Formation and decay of polyribosomes and ribosomes during the inhibition of protein synthesis and recovery

BIOCHIMIE, 1971, 53, 987-1q~00.

Formation and decay of polyribosomes and ribosomes during the inhibition of protein synthesis and recovery C. (:OCIT().

Dept. o[ General Microbiology and Molecular Genetics, University of Louvain, Bruxelles 1206, Belgium. (18-10-1971). Summar!l. -- The two components of virginialnycin, M and S, when added separately to exponentia'l cultures of Bacillus subtilis, stop in a reversible f a s h i o n cell gro~vth and protein formation. When bacteria are incubated with both components, a loss of viability occurs. The assembly of polyribosomes is not altered by virginiamycin, but their decay is prevented. This observation accounts for the sudden halt in protein synthesis and for the protection of messenger RNA, which were observed in the presence of the antibiotic. Formation of ribosomes is blocked reversibly by single virginiamycin components. Ribosomal RNA which is synthesized u n d e r these conditions associates with previously synthesized non ribosomal proteins and form labile ribonucleoproteins, which can be sedimented by ultraeentrifugatiou. Upon removal of the drug such complexes dissociate, and their RNA component binds to newly formed ribosomal proteins and yields normal ribosomal snhnnits. When t)oth virginiamyein components are present, the inhibition 6f protein formation is not relieved, and no ribosomal subunits are formed, upon t r a n s f e r of the cells to antibiotic-free medium. This observation explains the lethal action of the association of virginialnycin M anti S.

INTRODUCTION. T h e p e c u l i a r f e a t u r e of t h e a n t i b i o t i c v i r g i n i a m y c i n (cf. t h e r e v i e w s of VASQUF.Z f_l, 2], a n d of WnISBLUM a n d DAVIES [3]) is t h a t it c o n t a i n s t w o c o m p o n e n t s , M a n d S, w h i c h h a v e a s y n e r g i s t i c effect. E a c h c o m p o n e n t is c a p a b l e o f l o w e r i n g t h e m i n i m a l i n h i b i t o r y c o n c e n t r a t i o n of its p a r t n e r . I n a d d i t i o n , w h i l e M a n d S s e p a r a t e l y do n o t a l t e r cell v i a b i l i t y , t h e i r a s s o c i a t i o n d e c r e a s e s t h e c o l o n y f o r m i n g c a p a c i t y of s e n s i t i v e m i c r o o r g a n i s m s [41.

Abbreviations used : rRNA, mRNA and tRNA = rii)osolne, messenger anti t r a n s f e r RNA ; HMW-RNA and LMW-RNA = highand law-molecular weight RNA (chromatography fractions) ; Leu = leucine ; Tro = t r y p t o p h a n ; Glc = g l u c o s e ; U = uridine ; T = thymidine ; ECD = enzymic digest of casein ; BSA = bovine serum alhumin ; TCA = trichloroaeetic acid ; EDTA : ethylene diaminetctraacetic acid ; SDS : sodium dodecyl s u l p h a t e ; DOG = sodium deoxycholate ; MAK : kieselguhr coated with methylated albumin ; Klett Units (K. U.) = A~,,m:* × 103/2 : L and OS = media for Bacillus subtilis ; TMNM and TMKM : buffers for particles ; M, S, K, and K: = virginiamyein components.

T h e m e t a b o l i s m of m a c r o m o l e c u l e s is q u i c k l y and Frofoundly altered by virginiamycin. Appar e n t l y , p r o t e i n f o r m a t i o n is t h e m a i n t a r g e t , as s h o w n b y its i m m e d i a t e h a l t u p o n a d d i t i o n of e i t h e r M o r S. T h e m a i n a l t e r a t i o n s of n u c l e i c a c i d m e t a b o l i s m a r e as f o l l o w s : a) t h e h a l f - l i f e of m e s s e n g e r RNA is i n c r e a s e d ; b) r i b o s o m a l RNA is n n d e r m e t h y l a t e d a n d m e t a b o l i c a l l y u n s t a b l e ; c) f o r m a t i o n of 23 S r i b o s o m a l RNA is p r e f e r e n t i a l l y i n h i b i t e d [4, 5]. The m e c h a n i s m by w h i c h v i r g i n i a m y c i n M stops protein formation has been elucidated by recent works with cell-free systems. The antibiotic b l o c k s s p e c i f i c a l l y t h e a c c e p t o r site of r i b o s o m e s a n d , t h r o u g h an e f f e c t of s t e r i c h i n d r a n c e , it p r e v e n t s t h e d o n o r site f r o m f u n c t i o n i n g E6!. C o n v e r sely, the m e c h a n i s m o f a c t i o n of v i r g i n i a m y c i n S is u n k n o w n . T h e p r e s e n t i n v e s t i g a t i o n is a n a t t e m p t at c o r r e l a t i n g the a l t e r a t i o n s w h i c h w e r e p r e v i o u s l y o b s e r v e d in n u c l e i c a c i d a n d p r o t e i n m e t a b o l i s m . In a d d i t i o n , t h e b a c t e r i c i d a l e f f e c t of t h e a s s o c i a tion of M a n d S w i l l be i n v e s t i g a t e d at a m o l e c u l a r level. F o r t h i s p u r p o s e , t h e f o r m a t i o n of r i b o s o m e s as w e l l as t h e a s s e m b l y a n d f u n c t i o n of p o l y r i b o s o m e s h a s b e e n e x p l o r e d in Bacillus subtilts w h i c h w a s g r o w n in t h e p r e s e n c e of v i r g i n i a mycin.

C. Cocito.

988 MATERIALS AND METHODS.

Concentration of polyribosomes and ribosomes, and preparation of particle-free cytoplasm. To

Bacterial strains, .qrowth media and labelling technlques. In different experiments a prototroph strain of B. subtilis (168/6) and two auxotrophs,

homogenates of labelled cells a ten-fold larger a m o u n t of lysed unlabelled cells was added, and the mixtures were layered over a 35 p. cent (w/v) solution of sucrose in TMNM buffer. Upon centrifugation for 1 to 2 h r at 150,000 g in an angular Ti-50 rotor (Spinco), pellets of polyribosomes and 70 S active units were separated from subunits r e m a i n i n g in the supernatants. When the sucrose was omitted, ultracentrifugation u n d e r the same c o n d i t i o n s yielded a pellet c o n t a i n i n g the whole set of cytoplasmic particles and a supernatant. The u p p e r two-third of the latter were w i t h d r a w n with curved-tip pipettes for analysis of <> cytoplasm. In some experiments polysomes plus ribosomes were harvested by filtration through 40-~micropore membranes. Membranes were washed 3 times with ice-cold buffers without d r y i n g and eluted with TMNM and TMKM. The suspensions of particles were processed without delay. These procedures were based on studies from other laboratories [13, 14, 15].

168/2 (Leu-Trp-) and A26 (U-Trp-), were employed. Techni(lues for growing different strains, labelling proteins and nucleic acids, harvesting and d i s r u p t i n g cells, are detailed in a previous p u b l i c a t i o n of this series [4q.

Buffered sohltions used for particles. <> : 0.01 tris-HCl pH 7.4, 0.01 M Mg acetate, 0.05 M NH4C1, 6 mM ~-mercapto-ethanol. <> : 0.01 M tris-HCl, pH 7.4, 0.01 M MgC12, 0.08 M KC1, 6 mM ~-mercapto-ethanol. Some experiments with labelled v i r g i n i a m y c i n s were carried nut in the absence of [3-mercapto-ethanol (<< TMN >> and ¢ TMK >> buffers). For p r e p a r a t i o n and analysis of ribosome subunits, low-Mg solutions obtained by diluting 20-fold all the TMNM and TMKM c o m p o n e n t s but iris (¢TMNM-I/20>> and <> buffers) were employed. Preparation and disruption of protoplasts. Samples of labelled cells were r a p i d l y chilled to neat" the freezing point by addition of crushed ice and immersion in an ethanol-CO 2 bath, ten- to 100fold larger amounts of unlabelled bacteria were added, and cells were collected by centrifugation at 130,000 g-rain. Pellets were suspended in a 25 p. cent (w/v) sucrose solution in TMKM, and incubated for 5 m i n either at 4 ° or at 23 ° with 1 mg lysozyme/10 's cells/ml. Protoplasts were harvested at 190,000 g-rain anti shocked in ice-cold TMNM c o n t a i n i n g 1 t~g/ml of purified DNAase. Membranes were sedimented at 350,000 g-rain, and the s u p e r n a t a n t s carefully w i t h d r a w n with curved-tip pipettes were processed immediately. Another procedure was used for r a p i d lysis of small a m o u n t of labelled cells. Aliquots of bacterial cultures were rapidly chilled with ice and solid CO2 and filtered through 40-~L micropore filters u n d e r high pressure. Harvested cells (about 10 s bacteria) were immediately transferred to an ice-cold <>, Mann), 1.5 p. cent Na-deoxycholate (BDH) and 1 to 10 ~g of electrophoretically purified DNAase (Worthington). After 5-rain i n c u b a t i o n in melting ice, the homogenates were centrifuged at 350,000 g-rain, and the s u p e r n a t a n t used for preparation of polysomes. Similar procedures of cell d i s r u p t i o n have been developed by others [7, 8, 9, tO, ll, 12].

BIOCHIMIE, 1971, 53, n ° 9.

Ultracentrifuyal fractionation of polyribosomes: ribosomes and particle-free cytoplasm. Whole homogenates and concentrated p r e p a r a t i o n s of particles from labelled cells were fractionated in 15 to 30 p. cent (w/v) gradients of sucrose in one of the <>. Average centrifugation times were : 2 to 3 hr for polyribosomes, 6 to 14 h r for monosomes and ribosomal subunits, and 2 to 3 days for particle-free cytoplasms. Spinco (SW 25.1 rotor at 23,000 r e v / m i n ) and MSE (swing-out rotor 30 K at 30,000 r e v / m i n ) were employed for density gradient fractionation. Gradients were s c a n n e d by use of a continuous-flow ultraviolet absorption meter (Gilford 2000), and fractions were collected for radioactivity measurement.

Preparation and analysis of RNA from subcellular fractions. In some cases the RNA was released from polyribosomes and ribosomes by addition of 1 p. cent (w/v, f.c.) SDS. In most cases, however, suspensions of particles and cell homogenates were extracted with watersaturated phenol and 0.5 p. cent SDS at 4 °. After centrifugation, the aqueous phases were reextracted with phenol and ether, and the RNA was precipitated with-ethanol at - - 1 8 ° C for 18 hr. Solutions of RNA p r e p a r e d with either method, were layered on the top of 15 to 30 p. cent (w/v) sucrose gradients in 0.10 M NaC1 0.2 M acetic acid/Na-acetate buffer pH 5.2 c o n t a i n i n g 2 mg SDS per ml, and centrifuged for 24 hr in a swingout rotor SW 25.1 (Spinco) at 23,000 r e v / m i n .

Polgribosomes

during protein inhibition.

and Ribosomes

Puri[ication of unlabelled and labelled vir~iniamycin components. Separation and purifica-

Radioactivity determination of particles and molecules. S u s p e n s i o n s of p o l y s o m e s and ribos o m e s in either TMKM or TMNM bufl'ers w e r e

tion of M and S factors from crude virginiamycin preparations were achieved on column of silica gel [16~. Crystalline preparations of the two factors [!17J were employed through the entire work. The corresponding aqueous solutions were freshly prepared before each experiment. ~H-labelled dihydrovirginiamycin S (factors K~ and K_2) was obtained by reduction of crystalline virginiamycin S with Na (all) borohydride. The two epimers were separated by chromatography on silica gels unpublished data of G. JANSSEN and H. VANDnnHAE6n. Preliminary experiments have shown that virginiamycin factors S, K~, and K~ possess comparable activities on B. subtitis. :~H-labelled virginiamycin M was obtained by irradiation of crystalline preparation of the anti-

f i l t e r e d o n 40m m i c r o p o r e m e m b r a n e s p r e - s o a k e d in the s a m e solution. Collected particles w e r e w a s h e d 3 t i m e s w i t h i c e - c o l d b u f f e r , a i r d r i e d and

counted in a scintillation counter. Alternatively,

ice-cold

suspensions

of l a b e l l e d

particles and molecules were chilled in ice, 50 ~g of b o v i n e s e r u m a l b u m i n and TCA to 0.5 M (fie.) w e r e a d d e d , a n d s a m p l e s w e r e f i l t e r e d 30 m i n

later through micropore membranes pre-soaked in TCA f o r 18 hr at 4°C. P r e c i p i t a t e s w e r e w a s h e d on filters w i t h i c e - c o l d 0.3 M TCA and air d r i e d .

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C. Cocito.

990

biotic with t r i t i u m gas (Wilzbach method). (all) M~ was then purified by preparative thin layer chromatography [191. Few experiments were carried out with 14C-labelled v i r g i n i a m y c i n M~, w h i c h was obtained by growing the p r o d u c i n g strain of S t r e p t o m y c e s virginiae in the presence of 04C) acetate [ 1 9 ] .

RESULTS.

I. Formation of ribosomal subunits in virginiamycin-treated cells. Since the two v i r g i n i a m y c i n components M and S, separately and in c o m b i n a t i o n , i n h i b i t sharply

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Biochemical and biophysical determinations. Procedures for colorimetric, radioactivity and spectrophotometric d e t e r m i n a t i o n s have been described, and the source of chemicals and radioisotopes have been mentioned, in a previous publication [4]. BIOCH1MIE, 1971, 53, n ° 9.

the i n c o r p o r a t i o n of labelled amino acids into polypeptides [4], also the synthesis of ribosomal proteins should be stopped by the drug. In this case, v i r g i n i a m y c i n is expected to i n h i b i t the formation of ribosomes, unless a large pool of ribosomal proteins exists, w h i c h still allows the

Polyribosomes and Ribosomes during protein inhibition. assembly of particles to take place when the synthesis of their precursors is shut off.

v i r g i n i a m y c i n M, no 50 S subunits, and little if any 30 S ribosome subunits, became labelled (Fig. 2A). After chasing in the presence of the inhibitor, most of the radioactivity was found associated w i t h particles less than 25 S (Fig. 2B). When the drug was removed, the RNA of these particles entered normal ribosomal subunits, as shown by the overlapping of the peaks of radioactivity and a b s o r b a n c e (Fig. 2C). A similar reversible i n h i b i t i o n of ribosome formation occured after i n c u b a t i o n w i t h v i r g i n i a m y c i n S (Fig. 3A, B, C). However, if the cells were previously incu.. bated with a twenty-fold lesser amount of the two

To test this hypothesis, ceils were harvested by centrifugation after 15 min of labelling with (all) uracil in the presence of v i r g i n i a m y c i n . It has been shown previously that most of the radioactivity of 15-min pulse was present in 23 S and 16 S rRNA [4]. Labelled bacteria were divided into three aliquots : one sample was disrupted immediatly, whereas the other two samples were grown for 30 m i n in media c o n t a i n i n g an excess of (1H) uracil (chasing), xvifll or without virgin i a m y c i n . Upon addition of large amount of

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unlabelled cells (marker), the samples were disrupted by compression, and the ribosomes were sedimented by nltracentrifugation, resuspended in low-Mg buffer and fractionated in a sucrose density gradient. In the control samples, two peaks of radioactivity overlapping the 50 S and 30 S ribosome subunits were found, and no t u r n o v e r was observed upon chasing (Fig. 1A, B). In the presence of

BIOCHIMIE, 1971, 53, n ° 9.

v i r g i n i a m y c i n components, no sign of recovery appeared after removal of the drug (Fig. 4A, B, C) : the block of ribosome formation was, thus, irreversible. Data presented in Fig. 1 to 4 indicated that single v i r g i n i a m y c i n components blocked in a reversible fashion the formation of ribosomal subunits, whereas their association resulted in a p e r m a n e n t halt of ribosome formation. 67

C. C o c i l o .

992 1I. S y n t h e s i s and ribosomal RNA.

turnover

o[ p a r t i c l e - b o u n d

C o n t r o l s s h o w e d t w o m a j o r p e a k s of 23 S a n d 16 S r R N A , a n d o n e m i n o r p e a k of 5 S RNA (Fig. 5A, B), w i t h no r a d i o a c t i v i t y in t h e 10 S r e g i o n a n d n o s i g n of t u r n o v e r u p o n c h a s i n g . T h e RNA w h i c h w a s l a b e l l e d in t h e p r e s e n c e of v i r g i -

To g a t h e r a d d i t i o n a l i n f o r m a t i o n a b o u t t h e RNA c o m p o n e n t s of t h e p a r t i c l e s w h i c h w e r e m a d e in t h e p r e s e n c e of v i r g i n i a m y c i n a n d d u r i n g r e c o -

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Formation and late o[ ribosomal subunils and their rRNA component in the presence oI uirfliniamycin.

Strain : 168/6. Medium : OS, supplemented w i t h 5 mg Glc and 800 ,Ixg ECD per ml. Virginiamycin : none (controls) Figs 1 and 5, A and B), 10 ~g factor M (Figs 2 and 6, A, B, C), 10 Ilxg factor S (Figs 3 and 7, A, B, C) and 1 I~g of both factors (Figs 4 and 8, A, B, C), added to the cultures 2 min before the radioisotopes. L a b e l l i n g : ( 6 - a l l ) u r a c i l (spec. act. 6 C / m M ) f o r 10 rain. C h a s i n g : samples of labelled cells were rapidly harvested by filtration and divided into two aliquots w h i c h were grown in two media containing either 100 ttg of (1H) uracil and the same concentration of virginiamycin as before (samples B) or uracil but no inhibitors (samples C). Samples A were harvested after 0 rain, and samples B and C after 20 min of chasing. Cells were suspended in 1/20 TMNM and disrupted in a French pressure cell. Ribosomes were centrifuged and fractionated in sucrose gradients (Figs 1 to 4). Aliquots of the particle suspensions were extracted with 1 p. cent SDS and phenol, and the RNA was precipitated w i t h ethanol and fractionated in density gradients (Figs 5 to 8). Absorbance : continuous l i n e s ; Radioactivit~r (TCA-insoluble e o u n t s / m i n / fraction) : dotted lines.

very, r i b o s o m e pellets w e r e extracted w i t h phenol, a n d t h e RNA w a s p r e c i p a t e d w i t h e t h a n o l a n d f r a c t i o n a t e d by ultracentrifugation.

BIOCHIMIE, 1971, 53, n o 9.

n i a m y c i n M f o r m e d an h e t e r o g e n o u s s p e c i e s w i t h s e d i m e n t a t i o n c o e f f i c i e n t s 16 to 4 S (Fig. 6A). S u c h a p a t t e r n r e m a i n e d u n c h a n g e d u p o n c h a s i n g in t h e

Polgribosomes mzd Ribosomes during protein inhibition. [H~]

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Polyribosomcs and Ribosomes during protein inhibition. t)resence t)f the drug (Fig. 6B). When the lalter was removed, most of the radioactivity entered 23 S and 16 S rRNAS, while the 10 S RNA tended to disappear (Fig. 6C). Similar pictures were obtained in the presence of v i r g i n i a l n y c i n S (Figs. 7A, B, C). An irreversible alteration was observed --- Imwever - - upon i n c u b a t i o n of bacteria with M plus S, since quite similar patterns were obtained after periods of chasing in the presence or in the absence of the two virginiamycin coml)onents (Figs. 8A, B, C). This experiment indicated that : a) little or no 23 S rRNA was incorporated into the particles which were formed in the presence of virginiam y c i n ; b) in v i r g i n i a m y c i n - t r e a t e d cells, some RNA 4 to 16 S was made, w h i c h was sedimented by tdtracentrifugation at 100,000 g ; c) the metabolic alterations occuring after incut)ation with either M or S were reversible, whereas they became irreversible when both v i r g i n i a m y c i n components were present. III. Formation and decay of polgribosomes after treatment with virqiniamycin. The halt of ribosome formation, though accounting fur an alteration of cell viability, could not explain the block of protein synthesis. As a matter of fact, in cells infected with phage T2, t r a n s c r i p t i o n and translation of the cell message are prevented, and ribosome formation is b l o c k e d : nevertheless, viral messenger can still hire pre-existing ribosomes to make proteins (cf. e.g., the review of COHEN [!181). Hence, the rapid block of polypeptide synthesis by virgin i a m y c i n (which occurred in less than 1/25th of generation time [4], could only be explained by an interference with the translation m e c h a n i s m itself. In other words, the kinetics of protein formation in the presence of v i r g i n i a m y c i n can only be accounted for by an interference of the antibiotic with either the assembly or the function of polyribosomes. A study of the formation and decay of polysome complexes in the presence of the antibiotic would, then, be an approach to this problem. T h i r t y - s e c o n d pulses of (3H) u r i d i n e were a d m i n i s t e r e d to v i r g i n i a m y c i n - t r e a t e d cells, to label mBNA. Some cells were chilled, collected and disrupted w i t h i n few seconds. The r e m a i n i n g cultures were chased with an excess of unlabelled nridine, and aliquots w i t h d r a w n after various periods were lysed in the same way. Polysomes were concentrated, freed from subunits by centrifugation, and further fractionated in density

BIOCHIMIE, 1971, 53, n ° 9.

995

gradients. Absorbance anti radioactivity were monitored, and the c o r r e s p o n d i n g tracing are reported in Figs. 9 to 11. The pattern of polyribosomes decay in control ceils showed a progressive d i s a p p e a r a n c e of the heavy species, with simultaneous increase of the radioactivity overlapping the 70 S peak of absorbance (Figs. 9A, B, C). In Fig. 10D the effect of a treatment with 1 vg of ribonuclease is depicted. Radioactive and UV-absorbing material disappeared from the polyribosome region : the latter was found w i t h i n the 70 S peak, whereas the former appeared on the top of the gradient. When the pulse of u r i d i n e was a d m i n i s t r a t e d to cells treated with v i r g i n i a m y c i n M, the decay of labelled polyribosomes, p a r t i c u l a r l y the heaviest ones, was sharply reduced (Figs. 10A, B). After short i n c u b a t i o n with v i r g i n i a m y c i n S, the radioactivity tracing in the polysome region was shifted toward the highest molecular weight species, as compared to the controls (Fig. l l A ) , and the decay of polyribosomes slowed down (Fig. l l B , C). These experiments indicated that v i r g i n i a m y c i n did not prevent the assembly of polyribosomes from the pre-existing ribosomes and the mRNA which was made in the presence of the drug. However, the decay of the complexes - - w h i c h is k n o w n to parallel tile process of translation - was sharply i n h i b i t e d by the antibiotic. This was shown by the shift of the radioactivity tracing toward the heaviest polyribosomes after labelling, anti by the reduction of their decay after chasing. Thus, the functioning, not the assembly, of polyribosomes was blocked by v i r g i n i a m y e i n . IV. Incorporation o[ labelled into polyribosomes.

viryiniamycin

Block of translation at a step subsequent to that of polysome assembly could be easily explained, if factors M and S were found linked to polyribosome complexes. Labelled v i r g i n i a m y c i n preparations were employed to salve this problem. E x p o n e n t i a l l y growing cells were incubated for 20 see w i t h 3H-labelled v i r g i n i a m y c i n components. Bacteria were quickly chilled, harvested and lysed w i t h i n 40 see. Polysomes were concentrated free of monosomes and fraetionated in snerose density gradients. Small but consistent amounts of labelled virgin i a y i n c i n M and S were recovered indeed w i t h i n the l)olysome region, as indicated by the overlap-

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FIG. 12 A, B, C, D. - - Incorporation of labelled virginiamycin into polyribosomes. S t r a i n and m e d i u m as in the legend f o r Figs 9 to 11. Labelling : 3 0 - sec p u l s e s of e i t h e r (3H) v i r g i n i a m y c i n M, spec. act. 5.3 × 10-2 ~C/:~g, f o r s a m p l e s 12A and B, or (3H) v i r g i n i a m y c i n S, spee. act. 100 C / m M , f o r s a m p l e s 12C and D. Lysis : c e l ] s chi]led, h a r v e s t e d and lysed b y the <>. P o l y r i b o s o m e s w e r e c o n c e n t r a t e d and f r a c t i o n a t e d as o u t l i n e d in <>. One a l i q u o t of each of t h e two p r e p a r a t i o n s w a s layered on s u c r o s e g r a d i e n t s w i t h o u t f u r t h e r t r e a t m e n t ( s a m p l e s 12A and C) ; t h e o t h e r a l i q u o t w a s i n c u b a t e d f o r 30 sec at 4 ° w i t h L-cysteine (1 mM) b e f o r e f r a c t i o n a t i o n ( s a m p l e s 12B and D). A b s o r b a n c e at 260 m~ w a s c o n t i n u o u s l y m o n i t o r e d ( c o n t i n u o u s lines) and TCA- i n s o l u b l e r a d i o a c t i v i t y of single f r a c t i o n s w a s counted (dotted lines).

BIOCH1MIE, 1971, 53, n ° 9.

998

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p i n g of absorbance and radioactivity tracings (Figs. 12A and C), and by the d i s a p p e a r a n c e of the latter upon treatment with RNAase (not shown). Upon i n c u b a t i o n of polyribosomes with L-cysteine, both v i r g i n i a m y c i n s became dissociated from polysomes and monosomes (Figs. 12B and D) (L-cysteine alone did not alter the complexes). This study proved that both v i r g i n i a m y c i n s were incorporated into polyribosomes, and that the linkage of these antibiotics to hypothetical receptor sites w i t h i n the complexes was stable to low pH but sensitive to sulfhydryl reagents. DISCUSSION AND CONCI.USION. It has been previously reported that rRNA, w h i c h is made in chloramphenicol-treated cells is i n c o r p o r a t e d into r i b o n u c l e o p r o t e i n particles 18 to 25 S. The nucleic acid content of these particles was found to be higher than that of n o r m a l ribosomes, and the protein c o m p o n e n t to be withd r a w n from a pool of ribosomal proteins built up before the addition of the drug [20, 21, 22, 23]. Upon removal of the antibiotic, apparently, chlora m p h e n i c o l particles are converted into normal ribosome subunits : this was a reason for consid e r i n g such particles as normal intermediates in ribosomal formation. It has been observed, however, that in exponentially growing cells a nearly unsizeable pool of free ribosomal proteins exists [24]. It is likely, therefore, that the rRNA w h i c h is synthesized in the presence of chlorarnphenico! might associate with a pre-existing pool of non ribosomal proteins tn from an heterogeneous population of ribonucleoproteins. Upon shift to antibiotic free-medium, such complexes dissociate and the nucleic acid - - but not the protein component - - a p p a r e n t l y is incorporated into newly-made ribosomes [25, 26]. This interpretation is in good agreement with the results shown in the present work, anti those previously obtained with <
their association has a bactericidal effect [1, 2, 4]. Data presented in this paper offer a molecular explanation to the differential i n h i b i t o r y action of these antibiotics. In fact, the block of the assembly of 23 S rRNA and 50 S ribosomal subunits, which occurs in the presence of single virgin i a m y c i n components, progressively disappears after removal of the drugs. Conversely, these metabolic alterations become irreversible when both v i r g i n i a m y c i n components are present. Data reported in Figs. 9 to 11 indicate that virg i n i a m y c i n interferes with the decay of polyribosomes : the rate of disappearance of the complexes is, in fact, reduced by factors M and S. A quite similar observation was reported for anisomycin, an antibiotic w h i c h i n h i b i t s protein synthesis in m a m m a l i a n cells and fungi, but is inactive against bacteria. A n i s o m y c i n blocks the i n c o r p o r a t i o n of labelled amino acids from AAtRNA into polypeptide chains and prevents the release of nascent p r o t e i n s : the ratio of polysomes to monosomes, thus, increases in the presence of the i n h i b i t o r [28]. On the other hand, the protection of polysomes w h i c h is afforded by a n i s o m y c i n in m a m m a l i a n cells, and by virgin i a m y c i n in bacteria, contrasts with the increased decay of polyribosomes w h i c h was observed in tile presence of another i n h i b i t o r of protein synthesis, p u r o m y c i n . In tile latter case, a p r e m a t u r e detachment of incomplete polypeptide chains occurs, which is followed by a collapse of the polysome structure. It has been observed that the half life of cellular and viral mRNA is increased in v i r g i n i a m y c i n treated cells [4, ,5]. It is k n o w n also that the inhibitors of protein formation, such as chloramphenicol and p u r o m y c i n , may have opposite effects on the life of messenger RNA, d e p e n d i n g on whether tlle latter is detached from, or held by, tile translation complex. Moreover, chloramphenicol if previously b o u n d to ribosomes, i n h i b i t s p u r o m y c i n reaction in vitro [29]. Thus, the conclusion w h i c h can be d r a w n from the present and previous observations is that v i r g i n i a m y c i n , by p r e v e n t i n g the dissociation of the polysome complex, increase the half life of mRNA. The i n c o r p o r a t i o n of labelled v i r g i n i a m y c i n into polyribosomes has been proved by showing that : a) the i n c o r p o r a t i o n of factors M and S occurs after short pulse and decays in similar fashion as p o l y r i b o s o m e s ; b) the label can be sedimented with particles > 70 S ; c) the radioactivity t r a c i n g overlaps that of polyribosomes in sucrose gradients, and d) is sensitive to RNAase. The absolute amount of label recovered w i t h i n

Polyribosonles and Ribosomes during protein inhibition. t h e p o l y s o m e r e g i o n w a s s m a l l , as e x p e c t e d in e x p e r i m e n t s of t h i s sort, w h i c h a r e s u b j e c t e d to t h r e e k i n d s of l i m i t a t i o n s : t h e l e n g t h of t h e p u l s e , t h e c o m p l e t i o n of p o l y p e p t i d e c h a i n s a n d r u n n i n g off of p o l y r i b o s o m e s ( w h i c h p r o c e e d e v e n at 0°C [9, 10]), a n d t h e i n s t a b i l i t y of t h e l i n k a g e b e t w e e n i n h i b i t o r s a n d p o l y s o m e s . As a m a t t e r of fact, it h a s b e e n s h o w n t h a t e x t e n s i v e w a s h i n g s of c h l o r a m p h e n i c o l - - r i b o s o m e c o m p l e x e s , as w e l l as their centrifugation through sucrose gradients, r e m o v e c o m p l e t e l y t h e i n h i b i t i o n of t h e p u r o m y cin r e a c t i o n [29]. M o r e o v e r , a v e r y r e c e n t w o r k s h o w s t h a t t h e a s s o c i a t i o n of v e r n a m y c i n A ( w h i c h is s i m i l a r to virginiamycin M) w i t h 70 S r i b o s o m e s in vitro is u n s t a b l e a n d c a n b e b r o k e n d o w n , e.g., b y l o w e r i n g t h e c o n c e n t r a t i o n of K ~ [30].

O u r e x p e r i m e n t s s h o w t h a t b i n d i n g of v i r g i n i a m y c i n to p o l y s o m e s is a f f e c t e d b y c o m p o u n d s b e a r i n g f r e e s u l f h y d r y l g r o u p s , s u c h as c y s t e i n e , glutathione or ~-mercapto-ethanol. The simplest e x p l a n a t i o n is t h a t t h i o l s c o m p e t e w i t h v i r g i niamycin for hypothetical binding sites within polyribosomes. This view agrees with the report that sulfhydryl reagents abolish the poly-U directed b i n d i n g of AA-tRNA to r a b b i t r e t i c u l c y t e rib o s o m e s [31] : t h e site of i n h i b i t i o n is t h e r i b o s o m a l p r o t e i n f r a c t i o n [32]. An e x t r a p o l a t i o n of t h e s e o b s e r v a t i o n s l e a d s to c o n c e i v e s o m e r i b o s o m a l p r o t e i n c o m p o n e n t s as tim t a r g e t s of v i r g i niamycin within polysomes, a possibility which is t e s t e d at t h e p r e s e n t t i m e . We can conclude that virginiamycin does not p r e v e n t t h e a s s e m b l y of p o l y s o m e s , i n w h i c h it becomes incorporated. Such incorporation prev e n t s t h e g r o w t h a n d d e t a c h m e n t of p o l y p e p t i d e chains from translation complexes. Polysomes and m R N A are, t h u s , s t a b i l i z e d , a n d t h e i r h a l f life p r o l o n g e d . In t u r n , t h e b l o c k of p r o t e i n b i o s y n t h e s i s p r e v e n t s t h e a s s e m b l y of 23 S r R N A f r o m h y p o t h e t i c a l rRNA p r e c u r s o r s , a n d t h e f o r m a t i o n of r i b o s o m a l s u b u n i t s f r o m r R N A a n d r i b o s o m a l 1)roteins. T h e s e a l t e r a t i o n s , r e v e r s i b l e i n t h e p r e s e n c e of s i n g l e v i r g i n i a m y c i n s , a r e r e n d e r e d i r r e versible by their association.

Les deux composants de la virginiamycine, M et S, lorsqu'ils sont ajout6s s6par6ment h des cultures de Bacillus subtilis en phase exponentielle, b l o q u e n t de fagon t e m p o r a i r e la croissance cellulaire et la synth6se prot6ique. L'incubation des bact6ries avec les deux composants M et S ensemble, e n t r a i n e une perte pr6coce de viabilitC Non seulement la f o r m a t i o n de polyribosomes n'est pas inhib6e par la virginiamycine, mats la demi-vie

BIOCHIMIE, 1971, 53, n ° 9.

999

de ces complexes est augment6e. Dans des conditions physiologiques, la dissolution des polysomes va de paire avee la t r a d u c t i o n des messages. L'augmenta;tion de la demi-vie de ces complexes explique done l ' i n h i bition de la synth6se prot6ique et la protection de I'ARN messager, ce qui avait dtd observ6 pr6c6demment en pr6sence de l'antibiotique. La f o r m a t i o n de ribosomes est bloqu6e de faqon temporaire par les eomposants M e t S s6par6s. L'ARN r i b o s o m i q u e qui se forme dans ees conditions s'associe h des prot6ines n o n - r i b o s o m i q u e s pr6existantes, et les ribonucl6oprot6ines qui en r6sultent p e u v e n t ~tre isol6es p a r u l t r a c e n t r i f u g a t i o n . Suite h l'enl6vement de I'antibiotique, ces ribonucl6oprot6ines labiles se dissocient, et leur ARN se lie avec des prot6ines ribosomiques n6oform6es : des subunit6s ribosomiques a p p a r e m m e n t normales sont ainsi assembl6es. Si les bact~ries sont trait6es s i m u l t a n 6 m e n t avec les deux composants de la virginiamyeine, it en r6sulte une i n h i b i t i o n p e r m a n e n t e de la synthbse des subunitds ribosomiques : ees derni6res ne se f o r m e n t plus, lorsque les cellules sont transf6r6es darts u n milieu d6ponrvu d'antibiotiques. Cette observation f o u r n i t une explication mo16eulaire h la perte de viabilit6 qui accompagne u n t r a i t e m e n t avec tes deux composants de la virginiamycine. ZUSAMMENFASSUNG.

Die beiden Bestanteile des Virginamycins, M u n d S, u n t e r b r e c h e n auf reversible Weise das Z e l l w a c h s t u m und die Proteinbildung, w e n n sic einzeln zu Expon e n t i a l k u l t u r e n yon Bacillus subtilis hinzugefiigt werden. Wenn die Bakterien m i t beiden F r a k t i o n e n inkubiert werden, so erfolgt ein Viabilitiitsverlust. Die Bildung der P o l y r i b o s o m e n wird d u r c h Virginamycin nicht veriindert, aber ihr Zerfall w i r d verhindert. Diese Beobachtung erkliirt das plStzliche Aufh6ren der P r o t e i n s y n t h e s e und den Schutz der Messenger-RNS, welche m a n in Gegenwart des A n t i b i o t i k u m s feststcllt. Die Bildung der Ribosomen w i r durch einzeln,e Virg i n a m y c i n - b e s t a n d t e i l e reversibel gehemmt. RibosomRNS, welche u n t e r diesen Bedingungen hergestellt wird, vereinigt sich mit n i e h t - r i b o s o m a l e n Prote:nen und bildet labile Ribonukleoproteine, welche durch Ultracentrifugieren sedimentiert werden kSnnen. Nach E n t f e r n e n der Droge, dissoziieren sich solehe Komplexe, und ihre RNS-Bestandteile b i n d e n sich mit den neu gebildeten Ribosomen-proteinen und ergeben normale R i b o s o m e n - U n t e r e i n h e i t e n . Wenn beide Virginamycin-Bestanteile v o r h a n d e n sind, wird die I n h i b i t i o n der P r o t e i n b i l d u n g nicht aufgehoben, und keine R i b o s o m e n - U n t e r e i n h e i t e n werden nach Ubertragen der Zellen in ein A n t i b i o t i k u m - f r e i e s Medium gebildet. Diese Beobachtung erkHirt die lethale W i r k u n g der Assoziation yon Virginamycin M und S. REFERENCES. 1. VASQUEZ D., Syrup. Soc. Gem Microb., 1966, 16, 169. 2. VASQUEZ D., Antibiotics (Ed. D. Gottlieb and P. D. Shaw) New York, Springer Verlag, 1967, 1, 387. 3. WEmBLUM B. and DAVIES J., Bacter. Revs., 1968, 32, 4~3.

1000 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

C. C o c i t o .

COCITO C., J. GeE. Microbiol., 1969, 57, 179. COCITO C., J. GeE. Microbiol., 1969, 57, 195. COCtTO C. a n d KAJI A., Biochimie, 1971, 53, 763. MANGIAROTTI G. a n d SCHLESSINGI~H D., J. Mol. Biol., 1966, 20, 123. FLESSEL C. P., RALPH P. a n d RICH A., Science, 1967, 158, 658. GODSON G. N. a n d SINSH~IMEn R. L., Biochim. Biophys. Acta, 1967, 149, 476. GODSON G. N. a n d SINSHEIMER R. L., Biochim. Biophys. Acta, 1969, 149, 489. KOHLER R. E., RON E'. Z. a n d DAVIS B. D., J. Mol. Biol., 1968, 36, 71. R O N E . Z., KOHI.En R. E. a n d DAVIS B. D, J. Mol. Biol., 1968, 36, 83. NInENBEnc M. a n d LEImn P., Science, 1964, 145, 1399. MAXWELL I. H., Motet. Pharmaeol., 1968, 4, 25. TAKEDA Y., SUZUEA I. a n d KAJI A., J. Biol. Chem., 1968, 243, 1075. VANDEnHAEC,E H., VAN DIJCK P., PARMENTIER G. a n d DE SOMER P , Anlib. Chemother., 1957, 3, 606. GOSSE.LINCKS 1., Scheikundige studie van her antibioticum staphylomycine. T h e s i s , U n i v e r s i t y of Louvain (Belgium).

BIOCHIMIE, 1971, 53, n ° 9.

18. COHEN S. 8., Ann. Bey. Biochem., 1963, 32, 83. 19. PONCELET F., ROnEnFROID M. a n d DCMONT P., J. Pharm. Belg., 1967, 11, 395. 20. NOMURA M. a n d D,TATSON J. D., J. Mol. Biol., 1959, 1, 204. 21. KURLAND C. G. a n d MAALOE 0., J. Mol. Biol., 1962, 4, 193. 22. KURLAND C. G. a n d WATSON J. D., J. Mol. Biol., 1962, 4, 388. 23. HOSOKAWA K. a n d NOMURA M., J. Mol. Biol., 1965, 12, 225. 24. SCHLEIF R., J. Mol. Biol., 1967, 27, 41. 25. SCHLEIF R., J. Mol. Biol., 1968, 37, 119. 26. YOSHIDA K. a n d OSA~'A S., J. Mol. Biol., 1968, 33, 559. 27. NAKADA D., ANDERSON J. A. C. a n d MAGASANIK B., J. Mol. Biol., 1964, 9, 472. 28. GnOLLMAN A., J. Biol. Chem., 1967, 242, 3226. 29. CA~NO~ M., Europ. J. Biochem., 1968, 7, 137. 30. ENNIS H. L., Biochemistry, 1971, 10, 1265. 31. HE1NTS R. L., MeALLISTER H. C., AnLINGHAUS R. a n d SCHWEEX R. S., Cold Spring Harbor Symp. Quant. Biol., 1966, 31, 633. 32. McALLISTER H. (~. a n d SCHWEET R. S., J. Mol. Biol., 1968, 34, 519.