRNA . . . . . . . . . . . . . . . . -- initiation factors ........... - - p o l y ( A , G, U ) . . . . . . . . . . . . . .
19.2 26.1
14.5
32.5
4.3
9.7 3.0
complete (Phe-tRNA) . . . . . . . . . . (Met-tRNA) . . . . . . . . . .
18.2 23.0
2.3 2.7
Assembly of the i n i t i a t i o n complex, ~w a s h i n g a n d e l u t i o n were carried out. as described in figure 1 ; 30S r i b o s o m e s (3H) a n d fMet-tRNA (14C) or o t h e r aa-tRNA (14C) were used as indicated. Counts in the EDTA elnate were corrected for efficiencies a n d converted into picomoles.
puromycin containing buffer indeed removes m o r e t h a n 50 p. c e n t of t h e a v a i l a b l e fMet a t l o w t e m p e r a t u r e , w h e r e a s all t h e 50S p a r t i c l e s r e m a i n a s s o c i a t e d to t h e m e s s e n g e r - 3 0 S complex.
2. Binding of 70S ribosomes to resin coupled poly (U). I n t h e p r e s e n c e of p o l y (U) as m e s s e n g e r , N - a c e t y l P h e - t R N A b i n d s t i g h t l y to t h e p e p t i d y l site of 70S r i b o s o m e s p r o v i d e d t h e Mg 2÷ c o n c e n t r a t i o n is k e p t a b o v e 10 m M [12] ; i n a d e q u a t e i o n i c c o n d i t i o n s , it r e a c t s r e a d i l y w i t h p u r o m y c i n [13]. T o i l l u s t r a t e t h e g e n e r a l a p p l i c a b i l i t y of o u r m e thod, we performed this reaction using resin coup l e d p o l y (U) as m e s s e n g e r : N - a c e t y l P h e - t R N A ( P h e - 3 H ) w a s b o u n d to 70S r i b o s o m e s ( 1 4 C ) a s described in figure 3 ; the complex was iirst washed 56
Jean Petre, Alex Bollen, Pierre Nokin and Henri Grosjean.
826
with buffer 4 and then eluted with the same buffer containing puromycin. Finally, the non-reactive f o r m of N - a e e t y l P h e - t R N A a n d t h e b o u n d r i b o somes were removed with buffer 2 (EDTA). 4
I
I
I !~
I
,,l ,
m y c i n d e r i v a t i v e . L e t u s n o t e t h a t 50S r i b o s o m e s , t h e r e f o r e p r e s u m a b l y also 30S p a r t i c l e s , r e m a i n bound to messenger RNA during the puromycin step, unless a low magnesium or EDTA containing b u f f e r is u s e d . T h i s c o n f i r m s t h e d a t a of K a e m p f e r a n d M e s e l s o n [14] s h o w i n g t h a t n o e x c h a n g e of ribosomal subunits occurs during puromycin t r e a t m e n t at l o w t e m p e r a t u r e .
l
CONCLUSIONS.
",\
\
'
. . . .
O
10
20 30 F RACTIONS
40
bhc,. 2. - - Functional activity of the complex ; joining of the 50S parlicle and puromgcin reaction. The i n i t i a t i o n complex was p e r f o r m e d as described in fig. 1, b u t u n l a b e l l e d 30S r i b o s o m e s were used a n d a f t e r 10 rain i n c u b a t i o n , the m i x t u r e was supplem ented w i t h a n e q u i v a l e n t a m o u n t of 50S r i b o s o m e s (all) ; i n c u b a t i o n was c o n t i n u e d for l0 a d d i t i o n a l m i n u t e s . After t r a n s f e r into a c o l u m n a n d w a s h i n g w i t h buffer 1, e l u t i o n was carried out at 4 ° w i t h buffer 1 c o n t a i n i n g p u r o m y c i n (0.4 m g / m l ) , t h e n w i t h buffer 2 (EDTA) ; - - - 50S (3H) net counts, - - - - fMet (Met-14C) counts. Arrows indicate buffer changes.
The results clearly indicate that the isolated c o m p l e x r e t a i n s a h i g h c a p a c i t y to r e a c t w i t h p u r o m y c i n e v e n at l o w t e m p e r a t u r e , s i n c e a b o u t 80 p. c e n t of t h e b o u n d N - a c e t y l P h e e l u t e s as p u r o -
l
r
T
]
'T
ii'l 7 lo
i _z
OL._ ............ 0 10
20 30 FRACTIONS
40
Fro. 3. - - Binding of 70S ribosomes to resin coupled polg(U) ; N-acetglPhe-tRNA binding and puromycin reaction. 70S ribosomes a n d N-acetylPhe-tRNA b i n d i n g to r e s i n coupled poly(U) was p e r f o r m e d by 10 rain incub a t i o n at 37 ° in a final volume of 0.5 ml c o n t a i n i n g 10 mM Tris-C1 (pH 7.8) buffer, 10 mM Mg acetate, 160 mM NH4C1, 2 mM GTP, 6 m,M m e r c a p t o e t h a n o l , 600 I~g 30S ribosomes, one e q u i v a l e n t a m o u n t of 50S ribosomes (14C), 300 .~g N-acetylPhe-tRNA (Phe-aH) a n d 400 tLg poly(U) supplied as r e s i n coupled derivative. After w a s h i n g u n b o u n d m a t e r i a l w i t h buffer 4, elution was carried out w i t h buffer 4 c o n t a i n i n g purom y c i n (0.4 m g / m l ) , t h e n w i t h buffer 2 (EDTA) ; - - 50S 04C) counts, N-acetylPhe (Phe-aH) net counts. Arrows indicate buffer changes.
BIOCHIMIE, 1972, 54, n ° 7.
C o v a l e n t a t t a c h e m e n t of n u c l e i c a c i d s to i n s o luble supports has already been described by sever a l w o r k e r s E14, 15481 ; h o w e v e r , s u c h a p r o c e d u r e h a s n e v e r b e e n a p p l i e d to t h e i s o l a t i o n of intermediary complexes involved in the success i v e s t e p s of p o l y p e p t i d e s y n t h e s i s . The experiments described in the present work d e m o n s t r a t e t h a t m e s s e n g e r R N A c o u p l e d to a r i g i d m a t r i x r e t a i n s a h i g h c a p a b i l i t y of b i n d i n g and programming ribosomes in a specific mann e e r . T h e p r o c e d u r e e n a b l e s t h e i s o l a t i o n of 30S ribosomes engaged in an initiation complex encoded by a synthetic messenger RNA containing A U G a n d of 70,S r i b o s o m e s b o u n d to a p o l y ( U ) template ; it provides a direct method for measur i n g t h e s t o i c h i o m e t r y of t h e c o m p o n e n t s e n g a g e d in the complex. The method does not impair the a c t i v i t y of t h e c o m p l e x , s i n c e t h e l a t t e r r e t a i n s its a b i l i t y to t r a n s f e r b o u n d a m i n o a c y l - t R N A . t o p u r o m y c i n i n t h e p r e s e n c e of 50S p a r t i c l e s . Since any component interacting with ribosomes or messenger RNA may be added or washed off at a n y t i m e i n t h i s p r o c e d u r e , t h e m e t h o d s h o u l d p r o v i d e a n e w a p p r o a c h i n t h e s t u d y of sequential and coordinate events occurring during polypeptide synthesis. Furtherm:ore, the method affords a unique way to i s o l a t e a p o p u l a t i o n of r i b o s o m e s s t r i c t l y h o m o g e n e o u s w i t h r e s p e c t to t h e i r f u n c t i o n a l state, t h u s providing a direct biochemical approach to some a s p e c t s of t h e s t r u c t u r a l a n d f u n c t i o n a l h e t e r o g e n e i t y of t h e 30S r i b o s o m e s . W o r k i n t h i s l a s t field is n o w i n p r o g r e s s i n o u r l a b o r a t o r y .
Acknolwledgemenis. This work received support f r o m the Fonds de la Recherche F o n d a m e n t a l e et Collective, E u r a t o m a n d the Minist~re de la Politique et de ta P r o g r a m m a t i o n Scientifiques. J. P. and A. B. are respectively A s p i r a n t and Charg~ de Recherehes du F o n d s N a t i o n a l de la Recherche Scientifique. We are g r a t e f u l to Prof. H. C h a n t r e n n e f o r revising m a n u s c r i p t . R~suM~. La f o r m a t i o n d ' u n e h y d r a z o n e e n t r e du RNA oxydd au periodate et u n polym~re lin6aire d ' h y d r a z i d e acrylique pi6g~ dans u n gel d ' a g a r p e r m e t d ' o b t e n i r
Binding
of ribosomes
f a c i l e m e n t u n e r~sine p o r t a n t u n ARN m e s s a g e r s y n t h f t i q u e lid c h i m i q u e m e n t . L ' e m p l o i d ' u n tel d~riv6 de I'ARN p e r m e t d ' i s o l e r des r i b o s o m e s 30S ou 70S engag~s d a n s le eom,plexe & i n i t i a t i o n p r o g r a m m f i p a r d u poly(A,G,U), de m~rrte que l ' i s o l e m e n t de r i b o s o m e s 70S li6s h d u poly(U). Le coinplexe isolg est sp6cifique et, comxne le m o n t r e la rdaetion ~ la puroxnyeine, il c o n s e r v e u n e b o n n e aetivit6 d a n s le t r a n s f e r t d ' u n a m i n o a e y l - t A R N N-bloqu6. P a r eons~qnent, eette m~t h o d e c o n s t i t u e u n m o y e n o r i g i n a l p o u r la p r d p a r a t i o n de r i b o s o i n e s actifs prfisumds h o m o g ~ n e s q u a n t "~ leur 6tat f o n e t i o n n e l .
ZUSAMMENFASSUNG,
Die B i l d u n g eines H y d r a z o n s z w i s c h e n m i t P e r i o d a t o x y d i e r t e r RNS u u d e i n e m l i n e a r e n P o l y m e r yon Aerylhydrazid, das in Agargel e i n g e s c h l o s s e n ist, e r l a u b t , leieht ein Harz zu bilden, d a s eine e h e m i s c h g e b u n d e n e s y n t h e t i s c h e Messenger-RNS tr~gt. Die V e r w e n d u n g eines d e r a r t i g e n R N S - D e r i v a t s g e s t a t t e t 30 S oder 70 S - R i b o s o m e n , die in e i n e m u r s p r f i n g l i c h e n Kompiex, der d u r c h Poly (A, G, N) p r o g r a m m i e r t wird, g e b u n d e n sind, ebenso wie 70 S - R i b o s o m e n , die an P o l y (U) g e b u n d e n sind, zu isolieren. Der isolierte K o m p l e x ist spezifiseh u n d er b e i n h a l t e t , wie die R e a k t i o n m i t P u r o m y z i n zeigt, eine h o h e Aktivit~it b e i m T r a n s f e r einer N - b l o c k i e r t e n A m i n o a c y l - T r a n s f e r RNS. Folglieh stellt diese Methode e i n e n e i n z i g a r t i g e n Weg, aktive R i b o s o m e n , die als h o m o g e n v o r a u s g e setzt werden, m i t Riieksicht a u f i h r e n f u n k t i o n e l l e n Z u s t a n d , zu pr~iparieren.
BIOCHIMIE, 1972, 54, n" 7.
to matrix
bound
mRNA.
827
REFERENCES. 1. Knorre, D, G., Misina, S. D. & S a n d a c h t c h i e v , L. C. (1964) Izvestia Sibirskooo Otdelenia A k a d e m i y Nauk, Seria C h i m i c h e s k i c h Nauk, l l , 134. 2. No:kin, P., M a n u s c r i p t in p r e p a r a t i o n . 3. Nelidova, O. D. & Kiselev, L. L. (1968) Molecular Biology, 2, 47 ( t r a n s l a t e d f r o m R u s s i a n ) . 4. Kiselev, L. L. & A v d o n i n a , T. A. (1971) Molecular Biology, 3, 88 ( t r a n s l a t e d f r o m R u s s i a n ) . 5. N o n l u r a , M., Lowry, C. V. ~ Guthrie, C. (1967) Proc. Nail. Acad. Sci., 5,8, 1487. 6. Kikuehi, A. ,~ Monier, R. (1970) F.E.B.S. Letters, 11, 157. 7. Z a m i r , A., Misldn, R. & Elson, D. (1971) J . Mol. Biol., 60, 347. 8. Revel, M., B r a w e n n a n , G., Lelong, J.-C. ~ Gros, F. (1968) Nature, 219, 1016. 9. Matthaei, J. H., Voigt, H. P., Heller, G., Neth, 1~., Sch6ch, G., Kiibler, H., A m e l u n x e n , F., Sander, G. ,~ P a r m e g g i a n i , A. (19.66) Cold Spring H a r b o r S y m p o s i a on Q n a n t . Biol., XXXI, 25. 10. Lengyel, P. t, $611, D. (1969) Bacteriol. Revie:ws, 33, 264. 11. Gualerzi, C., Pon, C. L. & Kaji, A. (1971) Biophys. Biochem. Res. Comm., 45, 1312. 12. L u c a s - L e n a r d , J. & L i p m a n n , F. (1967) Proc. Natl. Acad. Sei., 57, 1050. 13. Teraoka, H. ~ T a n a k a , K. (1971) Biochim. Biophys. Acta, 247, 304. 14. K a e m p f e r , R. & Meselson, M. (1969) Cold Spring H a r b o r S y m p o s i a on Q u a n t . Biol., XXXIV, 209. 15. G i l h a m , P. T. (1968) B i o c h e m i s t r y , 8, 2809. 16. Cavalieri, L. F. ~ Carroll, E. (1970) Proc. Natl. Acad. Sei., 67, 807. 17. P o o n i a n , M. S., Sehlabaeh, A. J. & W e i s s b a c h , A. (1971) Biochemistry, 10, 424. 18. R o b b e r s o n , D. L. & D a v i d s o n , N. (1972) Biochemistry, 11, 533.
The binding of Escherichia coli ribosomes to matrix bound messenger RNA Jean PETriE, Alex BOLLEN, P i e r r e NOKIN an d H e n r i GROSJEAN.
D e p a r t m e n t o[ Molecular Biology. University of Brussels, Rue des Chevaux, 67-B-1640 Rhode Saint Genbse, Belgium. (17-6-1972).
Summary. - - The formation of a hydrazone derivative between periodate oxidized RNA and a linear polymer of acrylic hydrazide entrapped in an agar gel affords a convenient method to prepare a resin bearing chemically eoupled synthetic messenger RNA. The use of such an RNA derivative instead of the free messenger rrmkes it possible to isolate 30S or 70S ribosomes involved in the initiation complex programmed by poly(A,G,U) as messenger, and of 70S ribosomes bound to a poly(U) template. The complex isolated is specific and, as judged by the puromycin reaction, it retains a high activity to transfer bound N-blocked anainoaeyl-transfer RNA. Therefore, this method appears an unique way to prepare a population of active ribosomes presumably homogeDeous with respect to their functional state. INTRODUCTION. A c o n s i d e r a b l e a m o u n t of w o r k has been devoted for m a n y years to the k n o w l e d g e of the successive steps of p o l y p e p t i d e b i o s y n t h e s is , and the different c o m p l e x e s i n v o l v e d in the i n i ti a t io n , elongation an d t e r m i n a t i o n m o d e s of the ribosomes have been d e s c r i b e d by m a n y w o r k e r s . Until now, the c h a r a c t e r i z a t i o n of these com.plexes and the d e m o n s t r a t i o n of t h e i r f u n c t i o n w a s a c h i e v e d e i t h e r by d e n s i t y g r a d i e n t u l t r a c e n t r i f u g a t i o n or by n i t r o c e l l u l o s e f i l t e r b i n d i n g assay. These t wo methods, in spite of t h e i r w i d e use and r e l i a b i l i t y , suffer f r o m some m a j o r limit a t i o n s ; a l t h o u g h the f o r m e r allows s o m e t i m e s the isolation of c o m p l e x e s for f u r t h e r p r o c e s s i n g , the latter i r r e v e r s i b l y destroys them. F u r t h e r more, none of these m e t h o d s enables the separation of the active c o m p l e x , w h i c h usually i n v o l v e s only a few p e r c e n t s of the r i b o s o m e s engaged, f r o m the bulk of p a r t i c l e s that did not p a r t i c i pate to the r e a c t i o n assayed. C o n s i d e r i n g these facts, one of ns (H.G.) propos ed a n e w m e t h o d of i n v e s t i g a t i o n based on the p r o p e r t i e s of m e s s e n g e r RNA c h e m i c a l l y c ou p l ed to an insoluble support.
Abbreviations : EDTA, ethylenediamine tetra-acetic acid. poly(U), polvuridylic acid. poly(A,G,U),'poly adenylic, guanylie, uridylie aeid. Met-tRNA, methionyl-transfer RNA. Phe-tRNA, phenylalanyl-transfer RNA. fMet-tRNA, N-formylmethionyl-transfer RNA. N-aeetylPhe-tRNA, N-aeetylphenylalanyl-tran sfev RNA. GTP, guanosine triphosphate.
The basic p r o c e d u r e , d e s c r i b e d by K n o r r e and c o w o r k e r s [1~, i n v o l v e s the f o r m a t i o n of a h y d r a zone d e r i v a t i v e b e t ~ ' e e n p e r i o d a t e o x i d i z e d RNA and a l i n e a r p o l y m e r of a c r y l i c h y d r a z i d e e n t r a p p ed in an agar gel. This c h e m i c a l c o u p l i n g features a v e r y high stability in d i f f er en t pH, i o n i c strcnth and t e m p e r a t u r e c o n d i t i o n s [2] and the RNA d e r i v a t i v e r e m a i n s c o m p l e t e l y u n d a m a ged after i n c u b a t i o n in the s t a n d a r d buffers used for assays in p o l y p e p t i d e s b i o sy n t h esi s. The m e t h o d has been a p p l i e d before to the p a r t i a l p u r i f i c a t i o n of an a m i n o a c y l - t R N A ligase by affinity c h r o m a t o g r a p h y on a c o l u m n c h a r g e d w i t h a speeific t r a n s f e r RNA [3]. A c o l u m n charged w i t h polyIU) w a s also used to d e m o n s t r a t e the specific r e t a r d a t i o n of Phe-tRNA by codona n t i e o d o n p a i r i n g [4]. In this p a p e r , we s h o w that this resin c h a r g e d w i t h s y n t h e t i c m e s s e n g e r RNA m a y actually be used i n st ead of the free template to p r o g r a m the a c t i v i t y of ribosomes. Since in this ease, the only r i b o s o m e s that are b o u n d to the linked m e s s e n g e r are r e t a i n e d on the resin, this p r o c e d u r e p r o v i d e s a v e r y efficient w a y to isolate active c o m p l e x e s i n v o l v i n g m e s s e n g e r RNA, 30S or 70S r i b o s o m e s and o t h e r ligands.
MATERIALS AND METHODS.
Escherichia colt (strain B) r i b o s o m e s and initiation factors from strain MRE 600 w e r e p r e p a red a c c o r d i n g to N o m u r a et al. [5J ; labelled ribosomes w e r e o b t a i n e d f r o m cells g r o w n on m i n i -
824
Jean Petre, A l e x Bollen, Pierre N o k i n a n d H e n r i Grosjean.
mal m e d i u m s u p p l e m e n t e d w i t h labelled a m i n o acids (14C or all). Ribosomal s u b u n i t s w e r e o b t a i n e d by g r a d i e n t c e n t r i f u g a t i o n in low Mg2÷ buffer, p r e c i p i t a t e d w i t h e t h a n o l [6] a n d dialyzed against buffer 1. They were r o u t i n e l y reactivated in the p r e s e n c e of a m m o n i u m chloride before use [7].
E. colt B t r a n s f e r RNAs (Schwarz Bioresearch) were r o u t i n e l y acylated w i t h m e t h i o n i n e (14C) or p h e n y l a l a n i n e (14C) ; fMet-tRNA (Met-14C) was o b t a i n e d b y e n z y m i c f o r m y l a t i o n of Met-(14C)tRNA [8], a n d the u n f o r m y l a t e d p r o d u c t s were deacyluted by t r e a t m e n t w i t h 1 mM CuSO 4 [9]. The final p r o d u c t s were purified by DEAE-celhdose c h r o m a t o g r a p h y and ethanol p r e c i p i t a t i o n . Na c e t y l P h e 4 R N A (Phe-SH) was k i n d l y supplied by Dr. J.-C. L.elong. Adenosine was oxidized by s o d i m n m e t a p e r i o date a n d passed t h r o u g h an a n i o n exchange resin to remove the excess reagents. Poly(U) and poly(A,G,U) (Boehringer M a n n h e i m ) were oxidized by the method of K n o r r e el al. [1], p r e c i p i t a ted w i t h e t h a n o l a n d stored in 10 mM s o d i u m acetate buffer (pH 4.8). The synthesis of p o l y a c r y l i c h y d r a z i d e and the i n c l u s i o n of the p o l y m e r into an agar gel was p e r f o r m e d as d e s c r i b e d by K n o r r e el al. [1?, but the final p o l y m e r c o n c e n t r a t i o n was b r o u g h t to 1 p. cent ( w / w ) . The r e s i n was sieved twice t h r o u g h a 60 mesh screen a n d stored in cold water. It w a s washed for at least 14 hours before use, until no free h y d r a z i n e could be detected i n the eluate. The c o u p l i n g reaction w i t h oxidized RNA was c a r r i e d out in the following c o n d i t i o n s : 0.2-0.35 g (wet weight) bed was dispersed inta a final volume of 1 ml c o n t a i n i n g 0.2 M sodium acetate buffer (pH 4.8), 20 mM m a g n e s i u m acetate and 1214 a b s o r b a n c y u n i t s (258 nm) of the oxidized RNA. The m i x t u r e was vigorously stirred at 4 ° for at least 3 h o u r s and p r e f e r a b l y overnight ; 8090 p. cent of the RNA was u s u a l l y coupled to the resin u n d e r these conditions. In o r d e r to block the excess of h y d r a z i d e groups, the resin was then s t i r r e d in 1 ml of the above buffer c o n t a i n i n g 250 a b s o r b a n c y u n i t s of oxidized a d e n o s i n e ; it was finally w a s h e d a n d e q u i l i b r a t e d before use in buffer 1 or 4. The f o r m a t i o n of the i n i t i a t i o n complex on resin coupled poly(A,G,U) a n d the b i n d i n g of 70S ribosomes to the poly(U) derivative were performed as described in the text ; following incubation, the sample was chilled, t r a n s f e r r e d into a small c o l u m n and washed w i t h 10-15 ml of buffer 1 or 4. The complex was r o u t i n e l y eluted
BIOCH1MIE, 1972, 54, n ° 7.
by buffer 2 (15 mM EDTA) at 45 m l / h flow rate, or by buffer 3 (0.1 mM Mg2+) ; in the latter case, the flow rate was r e d u c e d to 4.5 m l / h to allow complete recovery of the ribosomes from the column. 0.6 ml fractions were c o u n t e d in T r i t o n X-100 based scintillation fluid.
Buffers. Buffer 1 : Tris-C1 10 mM (pH 7.8), NH4C1 30 mM, Mg acetate 10 mM, m e r c a p t o e t h a n o l 6 mM. Buffer 2 : Tris-C1 10 mM (pH 7.8), NH4C1 30 mM, EDTA 15 raM, m e r c a p t o e t h a n o l 6 raM. Buffer 3 : Tris-C1 10 mM (pH 7.8), NH4C1 30 raM, Mg acetate 0.1 mM, m e r c a p t o e t h a n o l 6 mM. Buffer 4 : T r i s - C 1 10 mM (pH 7.8), NH4C1 160 mM, Mg acetate 10 raM, m e r c a p t o e t h a n o l 6 raM. RESULTS AND DISCUSSION.
1. Formalion and specificity of the iniliulion complex on resin bound poly (A, G, U). The f o r m a t i o n of the i n i t i a t i o n c o m p l e x requires the p r e s e n c e of p r o t e i n factors a n d involves a specific i n t e r a c t i o n b e t w e e n messenger RNA a n d 30S subparticles, followed by the b i n d i n g of i n i t i a t o r tRNA a n d GTP. The large subparticle then joins this complex a n d makes it competent to accept an aminoacyl-tRNA a n d to achieve the s y n t h e s i s of the first p e p t i d e b o n d [for a review, see ref. 10]. The first step i n v o l v i n g the i n t e r a c t i o n between 30S ribosomes and messenger RNA, it is expected that all the i n t e r m e d i a t e s o c c u r r i n g in the above process should be isolable as resin b o u n d products b y the bias of messenger RNA. In o r d e r to check this point, the a b i l i t y of resin b o u n d poly (A, G, U) to p r o g r a m the formation of the messenger RNA : 30S : GTP : fMet-tRNA complex i n the presence of crude i n i t i a t i o n factors w a s assayed. After w a s h i n g any u n b o u n d material, the complex was recovered by elution with a low m a g n e s i u m buffer (fig. la) or b y EDTA t r e a t m e n t (fig. l b ) ; the latter method, w h i c h is expected to p r o d u c e some more drastic effects on the breakage of tile complex, did not release more r a d i o a c t i v i t y t h a n the low m a g n e s i u m b u f f e r , although a n o t i c e a b l e a m o u n t of r a d i o a c t i v e material r e m a i n e d b o u n d after the successive elution steps. F i g u r e l b clearly shows that the residual r a d i o a c t i v i t y is released only after r i b o n u c l e a s e t r e a t m e n t ; however, since it is o b t a i n e d in irrep r o d u c i b l e amounts even i n columns l a c k i n g messenger RNA (fig. lc), we suspect that it corresp o n d s to unspecific aggregation product~ of degraded or n o n r i b o s o m a l c o n t a m i n a t i n g material.
B i n d i n g of r i b o s o m e s to m a t r i x b o u n d m R N A . I n o r d e r to p r o v e t h a t t h e c o m p l e x f o r m e d o n t h e r e s i n b o u n d m e s s e n g e r R N A c o r r e s p o n d s to a true initiation intermediate, we checked several of i t s c h a r a c t e r i s t i c s , n a m e l y t h e r e q u i r e m e n t f o r initiation factors, initiator tRNA and AUG codon.
825
Since ribosomes used in this experiment were not salt washed, they do not exhibit a strong requirement for added initiation factors for bind i n g f M e t - t R N A . H o w e v e r , m u c h m o r e 30S p a r t i c l e s b i n d to t h e m e s s e n g e r i n t h e a b s e n c e of i n i tiation factors than in the complete system, wher e a s t h e l e v e l of t R N A b i n d i n g is d e c r e a s e d u n d e r these conditions. Both observations suggest that t h e i n h i b i t i o n of n o n - i n i t i a t o r t R N A b i n d i n g i n d u c e d b y i n i t i a t i o n f a c t o r s [11] m i g h t o r i g i n a t e i n t h e s e l e c t i v e i n h i b i t i o n of r i b o s o m e a t t a c h e m e n t to n o n - i n i t i a t o r c o d o n s . To ascertain whether the isolated complex r e t a i n s i t s f u l l a c t i v i t y , t h e a b i l i t y of b o u n d fMett R N A to r e a c t w i t h p u r o m y c i n i n t h e p r e s e n c e of G T P a n d 50 S p a r t i c l e s w a s a s s a y e d (fig. 2). T h e
0 r ~< L,
a
Z
ft.. 0
TABLE I . o
Specificity of the initiation complex formed on resin bound poly(A,G,U). Eluted (picomoles) !1
0
0
w
20
r¢~i 40
FRACTIONS
FIG. 1.
--
Isolation of the initiation complex on resin bound poly(A,G,U).
The i n i t i a t i o n complex w a s p e r f o r m e d i n a polya l l o m e r tube b y 10 m i n i n c u b a t i o n at 37 ° in a final volume of 0.5 m l c o n t a i n i n g 10 mM Tris-C1 buffer (pH 7.8), 8 mM Mg acetate, 30 mM NH~C1, 2 mM GTP, 6 mM m e r c a p t o e t h a n o l , 350 ilxg crude i n i t i a t i o n factors, 250-300 I~g fMet-tRNA ( M e t - 1 4 C ) , 600 'ltg 30S ribgsomes ( u n l a b e l l e d or 3H) a n d 400 l~g poly(A,G,U) supplied as resin coupled derivative. After chilling, the sample was t r a n s f e r r e d into a s m a l l column, washed at 4 ° w i t h buffer 1, t h e n eluted w i t h buffer 2, buffer 3 or buffer 2 c o n t a i n i n g p a n c r e a t i c r i b o n u c l e a s e (10 u g / m l ) as indicated i n the figures. Fig. l a a n d l b : complete s y s t e m ; fig. l c : poly(A,G,U) o m i t t e d (resin s a t u r a t e d w i t h oxidized adenosine) ; - - - 30S (3H) net counts, fMet-tRNA (Met-14C) counts. Arrows indicate buffer changes.
M o r e o v e r , w e t e s t e d w h e t h e r P h e - t R N A o r MettRNA (unformylated) could replace fMet-tRNA in the assay. These operations were all made with t r i t i u m l a b e l l e d r i b o s o m e s a n d a m i n o a c y l (14C)tRNA. The data are summarized in table I ; they show clearly that we are dealing with a true initiation c o m p l e x . I n d e e d , it a p p e a r s t h a t m e s s e n g e r R N A is n e e d e d to a l l o w a s i g n i f i c a n t b i n d i n g of r i b o somes and tRNA ; furthermore, the'initiator tRNA engages in the complex with a stoichiometry close to 1 : 1, a n d it c a n n o t b e r e p l a c e d b y P h e - o r MettRNA.
BIOCHIM1E, 1972, 54, n ° 7.
System 30S
aa-tRNA
complete (fMet-tRNA) . . . . . . . . . . - - fMet-tRNA . . . . . . . . . . . . . . . . -- initiation factors ........... - - p o l y ( A , G, U ) . . . . . . . . . . . . . .
19.2 26.1
14.5
32.5
4.3
9.7 3.0
complete (Phe-tRNA) . . . . . . . . . . (Met-tRNA) . . . . . . . . . .
18.2 23.0
2.3 2.7
Assembly of the i n i t i a t i o n complex, ~w a s h i n g a n d e l u t i o n were carried out. as described in figure 1 ; 30S r i b o s o m e s (3H) a n d fMet-tRNA (14C) or o t h e r aa-tRNA (14C) were used as indicated. Counts in the EDTA elnate were corrected for efficiencies a n d converted into picomoles.
puromycin containing buffer indeed removes m o r e t h a n 50 p. c e n t of t h e a v a i l a b l e fMet a t l o w t e m p e r a t u r e , w h e r e a s all t h e 50S p a r t i c l e s r e m a i n a s s o c i a t e d to t h e m e s s e n g e r - 3 0 S complex.
2. Binding of 70S ribosomes to resin coupled poly (U). I n t h e p r e s e n c e of p o l y (U) as m e s s e n g e r , N - a c e t y l P h e - t R N A b i n d s t i g h t l y to t h e p e p t i d y l site of 70S r i b o s o m e s p r o v i d e d t h e Mg 2÷ c o n c e n t r a t i o n is k e p t a b o v e 10 m M [12] ; i n a d e q u a t e i o n i c c o n d i t i o n s , it r e a c t s r e a d i l y w i t h p u r o m y c i n [13]. T o i l l u s t r a t e t h e g e n e r a l a p p l i c a b i l i t y of o u r m e thod, we performed this reaction using resin coup l e d p o l y (U) as m e s s e n g e r : N - a c e t y l P h e - t R N A ( P h e - 3 H ) w a s b o u n d to 70S r i b o s o m e s ( 1 4 C ) a s described in figure 3 ; the complex was iirst washed 56
Jean Petre, Alex Bollen, Pierre Nokin and Henri Grosjean.
826
with buffer 4 and then eluted with the same buffer containing puromycin. Finally, the non-reactive f o r m of N - a e e t y l P h e - t R N A a n d t h e b o u n d r i b o somes were removed with buffer 2 (EDTA). 4
I
I
I !~
I
,,l ,
m y c i n d e r i v a t i v e . L e t u s n o t e t h a t 50S r i b o s o m e s , t h e r e f o r e p r e s u m a b l y also 30S p a r t i c l e s , r e m a i n bound to messenger RNA during the puromycin step, unless a low magnesium or EDTA containing b u f f e r is u s e d . T h i s c o n f i r m s t h e d a t a of K a e m p f e r a n d M e s e l s o n [14] s h o w i n g t h a t n o e x c h a n g e of ribosomal subunits occurs during puromycin t r e a t m e n t at l o w t e m p e r a t u r e .
l
CONCLUSIONS.
",\
\
'
. . . .
O
10
20 30 F RACTIONS
40
bhc,. 2. - - Functional activity of the complex ; joining of the 50S parlicle and puromgcin reaction. The i n i t i a t i o n complex was p e r f o r m e d as described in fig. 1, b u t u n l a b e l l e d 30S r i b o s o m e s were used a n d a f t e r 10 rain i n c u b a t i o n , the m i x t u r e was supplem ented w i t h a n e q u i v a l e n t a m o u n t of 50S r i b o s o m e s (all) ; i n c u b a t i o n was c o n t i n u e d for l0 a d d i t i o n a l m i n u t e s . After t r a n s f e r into a c o l u m n a n d w a s h i n g w i t h buffer 1, e l u t i o n was carried out at 4 ° w i t h buffer 1 c o n t a i n i n g p u r o m y c i n (0.4 m g / m l ) , t h e n w i t h buffer 2 (EDTA) ; - - - 50S (3H) net counts, - - - - fMet (Met-14C) counts. Arrows indicate buffer changes.
The results clearly indicate that the isolated c o m p l e x r e t a i n s a h i g h c a p a c i t y to r e a c t w i t h p u r o m y c i n e v e n at l o w t e m p e r a t u r e , s i n c e a b o u t 80 p. c e n t of t h e b o u n d N - a c e t y l P h e e l u t e s as p u r o -
l
r
T
]
'T
ii'l 7 lo
i _z
OL._ ............ 0 10
20 30 FRACTIONS
40
Fro. 3. - - Binding of 70S ribosomes to resin coupled polg(U) ; N-acetglPhe-tRNA binding and puromycin reaction. 70S ribosomes a n d N-acetylPhe-tRNA b i n d i n g to r e s i n coupled poly(U) was p e r f o r m e d by 10 rain incub a t i o n at 37 ° in a final volume of 0.5 ml c o n t a i n i n g 10 mM Tris-C1 (pH 7.8) buffer, 10 mM Mg acetate, 160 mM NH4C1, 2 mM GTP, 6 m,M m e r c a p t o e t h a n o l , 600 I~g 30S ribosomes, one e q u i v a l e n t a m o u n t of 50S ribosomes (14C), 300 .~g N-acetylPhe-tRNA (Phe-aH) a n d 400 tLg poly(U) supplied as r e s i n coupled derivative. After w a s h i n g u n b o u n d m a t e r i a l w i t h buffer 4, elution was carried out w i t h buffer 4 c o n t a i n i n g purom y c i n (0.4 m g / m l ) , t h e n w i t h buffer 2 (EDTA) ; - - 50S 04C) counts, N-acetylPhe (Phe-aH) net counts. Arrows indicate buffer changes.
BIOCHIMIE, 1972, 54, n ° 7.
C o v a l e n t a t t a c h e m e n t of n u c l e i c a c i d s to i n s o luble supports has already been described by sever a l w o r k e r s E14, 15481 ; h o w e v e r , s u c h a p r o c e d u r e h a s n e v e r b e e n a p p l i e d to t h e i s o l a t i o n of intermediary complexes involved in the success i v e s t e p s of p o l y p e p t i d e s y n t h e s i s . The experiments described in the present work d e m o n s t r a t e t h a t m e s s e n g e r R N A c o u p l e d to a r i g i d m a t r i x r e t a i n s a h i g h c a p a b i l i t y of b i n d i n g and programming ribosomes in a specific mann e e r . T h e p r o c e d u r e e n a b l e s t h e i s o l a t i o n of 30S ribosomes engaged in an initiation complex encoded by a synthetic messenger RNA containing A U G a n d of 70,S r i b o s o m e s b o u n d to a p o l y ( U ) template ; it provides a direct method for measur i n g t h e s t o i c h i o m e t r y of t h e c o m p o n e n t s e n g a g e d in the complex. The method does not impair the a c t i v i t y of t h e c o m p l e x , s i n c e t h e l a t t e r r e t a i n s its a b i l i t y to t r a n s f e r b o u n d a m i n o a c y l - t R N A . t o p u r o m y c i n i n t h e p r e s e n c e of 50S p a r t i c l e s . Since any component interacting with ribosomes or messenger RNA may be added or washed off at a n y t i m e i n t h i s p r o c e d u r e , t h e m e t h o d s h o u l d p r o v i d e a n e w a p p r o a c h i n t h e s t u d y of sequential and coordinate events occurring during polypeptide synthesis. Furtherm:ore, the method affords a unique way to i s o l a t e a p o p u l a t i o n of r i b o s o m e s s t r i c t l y h o m o g e n e o u s w i t h r e s p e c t to t h e i r f u n c t i o n a l state, t h u s providing a direct biochemical approach to some a s p e c t s of t h e s t r u c t u r a l a n d f u n c t i o n a l h e t e r o g e n e i t y of t h e 30S r i b o s o m e s . W o r k i n t h i s l a s t field is n o w i n p r o g r e s s i n o u r l a b o r a t o r y .
Acknolwledgemenis. This work received support f r o m the Fonds de la Recherche F o n d a m e n t a l e et Collective, E u r a t o m a n d the Minist~re de la Politique et de ta P r o g r a m m a t i o n Scientifiques. J. P. and A. B. are respectively A s p i r a n t and Charg~ de Recherehes du F o n d s N a t i o n a l de la Recherche Scientifique. We are g r a t e f u l to Prof. H. C h a n t r e n n e f o r revising m a n u s c r i p t . R~suM~. La f o r m a t i o n d ' u n e h y d r a z o n e e n t r e du RNA oxydd au periodate et u n polym~re lin6aire d ' h y d r a z i d e acrylique pi6g~ dans u n gel d ' a g a r p e r m e t d ' o b t e n i r
Binding
of ribosomes
f a c i l e m e n t u n e r~sine p o r t a n t u n ARN m e s s a g e r s y n t h f t i q u e lid c h i m i q u e m e n t . L ' e m p l o i d ' u n tel d~riv6 de I'ARN p e r m e t d ' i s o l e r des r i b o s o m e s 30S ou 70S engag~s d a n s le eom,plexe & i n i t i a t i o n p r o g r a m m f i p a r d u poly(A,G,U), de m~rrte que l ' i s o l e m e n t de r i b o s o m e s 70S li6s h d u poly(U). Le coinplexe isolg est sp6cifique et, comxne le m o n t r e la rdaetion ~ la puroxnyeine, il c o n s e r v e u n e b o n n e aetivit6 d a n s le t r a n s f e r t d ' u n a m i n o a e y l - t A R N N-bloqu6. P a r eons~qnent, eette m~t h o d e c o n s t i t u e u n m o y e n o r i g i n a l p o u r la p r d p a r a t i o n de r i b o s o i n e s actifs prfisumds h o m o g ~ n e s q u a n t "~ leur 6tat f o n e t i o n n e l .
ZUSAMMENFASSUNG,
Die B i l d u n g eines H y d r a z o n s z w i s c h e n m i t P e r i o d a t o x y d i e r t e r RNS u u d e i n e m l i n e a r e n P o l y m e r yon Aerylhydrazid, das in Agargel e i n g e s c h l o s s e n ist, e r l a u b t , leieht ein Harz zu bilden, d a s eine e h e m i s c h g e b u n d e n e s y n t h e t i s c h e Messenger-RNS tr~gt. Die V e r w e n d u n g eines d e r a r t i g e n R N S - D e r i v a t s g e s t a t t e t 30 S oder 70 S - R i b o s o m e n , die in e i n e m u r s p r f i n g l i c h e n Kompiex, der d u r c h Poly (A, G, N) p r o g r a m m i e r t wird, g e b u n d e n sind, ebenso wie 70 S - R i b o s o m e n , die an P o l y (U) g e b u n d e n sind, zu isolieren. Der isolierte K o m p l e x ist spezifiseh u n d er b e i n h a l t e t , wie die R e a k t i o n m i t P u r o m y z i n zeigt, eine h o h e Aktivit~it b e i m T r a n s f e r einer N - b l o c k i e r t e n A m i n o a c y l - T r a n s f e r RNS. Folglieh stellt diese Methode e i n e n e i n z i g a r t i g e n Weg, aktive R i b o s o m e n , die als h o m o g e n v o r a u s g e setzt werden, m i t Riieksicht a u f i h r e n f u n k t i o n e l l e n Z u s t a n d , zu pr~iparieren.
BIOCHIMIE, 1972, 54, n" 7.
to matrix
bound
mRNA.
827
REFERENCES. 1. Knorre, D, G., Misina, S. D. & S a n d a c h t c h i e v , L. C. (1964) Izvestia Sibirskooo Otdelenia A k a d e m i y Nauk, Seria C h i m i c h e s k i c h Nauk, l l , 134. 2. No:kin, P., M a n u s c r i p t in p r e p a r a t i o n . 3. Nelidova, O. D. & Kiselev, L. L. (1968) Molecular Biology, 2, 47 ( t r a n s l a t e d f r o m R u s s i a n ) . 4. Kiselev, L. L. & A v d o n i n a , T. A. (1971) Molecular Biology, 3, 88 ( t r a n s l a t e d f r o m R u s s i a n ) . 5. N o n l u r a , M., Lowry, C. V. ~ Guthrie, C. (1967) Proc. Nail. Acad. Sci., 5,8, 1487. 6. Kikuehi, A. ,~ Monier, R. (1970) F.E.B.S. Letters, 11, 157. 7. Z a m i r , A., Misldn, R. & Elson, D. (1971) J . Mol. Biol., 60, 347. 8. Revel, M., B r a w e n n a n , G., Lelong, J.-C. ~ Gros, F. (1968) Nature, 219, 1016. 9. Matthaei, J. H., Voigt, H. P., Heller, G., Neth, 1~., Sch6ch, G., Kiibler, H., A m e l u n x e n , F., Sander, G. ,~ P a r m e g g i a n i , A. (19.66) Cold Spring H a r b o r S y m p o s i a on Q n a n t . Biol., XXXI, 25. 10. Lengyel, P. t, $611, D. (1969) Bacteriol. Revie:ws, 33, 264. 11. Gualerzi, C., Pon, C. L. & Kaji, A. (1971) Biophys. Biochem. Res. Comm., 45, 1312. 12. L u c a s - L e n a r d , J. & L i p m a n n , F. (1967) Proc. Natl. Acad. Sei., 57, 1050. 13. Teraoka, H. ~ T a n a k a , K. (1971) Biochim. Biophys. Acta, 247, 304. 14. K a e m p f e r , R. & Meselson, M. (1969) Cold Spring H a r b o r S y m p o s i a on Q u a n t . Biol., XXXIV, 209. 15. G i l h a m , P. T. (1968) B i o c h e m i s t r y , 8, 2809. 16. Cavalieri, L. F. ~ Carroll, E. (1970) Proc. Natl. Acad. Sei., 67, 807. 17. P o o n i a n , M. S., Sehlabaeh, A. J. & W e i s s b a c h , A. (1971) Biochemistry, 10, 424. 18. R o b b e r s o n , D. L. & D a v i d s o n , N. (1972) Biochemistry, 11, 533.