- Email: [email protected]
Conversion of Escherichia coli 17S chloramphenicol RNA to a 16S RNA in vitro
BIOCHIMIE, 1976, 58, 1007-1009.
Conversion of Escherichia coli 17S ehloramphenieol RNA to a 16S RNA in dtro. J a c q u e s A l a i n TRI~TON 0 a n d F r a n ~ , o i s e HAYES. Laboratoire de Chimie Cellulaire, l n s t i t u t de Biologic Physieo-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris. (22-$-1976).
INTRODUCTION. We h a v e a l r e a d y described t h e in vitro m a t u r a t i o n of t h e 17S p r e c u r s o r of E. coli 16S rRNA by i n c u b a t i o n of 27S p r e c u r s o r p a r t i c l e s i s o l a t e d f r o m p u l s e labelled e x p o n e n t i a l l y g r o w i n g cells in a b a c t e r i a l $30 e x t r a c t [1]. T h e difficulty of p r e p a r i n g t h e 27S p r e c u r s o r in large a m o u n t h a s led u s to i n v e s t i g a t e the p o s s i b i l i t y of u s i n g i n s t e a d p a r t i c l e s r e c o n s t i t u t e d in vitro f r o m total 30S r i b o s o m a l p r o t e i n s a n d 17S c h l o r a m p h e n i c o l RNA. T h i s RNA w h i c h r e s e m b l e s t h e 17S p r e c u r s o r species p r e s e n t in t h e n a t u r a l 27S particle i n b e i n g submethylated and in possessing extra polynucleotide s e q u e n c e s at each of its e x t r e m i t i e s [2] is e a s i l y i s o l a t e d i n large q u a n t i t i e s . I n t h i s report, we p r e s e n t t h e r e s u l t s of e x p e r i m e n t s in w h i c h we h a v e s t u d i e d t h e in oitro processing of 17S-CM RNA b y r e c o n s t i t u t i n g r i b o n u c i e o p r o t e i n particles f r o m t h i s RNA a n d total 30S p r o t e i n s , a n d i n c u b a t i n g t h e m in a n $30 extract. U n d e r t h e s e c o n d i t i o n s t h e 17S-CM RNA in r e c o n s t i t u t e d r i b o s o m e like particles is p a r t i a l l y converted to a species ~ q t h t h e electrophoretic m o b i l i t y of 16S rRNA. MATERIALS AND METHODS.
Buffers : TM1, Tris-HC1 pH 7.5, 10 mM ; MgCl~, 10 mM ; NH~C1, 60 mM ; :~-mercaptoethanol, 6 raM.
Preparation of unlabelled and proteins.
[35S]
labelled 30S
Total 30S p r o t e i n s are i s o l a t e d f r o m u n l a b e l l e d or [35S] labelled 30S r i b o s o m a l s u b u n i t s b y modificat i o n [4] of t h e urea-LiC1 p r o c e d u r e [5].
Reconstitution of ribonucleoprotein particles front 17S CM t~NA and 30S proteins. U n l a b e l l e d 30S p r o t e i n s (100 f~g) a n d [3H] labelled 17:S CM IINA (40 u g ; 150 000 c p m [3H]) are i n c u b a t e d in a final v o l u m e of 0.2 m l of r e c o n s t i t u t i o n buffer according to T r a u b a n d N o m u r a [6] a n d r e c o n s t i t u t e d particles are purified b y s e d i m e n t a t i o n of the r e a c t i o n m i x t u r e t h r o u g h a 5 m l 5-20 per cent sucrose g r a d i e n t in TM2 ( g r a d i e n t f r a c t i o n s c o n t a i n i n g recons t i t u t e d particles are stored s e p a r a t e l y at - - 2 0 ° C ) . P a r t i c l e s c o n t a i n i n g [35S] labelled p r o t e i n s a n d u n l a belled RNA are p r e p a r e d in t h e s a m e m a n n e r .
Maturation in vitro. One v o l u m e of s u s p e n s i o n of r e c o n s t i t u t e d p a r t i c l e s c o n t a i n i n g [3H] -17S-CM RNA (aliquot of a sucrose g r a d i e n t f r a c t i o n c o n t a i n i n g TM2) is m i x e d w i t h 0.1 v o l u m e of a n $30 e x t r a c t of E. coli MRE 600 p r e p a r e d in TM1, t h e c o n c e n t r a t i o n of Mg ++ is a d j u s t e d to
TM2, as TM1, b u t 0.1 mM MgCl,~ R e c o n s t i t u t i o n buffer, K p h o s p h a t e , pH 7.8, 5 m M ; MgC12, 20 m M ; KCI, 0.3 M ; ~ - m e r c a p t o e t h a n o l , 6 raM. E l e c t r o p h o r e s i s buffer, T r i s acetate, pH 7.8, 40 r a M ; Na acetate, 20 mM ; Na EDTA, 3 raM, 0.2 per cent ' w / v SDS.
Preparation of [~H] labelled CM-17S IRNA. E. coli MRE 600 is g r o w ~ at 37"C in m i n i m a l m e d i u m 63 [3] to a n A 420 n m of 0.8, c h l o r a m p h e n i c o l (100 ~tg/ml) is t h e n follo~ved 3 m i n u t e s later by [5-3H] u r a c y l (3 !~Ci/ml) a n d i n c u b a t i o n is c o n t i n u e d for 30 m i n u t e s . Cells are collected, a crude e x t r a c t is prepared as p r e v i o u s l y described [1] a n d s e d i m e n t e d t h r o u g h a 25 m l 5-20 per cent sucrose g r a d i e n t prep a r e d in TM1 ; f r a c t i o n s c o n t a i n i n g 18S-CM p a r t i c l e s are pooled, a n d t h e i r c o n t e n t of 17S-CM RNA is recovered b y p h e n o l e x t r a c t i o n i n t h e presence of SDS, a n d e t h a n o l precipitation. Abbreviations : CM, c h l o r a m p h e n i c o l . CM-RNA, RNA s y n t h e s i z e d in CM t r e a t e d E. coli. SDS, s o d i u m dodecyl sulfate. To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d : Unit6 de R e c h e r c h e s G6rontologiques U. 118, I.N.S.E.R.M., 29, r u e ~Vilhem - - 75016 Paris.
,
iL5
?~
,C :z
2~
E
/i
i,
i'~
I '°
°¢ "\
o o
Ng FRACTION
Sedimentation properlies of reconslitution product. A s a m p l e c o n t a i n i n g 10 000 cpm of I32P] labelled 30S r i b o s o m e s was added to the p r o d u c t s of a s t a n d a r d r e e o n s t i t u t i o n r e a c t i o n (150 000 epm of [3HI labelled 17S CM RNA) a n d the m i x t u r e w a s a n a l y s e d b y 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 as described i n Materifils a n d Methods. - - O - - C ) - - I32p] --Q--O-- [3H] lq'I6. 1 . -
J. A. Trdton and F. Hayes.
1008
10 m M a n d t h e m i x t u r e is i n c u b a t e d a t 37°C f o r 30 m i n u t e s . To d e t e c t t r a n s f o r m a t i o n o f CM 17S R N A to a 16S s p e c i e s R N A i s o l a t e d f r o m a s a m p l e of t h e r e a c t i o n m i x t u r e b y p h e n o l - S D S d e p r o t e i n i s a t i o n is a n a l y sed by eleetrophoresis in a 4 per cent polyacrylamide gel i n t h e p r e s e n c e o f [32p1 l a b e l l e d n o r m a l E. colt 16S r R N A a s d e s c r i b e d e l s e w h e r e [1].
RNA content of reconstituted particles. [3H] l a b e l l e d R N A i s o l a t e d f r o m t h e r e c o n s t i t u t e d p a r t i c l e s i n p e a k 1 o f figure 1 ~vas m i x e d w i t h [32p] l a b e l l e d n o r m a l 16S r R N A a n d t h e m i x t u r e w a s e x a m i -
J, J
RESULTS.
AI
Reconstitution of 30S particles containing 17S CM RNA. F i g u r e 1 sho'ws t h e 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 profile o f t h e p r o d u c t s o b t a i n e d b y i n c u b a t i o n of 17S CM R N A a n d t o t a l 30S r i b o s o m a l p r o t e i n i n r e c o n s t i t u t i o n b u f f e r . A b o u t 50 p e r c e n t of t h e 17S CM R N A is p r e s e n t i n t h e f o r m of a r i b o n u e l e o p r o t e i n p a r t i c l e w h i c h c o s e d i m e n t s w i t h c o n t r o l 30S r i b o s o m e s . F r a c t i o n s i n d i c a t e d b y arro'w i n figure 1 a n d those in the corresponding peak in a sucrose gradient i n w h i c h r e c o n s t i t u t i o n p r o d u c t s c o n t a i n i n g [suS]
5
A
," t
,o
*
4
B
.... '
'
I!
: ?d
,,*"
!i~ , " I *-~,'
E
E
o '
i
c
i
~,
i 5 5
t
Y2
+ i
4j g
i
,/
j,
lO
i a L
!
,
,
#l#l~
I "il!'htl
**
1(3 cm
Fro. 2 . -
Electrophoretic analgsis of RNA from reconstituted particles.
isolated
C o n t r o l [3H]-17S CM R N A a n d [3] l a b e l l e d R N A i s o l a t e d f r o m t h e r e c o n s t i t u t e d p a r t i c l e s i n p e a k 1, figure 1 (150 000 c p m i n e a c h case) w e r e m i x e d 'with [32p] l a b e l l e d 16S r R N A (4 000 c p m ) a n d t h e m i x t u r e s 'were a n a l y s e d b y p o l y a c r y l a m i d e gel e l e e t r o p h o r e s i s , P a n e l A, c o n t r o l [SH] 17S CM RNA. P a n e l B, ZH R N A e x t r a c t e d f r o m r e c o n s t i t u t e d p a r eles. --O--O-[32p] --O--O-[3H]
l a b e l l e d 30S p r o t e i n s a n d a n d u n l a b e l l e d 17S C M - R N A had been analysed were pooled and used for further analyses.
BIOCHIM1E, 1976, 58, n ° 8.
s
¢m
FIG. 3. - - Electrophoretic analysis of RNA extracted from reconstituted particles after i n v i t r o maturation.
I
5
lJ
4
o
i
e
Panels A and B : reconstituted particles containing [8H] l a b e l l e d 17S CM R N A (20 000 c p m ) w e r e i n c u b a t e d in vitro i n t h e p r e s e n c e of a n $30 e x t r a c t ( p a n e l B) or of a n e q u a l v o l u m e of b u f f e r TM1 ( p a n e l A) t h e R N A c o n t e n t s of i n c u b a t i o n m i x t u r e s w e r e i s o l a t e d a n d s u b j e c t e d to p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( M a t e r i a l s and Methods). P a n e l C : [3H] l a b e l l e d 17S CM R N A (15 000 c p m ) w a s i n c u b a t e d i n t h e p r e s e n c e o f a n $30 e x t r a c t u n d e r t h e c o n d i t i o n s d e s c r i b e d f o r in vitro m a t u r a t i o n (Mater i a l s a n d M e t h o d s ) a n d t h e R N A c o n t e n t of t h e i n c u b a t i o n m i x t u r e w a s i s o l a t e d a n d a n a l y s e d as b e f o r e . --O--O--
[32p]
--0--0--
[3H]
n e d b y e l e c t r o p h o r e s i s . A s h o w n i n figure 2 t h e r e c o vered RNA possesses the same eleetrophoretie mobility a s t h e o r i g i n a l 17S CM RNA. T h e r e s u l t s of p r e l i m i n a r y e x p e r i m e n t s i n ' w h i c h [35S] l a b e l l e d p r o t e i n s isolated from reconstituted particles were analysed b y t w o - d i m e n s i o n a l p o l y a c r y l a m i d e gel e l e e t r o p h o r e s i s [7] i n d i c a t e t h a t t h e s e p a r t i c l e s c o n t a i n m o s t b u t p o s s i b l y n o t all t h e 21 p r o t e i n s o f t h e 30S r i b o s o m e (data not shown).
In vitro
maturation
I n v i t r o maturation o[ 17S CM RNA in reconstituted particles. F i g u r e 3 s h o w s t h e r e s u l t s of p o l y a c r y l a m i d e gel a n a l y s e s of R N A s i s o l a t e d f r o m r e c o n s t i t u t e d p a r t i c l e s c o n t a i n i n g [3H]-17S CM R N A w h i c h h a d b e e n i n c u b a t e d in vitro i n t h e p r e s e n c e of a n $30 e x t r a c t or of a n e q u a l v o l u m e of b u f f e r . T h e m i g r a t i o n profiles i n figure 3 s h o w t h a t i n c u b a t i o n i n t h e p r e s e n c e of a n $30 e x t r a c t c o n v e r t s t h e a b s e n c e o f $30 c a u s e s n o c h a n g e i n i t s e l e c t r o p h o r e t i e m o b i l i t y . As s h o w n i n p a n e l C of figure 3 t r e a t m e n t of f r e e 17S CM R N A u n d e r t h e s a m e c o n d i t i o n s l e a d s to e x t e n s i v e n o n specific d e g r a d a t i o n w i t h o u t p r o d u c t i o n of s i g n i f i c a n t amounts of material with the electrophoretic mobility of 16S r R N A .
of CM-17S RNA.
1009
c o n v e r s i o n to a 16S f r o m in vitro t h a t of t h e n o r m a l 17S p r e - r i b o s o m a l R N A p r e s e n t i n 27S r i b o s o m a l p r e c u r s o r p a r t i c l e s . F u r t h e r w o r k 'will be n e c e s s a r y to establish the structural relationships between the r e c o n s t i t u t e d 30S p a r t i c l e s d e s c r i b e d h e r e a n d t h e 27S pre-ribosomal particles found in exponentially grow i n g E. coli [9] a n d b e t w e e n t h e 16S p r o d u c t f o r m e d f r o m 17S CM R N A i n o u r e x p e r i m e n t s a n d n o r m a l 16S r R N A (3'-OH a n d 5' p h o s p h a t e t e r m i n a l s e q u e n c e s m e t h y l a t e d b a s e s , ect...). Ho~wever o n t h e b a s e s of t h e results already obtained we believe that reconstituted p a r t i c l e s c o n t a i n i n g 17S CM R N A m a y p r o v e to be u s e f u l s n b s t r a t e s i n t h e s t u d y of t h e e n z y m o l o g y of ribosomal precursor RNA maturation.
A cknoiwledgments. DISCUSSION. T h e r e s u l t s d e s c r i b e d i n t h i s r e p o r t l e a d to t h e following conclusions : 1. W h e n E. coli 17S CM R N A is i n c u b a t e d w i t h t o t a l 30S p r o t e i n s u n d e r t h e r e c o n s t i t u t i o n c o n d i t i o n s defin e d b y T r a u b a n d N o m u r a [~] it is i n c o r p o r a t e d i n moderate yield into ribonucleoprotein particles which p o s s e s s a s e d i m e n t a t i o n c o e f f i c i e n t o f 30S a n d c o n t a i n m o s t t h o u g h p e r h a p s n o t all o f t h e p r o t e i n s o f t h e 30S r i b o s o m a l s u b u n i t . A n a l o g o u s r e s u l t s h a v e b e e n r e p e t e d p r e v i o u s l y b y W i r e m a n a n d S y p h e r d [81 w h o u s e d a p r e c u r s o r f r o m (< r e l a x e d >) m e t h i o n i n e a u x o t r o p h of E. coli g r o w n i n t h e a b s e n c e of m e t h i o n i n e . 2. I n c o r p o r a t i o n of 17S CM R N A i n t o 30S p a r t i c l e s does n o t c a u s e a n y a l t e r a t i o n i n its e l e c t r o p h o r e t i c mobility. 3. I n c u b a t i o n of r e c o n s t i t u t e d 30S p a r t i c l e s c o n t a i n i n g 17S CM R N A i n t h e p r e s e n c e of a n $30 e x t r a c t l e a d s to c o n v e r s i o n of t h i s R N A to a s p e c i e s w i t h a n e l e c t r o p h o r e t i e m o b i l i t y i d e n t i c a l to t h a t of n o r m a l 16S r R N A . T h e b e h a v i o u r o f 17S CM R N A i n t h e r e c o n s t i t u t i o n p r o c e s s t h u s m i m i c s t h a t of m a t u r e 16S r R N A a n d i t s
BIOCHIMIE, 1976, 58, n ° 8.
Financial support for this work was provided by t h e C e n t r e N a t i o n a l de la R e c h e r c h e s c i e n t i f i q u e (Sqnipe de R e c h e r c h e n ° 101), b y t h e N o r t h A t l a n t i c T r e a t y O r g a n i s a t i o n ( G r a n t 664), t h e C o m m i s s a r i a t ~_ l ' E n e r g i e A t o m i q u e , a n d t h e F o n d s de ]a R e c h e r c h e M(~dicale F r a n g a i s e . REFERENCES. 1. H a y e s , F. ~ V a s s e u r , M. (1976) Enr. J. Biochem., 61, 433-442. 2. L o w r y , C. V. ~ D a l h b e r g , J. E. (1971) Nature N. B., 232, 52-54. 3. In experiments in molecular genetics (1972). Ed. J e f f r e y H. Miller, Cold S p r i n g H a r b o r L a b o r a t o r y 431. 4. S p i t n i k - E l s o n , P. (1965) Biochem. Biophys. Res. Commun., 18, 557-562. 5. B a r r i t a u l t , D., E x p e r t - B e z a n g o n , A., Gu6ri.n, M. F. H a y e s , D. H. (1976) Eur. J. Bioehem., m p r e s s . 6. T r a u b , P. ~ N o m u r a , M. (1969) J. Mol. Biol., 40, 391-413. 7. K a l t s c h m i d t , E. ~ W i t t m a n n , H. G. (1970) Anal. Bioehem., 3~, 401-412. 8. W i r e m a n , J. ~ S y p h e r d , P. (1974) Biochemistry, 13, 1215-1221. 9. H a y e s , F. ~ H a y e s , D. H. (1971) Biochimie, 53, 369382.
Conversion of Escherichia coli 17S ehloramphenieol RNA to a 16S RNA in dtro. J a c q u e s A l a i n TRI~TON 0 a n d F r a n ~ , o i s e HAYES. Laboratoire de Chimie Cellulaire, l n s t i t u t de Biologic Physieo-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris. (22-$-1976).
INTRODUCTION. We h a v e a l r e a d y described t h e in vitro m a t u r a t i o n of t h e 17S p r e c u r s o r of E. coli 16S rRNA by i n c u b a t i o n of 27S p r e c u r s o r p a r t i c l e s i s o l a t e d f r o m p u l s e labelled e x p o n e n t i a l l y g r o w i n g cells in a b a c t e r i a l $30 e x t r a c t [1]. T h e difficulty of p r e p a r i n g t h e 27S p r e c u r s o r in large a m o u n t h a s led u s to i n v e s t i g a t e the p o s s i b i l i t y of u s i n g i n s t e a d p a r t i c l e s r e c o n s t i t u t e d in vitro f r o m total 30S r i b o s o m a l p r o t e i n s a n d 17S c h l o r a m p h e n i c o l RNA. T h i s RNA w h i c h r e s e m b l e s t h e 17S p r e c u r s o r species p r e s e n t in t h e n a t u r a l 27S particle i n b e i n g submethylated and in possessing extra polynucleotide s e q u e n c e s at each of its e x t r e m i t i e s [2] is e a s i l y i s o l a t e d i n large q u a n t i t i e s . I n t h i s report, we p r e s e n t t h e r e s u l t s of e x p e r i m e n t s in w h i c h we h a v e s t u d i e d t h e in oitro processing of 17S-CM RNA b y r e c o n s t i t u t i n g r i b o n u c i e o p r o t e i n particles f r o m t h i s RNA a n d total 30S p r o t e i n s , a n d i n c u b a t i n g t h e m in a n $30 extract. U n d e r t h e s e c o n d i t i o n s t h e 17S-CM RNA in r e c o n s t i t u t e d r i b o s o m e like particles is p a r t i a l l y converted to a species ~ q t h t h e electrophoretic m o b i l i t y of 16S rRNA. MATERIALS AND METHODS.
Buffers : TM1, Tris-HC1 pH 7.5, 10 mM ; MgCl~, 10 mM ; NH~C1, 60 mM ; :~-mercaptoethanol, 6 raM.
Preparation of unlabelled and proteins.
[35S]
labelled 30S
Total 30S p r o t e i n s are i s o l a t e d f r o m u n l a b e l l e d or [35S] labelled 30S r i b o s o m a l s u b u n i t s b y modificat i o n [4] of t h e urea-LiC1 p r o c e d u r e [5].
Reconstitution of ribonucleoprotein particles front 17S CM t~NA and 30S proteins. U n l a b e l l e d 30S p r o t e i n s (100 f~g) a n d [3H] labelled 17:S CM IINA (40 u g ; 150 000 c p m [3H]) are i n c u b a t e d in a final v o l u m e of 0.2 m l of r e c o n s t i t u t i o n buffer according to T r a u b a n d N o m u r a [6] a n d r e c o n s t i t u t e d particles are purified b y s e d i m e n t a t i o n of the r e a c t i o n m i x t u r e t h r o u g h a 5 m l 5-20 per cent sucrose g r a d i e n t in TM2 ( g r a d i e n t f r a c t i o n s c o n t a i n i n g recons t i t u t e d particles are stored s e p a r a t e l y at - - 2 0 ° C ) . P a r t i c l e s c o n t a i n i n g [35S] labelled p r o t e i n s a n d u n l a belled RNA are p r e p a r e d in t h e s a m e m a n n e r .
Maturation in vitro. One v o l u m e of s u s p e n s i o n of r e c o n s t i t u t e d p a r t i c l e s c o n t a i n i n g [3H] -17S-CM RNA (aliquot of a sucrose g r a d i e n t f r a c t i o n c o n t a i n i n g TM2) is m i x e d w i t h 0.1 v o l u m e of a n $30 e x t r a c t of E. coli MRE 600 p r e p a r e d in TM1, t h e c o n c e n t r a t i o n of Mg ++ is a d j u s t e d to
TM2, as TM1, b u t 0.1 mM MgCl,~ R e c o n s t i t u t i o n buffer, K p h o s p h a t e , pH 7.8, 5 m M ; MgC12, 20 m M ; KCI, 0.3 M ; ~ - m e r c a p t o e t h a n o l , 6 raM. E l e c t r o p h o r e s i s buffer, T r i s acetate, pH 7.8, 40 r a M ; Na acetate, 20 mM ; Na EDTA, 3 raM, 0.2 per cent ' w / v SDS.
Preparation of [~H] labelled CM-17S IRNA. E. coli MRE 600 is g r o w ~ at 37"C in m i n i m a l m e d i u m 63 [3] to a n A 420 n m of 0.8, c h l o r a m p h e n i c o l (100 ~tg/ml) is t h e n follo~ved 3 m i n u t e s later by [5-3H] u r a c y l (3 !~Ci/ml) a n d i n c u b a t i o n is c o n t i n u e d for 30 m i n u t e s . Cells are collected, a crude e x t r a c t is prepared as p r e v i o u s l y described [1] a n d s e d i m e n t e d t h r o u g h a 25 m l 5-20 per cent sucrose g r a d i e n t prep a r e d in TM1 ; f r a c t i o n s c o n t a i n i n g 18S-CM p a r t i c l e s are pooled, a n d t h e i r c o n t e n t of 17S-CM RNA is recovered b y p h e n o l e x t r a c t i o n i n t h e presence of SDS, a n d e t h a n o l precipitation. Abbreviations : CM, c h l o r a m p h e n i c o l . CM-RNA, RNA s y n t h e s i z e d in CM t r e a t e d E. coli. SDS, s o d i u m dodecyl sulfate. To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d : Unit6 de R e c h e r c h e s G6rontologiques U. 118, I.N.S.E.R.M., 29, r u e ~Vilhem - - 75016 Paris.
,
iL5
?~
,C :z
2~
E
/i
i,
i'~
I '°
°¢ "\
o o
Ng FRACTION
Sedimentation properlies of reconslitution product. A s a m p l e c o n t a i n i n g 10 000 cpm of I32P] labelled 30S r i b o s o m e s was added to the p r o d u c t s of a s t a n d a r d r e e o n s t i t u t i o n r e a c t i o n (150 000 epm of [3HI labelled 17S CM RNA) a n d the m i x t u r e w a s a n a l y s e d b y 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 as described i n Materifils a n d Methods. - - O - - C ) - - I32p] --Q--O-- [3H] lq'I6. 1 . -
J. A. Trdton and F. Hayes.
1008
10 m M a n d t h e m i x t u r e is i n c u b a t e d a t 37°C f o r 30 m i n u t e s . To d e t e c t t r a n s f o r m a t i o n o f CM 17S R N A to a 16S s p e c i e s R N A i s o l a t e d f r o m a s a m p l e of t h e r e a c t i o n m i x t u r e b y p h e n o l - S D S d e p r o t e i n i s a t i o n is a n a l y sed by eleetrophoresis in a 4 per cent polyacrylamide gel i n t h e p r e s e n c e o f [32p1 l a b e l l e d n o r m a l E. colt 16S r R N A a s d e s c r i b e d e l s e w h e r e [1].
RNA content of reconstituted particles. [3H] l a b e l l e d R N A i s o l a t e d f r o m t h e r e c o n s t i t u t e d p a r t i c l e s i n p e a k 1 o f figure 1 ~vas m i x e d w i t h [32p] l a b e l l e d n o r m a l 16S r R N A a n d t h e m i x t u r e w a s e x a m i -
J, J
RESULTS.
AI
Reconstitution of 30S particles containing 17S CM RNA. F i g u r e 1 sho'ws t h e 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 profile o f t h e p r o d u c t s o b t a i n e d b y i n c u b a t i o n of 17S CM R N A a n d t o t a l 30S r i b o s o m a l p r o t e i n i n r e c o n s t i t u t i o n b u f f e r . A b o u t 50 p e r c e n t of t h e 17S CM R N A is p r e s e n t i n t h e f o r m of a r i b o n u e l e o p r o t e i n p a r t i c l e w h i c h c o s e d i m e n t s w i t h c o n t r o l 30S r i b o s o m e s . F r a c t i o n s i n d i c a t e d b y arro'w i n figure 1 a n d those in the corresponding peak in a sucrose gradient i n w h i c h r e c o n s t i t u t i o n p r o d u c t s c o n t a i n i n g [suS]
5
A
," t
,o
*
4
B
.... '
'
I!
: ?d
,,*"
!i~ , " I *-~,'
E
E
o '
i
c
i
~,
i 5 5
t
Y2
+ i
4j g
i
,/
j,
lO
i a L
!
,
,
#l#l~
I "il!'htl
**
1(3 cm
Fro. 2 . -
Electrophoretic analgsis of RNA from reconstituted particles.
isolated
C o n t r o l [3H]-17S CM R N A a n d [3] l a b e l l e d R N A i s o l a t e d f r o m t h e r e c o n s t i t u t e d p a r t i c l e s i n p e a k 1, figure 1 (150 000 c p m i n e a c h case) w e r e m i x e d 'with [32p] l a b e l l e d 16S r R N A (4 000 c p m ) a n d t h e m i x t u r e s 'were a n a l y s e d b y p o l y a c r y l a m i d e gel e l e e t r o p h o r e s i s , P a n e l A, c o n t r o l [SH] 17S CM RNA. P a n e l B, ZH R N A e x t r a c t e d f r o m r e c o n s t i t u t e d p a r eles. --O--O-[32p] --O--O-[3H]
l a b e l l e d 30S p r o t e i n s a n d a n d u n l a b e l l e d 17S C M - R N A had been analysed were pooled and used for further analyses.
BIOCHIM1E, 1976, 58, n ° 8.
s
¢m
FIG. 3. - - Electrophoretic analysis of RNA extracted from reconstituted particles after i n v i t r o maturation.
I
5
lJ
4
o
i
e
Panels A and B : reconstituted particles containing [8H] l a b e l l e d 17S CM R N A (20 000 c p m ) w e r e i n c u b a t e d in vitro i n t h e p r e s e n c e of a n $30 e x t r a c t ( p a n e l B) or of a n e q u a l v o l u m e of b u f f e r TM1 ( p a n e l A) t h e R N A c o n t e n t s of i n c u b a t i o n m i x t u r e s w e r e i s o l a t e d a n d s u b j e c t e d to p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( M a t e r i a l s and Methods). P a n e l C : [3H] l a b e l l e d 17S CM R N A (15 000 c p m ) w a s i n c u b a t e d i n t h e p r e s e n c e o f a n $30 e x t r a c t u n d e r t h e c o n d i t i o n s d e s c r i b e d f o r in vitro m a t u r a t i o n (Mater i a l s a n d M e t h o d s ) a n d t h e R N A c o n t e n t of t h e i n c u b a t i o n m i x t u r e w a s i s o l a t e d a n d a n a l y s e d as b e f o r e . --O--O--
[32p]
--0--0--
[3H]
n e d b y e l e c t r o p h o r e s i s . A s h o w n i n figure 2 t h e r e c o vered RNA possesses the same eleetrophoretie mobility a s t h e o r i g i n a l 17S CM RNA. T h e r e s u l t s of p r e l i m i n a r y e x p e r i m e n t s i n ' w h i c h [35S] l a b e l l e d p r o t e i n s isolated from reconstituted particles were analysed b y t w o - d i m e n s i o n a l p o l y a c r y l a m i d e gel e l e e t r o p h o r e s i s [7] i n d i c a t e t h a t t h e s e p a r t i c l e s c o n t a i n m o s t b u t p o s s i b l y n o t all t h e 21 p r o t e i n s o f t h e 30S r i b o s o m e (data not shown).
In vitro
maturation
I n v i t r o maturation o[ 17S CM RNA in reconstituted particles. F i g u r e 3 s h o w s t h e r e s u l t s of p o l y a c r y l a m i d e gel a n a l y s e s of R N A s i s o l a t e d f r o m r e c o n s t i t u t e d p a r t i c l e s c o n t a i n i n g [3H]-17S CM R N A w h i c h h a d b e e n i n c u b a t e d in vitro i n t h e p r e s e n c e of a n $30 e x t r a c t or of a n e q u a l v o l u m e of b u f f e r . T h e m i g r a t i o n profiles i n figure 3 s h o w t h a t i n c u b a t i o n i n t h e p r e s e n c e of a n $30 e x t r a c t c o n v e r t s t h e a b s e n c e o f $30 c a u s e s n o c h a n g e i n i t s e l e c t r o p h o r e t i e m o b i l i t y . As s h o w n i n p a n e l C of figure 3 t r e a t m e n t of f r e e 17S CM R N A u n d e r t h e s a m e c o n d i t i o n s l e a d s to e x t e n s i v e n o n specific d e g r a d a t i o n w i t h o u t p r o d u c t i o n of s i g n i f i c a n t amounts of material with the electrophoretic mobility of 16S r R N A .
of CM-17S RNA.
1009
c o n v e r s i o n to a 16S f r o m in vitro t h a t of t h e n o r m a l 17S p r e - r i b o s o m a l R N A p r e s e n t i n 27S r i b o s o m a l p r e c u r s o r p a r t i c l e s . F u r t h e r w o r k 'will be n e c e s s a r y to establish the structural relationships between the r e c o n s t i t u t e d 30S p a r t i c l e s d e s c r i b e d h e r e a n d t h e 27S pre-ribosomal particles found in exponentially grow i n g E. coli [9] a n d b e t w e e n t h e 16S p r o d u c t f o r m e d f r o m 17S CM R N A i n o u r e x p e r i m e n t s a n d n o r m a l 16S r R N A (3'-OH a n d 5' p h o s p h a t e t e r m i n a l s e q u e n c e s m e t h y l a t e d b a s e s , ect...). Ho~wever o n t h e b a s e s of t h e results already obtained we believe that reconstituted p a r t i c l e s c o n t a i n i n g 17S CM R N A m a y p r o v e to be u s e f u l s n b s t r a t e s i n t h e s t u d y of t h e e n z y m o l o g y of ribosomal precursor RNA maturation.
A cknoiwledgments. DISCUSSION. T h e r e s u l t s d e s c r i b e d i n t h i s r e p o r t l e a d to t h e following conclusions : 1. W h e n E. coli 17S CM R N A is i n c u b a t e d w i t h t o t a l 30S p r o t e i n s u n d e r t h e r e c o n s t i t u t i o n c o n d i t i o n s defin e d b y T r a u b a n d N o m u r a [~] it is i n c o r p o r a t e d i n moderate yield into ribonucleoprotein particles which p o s s e s s a s e d i m e n t a t i o n c o e f f i c i e n t o f 30S a n d c o n t a i n m o s t t h o u g h p e r h a p s n o t all o f t h e p r o t e i n s o f t h e 30S r i b o s o m a l s u b u n i t . A n a l o g o u s r e s u l t s h a v e b e e n r e p e t e d p r e v i o u s l y b y W i r e m a n a n d S y p h e r d [81 w h o u s e d a p r e c u r s o r f r o m (< r e l a x e d >) m e t h i o n i n e a u x o t r o p h of E. coli g r o w n i n t h e a b s e n c e of m e t h i o n i n e . 2. I n c o r p o r a t i o n of 17S CM R N A i n t o 30S p a r t i c l e s does n o t c a u s e a n y a l t e r a t i o n i n its e l e c t r o p h o r e t i c mobility. 3. I n c u b a t i o n of r e c o n s t i t u t e d 30S p a r t i c l e s c o n t a i n i n g 17S CM R N A i n t h e p r e s e n c e of a n $30 e x t r a c t l e a d s to c o n v e r s i o n of t h i s R N A to a s p e c i e s w i t h a n e l e c t r o p h o r e t i e m o b i l i t y i d e n t i c a l to t h a t of n o r m a l 16S r R N A . T h e b e h a v i o u r o f 17S CM R N A i n t h e r e c o n s t i t u t i o n p r o c e s s t h u s m i m i c s t h a t of m a t u r e 16S r R N A a n d i t s
BIOCHIMIE, 1976, 58, n ° 8.
Financial support for this work was provided by t h e C e n t r e N a t i o n a l de la R e c h e r c h e s c i e n t i f i q u e (Sqnipe de R e c h e r c h e n ° 101), b y t h e N o r t h A t l a n t i c T r e a t y O r g a n i s a t i o n ( G r a n t 664), t h e C o m m i s s a r i a t ~_ l ' E n e r g i e A t o m i q u e , a n d t h e F o n d s de ]a R e c h e r c h e M(~dicale F r a n g a i s e . REFERENCES. 1. H a y e s , F. ~ V a s s e u r , M. (1976) Enr. J. Biochem., 61, 433-442. 2. L o w r y , C. V. ~ D a l h b e r g , J. E. (1971) Nature N. B., 232, 52-54. 3. In experiments in molecular genetics (1972). Ed. J e f f r e y H. Miller, Cold S p r i n g H a r b o r L a b o r a t o r y 431. 4. S p i t n i k - E l s o n , P. (1965) Biochem. Biophys. Res. Commun., 18, 557-562. 5. B a r r i t a u l t , D., E x p e r t - B e z a n g o n , A., Gu6ri.n, M. F. H a y e s , D. H. (1976) Eur. J. Bioehem., m p r e s s . 6. T r a u b , P. ~ N o m u r a , M. (1969) J. Mol. Biol., 40, 391-413. 7. K a l t s c h m i d t , E. ~ W i t t m a n n , H. G. (1970) Anal. Bioehem., 3~, 401-412. 8. W i r e m a n , J. ~ S y p h e r d , P. (1974) Biochemistry, 13, 1215-1221. 9. H a y e s , F. ~ H a y e s , D. H. (1971) Biochimie, 53, 369382.