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Streptomycin-induced conformational changes in the 70-S bacterial ribosome
413
Biochimica et Biophysica Acta, 521 (1978) 413--425 © Elsevier/North-Holland Biomedical Press
BBA 99332
STREPTOMYCIN-INDUCED CONFORMATIONAL CHANGES IN THE 70-S BACTERIAL RIBOSOME LI~A BRAKIER-GINGRAS a, GUY BOILEAU a, SOPHIE G L O R I E U X a and NORMAND BRISSON b
Ddpartements de a Biochimie et de b Chimie, Universitd de Montreal, Montreal, Quebec (Canada) (Received December 8th, 1977) (Revised manuscript received June 27th, 1978)
Summary Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and $18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.
Introduction
Streptomycin binds to the 30-S ribosomal subunit and inhibits several steps of protein biosynthesis [1,2]. The known effects of this antibiotic are induction of misreading, phenotypic suppression, destabilization of the initiation complex, interference with the dissociation of ribosome into subu~its, and a breakdown of polysomes. Several studies have demonstrated that the structure of the ribosome is altered upon binding of streptomycin. These included hydrogen-tritium exchange [3,4], spin labeling [5], reaction with kethoxal [6] and iodination [7]. Structural changes in the ribosome were also suggested by an examination MalNEt, N-ethylmaleimide; NO-MalNEt, nitxoxide analog of MalNEt; EPR, electron pazarnagnetie resonance. Abbreviations:
414
of the thermodynamic parameters which characterize the binding of streptomycin [8]. This study investigated the changes induced by streptomycin in the ribosome, using fluorescent, radioactive and spin-labeled probes. The fluorescent probe, ethidium bromide, was used since it intercalates preferentially into double-stranded regions of nucleic acids [9,10] and is therefore expected to reveal structural changes in ribosomal RNA. The radioactive marker, N-[14C]ethylmaleimide and the spin-labeled probe, a nitroxide analog of N-ethylmaleimide, which react selectively with sulfhydryl groups of ribosomal proteins [ 11 ] were chosen to monitor conformational changes at the protein level. Preliminary studies in which many ribosomal proteins were spin-labeled suggested that the binding of streptomycin loosens the structure of the ribosome [5]. In the present study, more specific labeling conditions have been used and the derivatized proteins identified with a radioactive label. These data, combined with the results on fluorescence, allow a more conclusive description of the structural changes in the ribosome induced by streptomycin. Materials and Methods Materials Unlabeled MalNEt was purchased from Eastman Corp., [14C]MalNEt (specific activity: 8.4 Ci/mol) was from New England Nuclear and NO-MalNEt, N-(l~xyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide, was obtained from Syva Associates. Ethidium bromide (3,8-diamino-5-ethyl-6-phenyl phenanthridinium bromide) was purchased from Sigma Chemical Co. Streptomycin was obtained from General Biochemicals and [3H]dihydrostreptomycin (3 Ci/mmol) was a product from Amersham Radiochemical Centre. Scintillation counting fluids for extracting 14C-labeled materials from acrylamide gels (Protosol-Econofluor) were purchased from New England Nuclear. Reagents for two
poly(U)~iirected polyphenylalanine synthesis. Their sedimentation constant and purity were checked by analytical ultracentrifugation at 30 000 rev./min
415 with a Beckman-Spinco ultracentrifuge model E, equipped with schlieren and ultraviolet optics.
Binding of streptomycin ethidium bromide
to ribosomes labeled with N-ethylmaleimide or
The influence of the labeling of ribosomes on [3H]dihydrostreptomycin binding was determined b y filtration on Millipore filters according to Chang and Flaks [8]. The concentration of ribosomes was 30 A 2 6 0 n m units/ml (0.72 • 10 -6 M) *. Ethidium bromide and [3H]dihydrostreptomycin were added to give a molar ratio relative to ribosomes of 250 and 100, respectively, and the labeling time was 15 min. Two types of labeling with MalNEt were performed: a limited labeling, in which the ratio of label to ribosome was 125 and the labeling time was 1.50 min and a longer labeling with a ratio of label to ribosome of 250 and an incubation period of 4 h. The antibiotic was added before or after labeling.
Radioactive labeling Labeling of ribosomes with [14C]MalNEt was performed in the absence or presence of streptomycin at 37°C in buffer C. The reaction volume was 0.1 ml, the concentration of ribosomes was 300 A2eonm units/ml; [14C]MalNEt (0.42 Ci/mol) was at a molar ratio of label to ribosome of 125 or 250, corresponding to a labeling of 1.50 min and 4 h respectively. Streptomycin, when present, was added at a molar ratio of antibiotic to ribosome of 100. Ribosomes were first preincubated for 15 min in the presence or absence of streptomycin at 37°C, before adding [14C]MalNEt. After an incubation period of 1.50 min or 4 h, the reaction was stopped b y gel filtration through Sephadex G-25. Ribosomes were precipitated with a cold solution of 5% trichloroacetic acid, filtered on Reeve Angel glass fiber filters, dried and counted in a Packard Tri-Carb Scintillation Counter using a toluene-based scintillation fluid. To identify which ribosomal proteins were labeled, labeling was carried o u t under the same conditions except that the specific activity of [14C]MalNEt was 8.4 Ci/mol. Ribosomal proteins were extracted by the acetic acid m e t h o d of Hardy et al. [ 15] and their separation effected by two-dimensional polyacrylamide gel electrophoresis according to Kaltschmidt and Wittmann [16]. Proteins were stained with 0.2% Coomassie Blue, the spots were cut o u t from the gels, crushed, incubated overnight at 37°C in 10 ml of a 5% Protosol solution in Econofluor, and then counted.
Spin labeling Labeling of the ribosomes with NO-MalNEt was performed under conditions similar to those described above for the radioactive label. Ribosomes were labeled either for 1.50 min or 4 h at 37°C. U n b o u n d NO-MalNEt was removed b y gel filtration through Sephadex G-25. Ribosomes were then incubated at 37°C for 15 min, with or without streptomycin. The EPR spectra were * O n e A 2 6 0 n m u n i t is t h e q u a n t i t y o f m a t e r i a l c o n t a i n e d i n 1 m l o f a s o l u t i o n w h i c h has an a b s o r b a n c e o f 1 a t 2 6 0 n m , w h e n m e a s u r e d i n a c e l l w i t h a 1 - c m p a t h length. One A 2 6 0 n m u n i t c o r r e s p o n d s t o 24 pmol of 70-S ribosomes [14].
416 recorded at 37°C _+ I°C on a Brucker 414S spectrometer operating at 9.33 GHz and equipped with a temperature control unit. The samples were examined in a small aqueous flat cell (Scanlon Co.). Changes in the EPR spectra arise from modifications in the mobility of the labels (for a discussion of the factors involved in EPR line shape analysis, see ref. 17). The labeling conditions were slightly different from those used previously [ 5], in t h a t a lower ratio of label to ribosome and a shorter period of treatment with NO-MalNEt (1.50 min or 4 h instead of 24 h) were used. Under these conditions, less dissociation was found to occur and ribosome degradation was avoided.
Fluorescence labeling Fluorescence labeling of ribosomes in the presence or in the absence of streptomycin with different concentrations of ethidium bromide was performed at 37°C in buffer C'. The reaction volume (3 ml) contained ribosomes (10 A260nm units/ml) and ethidium bromide at a molar ratio of label to ribosome of 50 to 250. The molar ratio of streptomycin to ribosome was 100. When appropriate, ribosomes were preincubated with streptomycin for 15 min before adding ethidium bromide. The fluorescence intensity of labeled ribosomes was measured at 37°C with a PMQ II Zeiss spectrophotometer equipped with a fluorescent attachment and a temperature control unit. The excitation wavelength was 540 nm, the emission was measured at 590 nm. Absorption studies were performed in order to determine the difference in the number o f ethidium bromide molecules bound to ribosomes in the presence or in the absence of streptomycin. The conditions for labeling were similar to those used in fluorescence labeling except for the amounts of ribosomes and ethidium bromide used: the reaction volume was 2 ml, the ribosome concentration was 100 A2e0nm units/ml, the concentration of ethidium bromide was in a molar ratio of label to ribosome varying from 5 to 100. The molar ratio of streptomycin to ribosome was 100. The absorption of ribosome-bound ethidium bromide was measured at 465 nm at 37°C with a C a r y - l l 8 recording spectrophotometer using a l~cm light path cell in a thermostated cell holder. The a m o u n t o f free and bound ethidium bromide was determined following the procedure o f Douthart et al. [18], which is based on the difference in the extinction coefficient of free and bound form. The association constant and the number o f binding sites for ethidium bromide were estimated using the Scatchard relationship [19]. Possible release of ribosomal proteins after treatment with ethidium bromide [20] was checked by two-dimensional polyacrylamide gel electrophoresis on the 150 000 X g supernatant of labeled ribosomes. Results
Effect o f labeling with N-ethylmaleimide or ethidium bromide on the binding o f streptomycin to ribosomes Labeling with MalNEt or with ethidium bromide did not interfere with the binding of [3H]dihydrostreptomycin to ribosomes as assayed by Millipore
417 TABLE I I D E N T I F I C A T I O N O F T H E 70-S R I B O S O M A L P R O T E I N S L A B E L E D W I T H N - [ 1 4 C ] E T H Y L M A L E I MIDE A F T E R A T R E A T M E N T OF 1.50 MIN P r o t e i n s w e r e e x t r a c t e d f r o m r i b o s o m e s l a b e l e d w i t h [ 1 4 C ] M a l N E t f o r 1 . 5 0 m i n as d e s c r i b e d in Materials and M e t h o d s . T h e y w e r e f r a c t i o n a t e d 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 c t r o p h o r e s i s and t h e a m o u n t o f r a d i o a c t i v i t y b o u n d t o e a c h p r o t e i n s p o t w a s d e t e r m i n e d . R e s u l t s are a v e r a g e s o f five d i f f e r e n t e x p e r i m e n t s . C o u n t s are u n c o r r e c t e d f o r t h e p e r c e n t a g e o f m a t e r i a l r e m a i n i n g at t h e origin o f p o l y a c r y l a m i d e gels. T o a c c o u n t for d i f f e r e n c e s in t h e a m o u n t o f p r o t e i n s s u b m i t t e d t o e l e e t r o p h o r e s i s , results are n o r m a l i z e d t o a t o t a l o f 1 6 0 0 c o u n t s m i n -x p e r gel. S t a n d a r d d e v i a t i o n o n i n d i v i d u a l c o u n t s p e r p r o t e i n is 10%. P r o t e i n s l a b e l e d w i t h less t h a n 5% o f t h e r a d i o a c t i v i t y f o u n d in t h e p r o t e i n w i t h m a x i m u m l a b e l i n g h a v e n o t b e e n listed. 30-S p r o t e i n s ( c o u n t m i n - 1 )
50-S p r o t e i n s ( c o u n t r a i n - 1 )
Withoutstreptomycin
With s t r e p t o m y c i n
Withoutstreptomycin
With s t r e p t o m y c i n
S1 S18
295 885
L10 LI7
156 264
285 880
167 268
filtration (data not shown) *. It is known that labeling with a higher concentration o f MalNEt does interfere with the binding of streptomycin [ 5,21], because of the blocking of the sulfhydryl group of protein S14 [22]. However, as will be shown later, labeling of protein S14 did not occur under the experimental conditions used in this study.
Binding of N-[14C]ethylmaleimide Depending on whether the incubation with MalNEt lasted 1.50 min or 4 h, an average of one or six sulfhydryl groups per ribosome were found to react with MalNEt. The presence of streptomycin did not modify the amount o f binding. To identify the proteins which are derivatized under our conditions, ribosomes were labeled with [14C]MalNEt, ribosomal proteins were extracted and fractionated b y two
418
labeled. This reflects the variability in the reactivity of the different sulfhydryl groups [23,24] and indicates that all the ribosomes are not equally labeled.
Spin labeling The EPR spectra of spin-labeled ribosomes obtained after a 1.50 min or a 4 h labeling period are presented in Fig. 1 (A--B). These spectra are typical for spin labels bound to proteins. They are t w o - c o m p o n e n t spectra consisting of a constrained (I) and a more mobile (M) part. The existence of the two components in the EPR spectra of spin-labeled ribosomes can be explained if there are two types of sulfhydryl binding sites corresponding to the mobile and the less mobile part of the spectra, or if the label, at each of the binding sites, can alternatively occupy t w o isomeric states, one mobile and one constrained, a situation frequently encountered with spin-labeled proteins [25--27].
iM
I
w
I+M Z 0
m
~M
f
~M
10G
MAGNETIC FIELD (G) Fig. I . A, E P R s p e c t r u m o f 70-S r i b o s o m e s spin-labeled f o r 1 . 5 0 m i n w i t h N O - M a l N E t in t h e a b s e n c e (solid line) o r in t h e p r e s e n c e o f s t r e p t o m y c i n ( d a s h e d line). T h e a r r o w s M a n d I r e f e r t o t h e m o b i l e a n d less m o b i l e p a r t o f t h e s p e c t r u m , r e s p e c t i v e l y . T h e i n s e r t is a n e n l a r g e d s e c t i o n o f t h e l o w field p a r t of t h e s p e c t n a m w i t h t h e s y m b o l s h I a n d h 2 r e p r e s e n t i n g t h e h e i g h t s o f t h e l o w field b a n d s c o r r e s p o n d i n g t o t h e m o b i l e a n d less m o b i l e p a r t o f t h e s p e c t r u m . E P R s p e c t r a w e r e m e a s u r e d w i t h m i c r o w a v e f r e q u e n c y , 9 . 3 3 G H z ; m o d u l a t i o n a m p l i t u d e , I G; m i c r o w a v e p o w e r , 1 0 m W a n d a m p l i f i c a t i o n 5 • 104. B, E P R spect r u m o f 70-S r i b o s o m e s spin-labeled f o r 4 h w i t h N O - M a l N E t . C o n d i t i o n s o f m e a s u r e m e n t are t h e s a m e as a b o v e e x c e p t f o r m o d u l a t i o n a m p l i t u d e , 0.6 G; m i c r o w a v e p o w e r , 5 m W a n d a m p l i f i c a t i o n , 4 • 104. T h e s p e c t r u m is n o t s i g n i f i c a n t l y a l t e r e d in t h e p r e s e n c e o f s t r e p t o m y c i n ( n o t s h o w n ) .
419 T A B L E II EFFECT OF STREPTOMYCIN ON THE EPR SPECTRA OF SPIN-LABELED RIBOSOMES T h e i n d e x o f m o b i l i t y , t h e h 1/h 2 r a t i o , is d e f i n e d as t h e r a t i o o f t h e h e i g h t s o f t h e l o w field p e a k s corresp o n d i n g t o t h e m o b i l e a n d less m o b i l e p a r t o f t h e s p e c t r a (Fig. 1). T h e h l / h 2 r a t i o v a r i e s slightly w i t h t h e d i f f e r e n t p r e p a r a t i o n s or b e c a u s e o f t h e d e l a y o c c u r r i n g b e f o r e r e c o r d i n g t h e s p e c t r a . T h e m o s t p r e v a l e n t eases are p r e s e n t e d h e r e . F o r e a c h e x p e r i m e n t , t h r e e s a m p l e s f r o m t h e s a m e p r e p a r a t i o n w e r e l a b e l e d und e r s i m i l a r c o n d i t i o n s a n d t h e E P R s p e c t r a w e r e r e c o r d e d in t h e a b s e n c e a n d t h e p r e s e n c e o f s t r e p t o m y c i n . T o i n c r e a s e t h e a c c u r a c y i n t h e m e a s u r e m e n t o f h l / h 2, t h e p a r t o f t h e s p e c t r u m c o r r e s p o n d i n g t o t h e l o w field lines w a s s c a n n e d w i t h a n i n c r e a s e d gain s e t t i n g . F o r a n y given set o f e x p e r i m e n t s , t h e v a l u e s o f h l / h 2 w e r e r e p r o d u c i b l e w i t h i n a s t a n d a r d d e v i a t i o n o f -+5%. Conditions
Experiment
I n d e x o f m o b i l i t y , hl/h 2 Without streptomycin
With s t r e p t o m y c i n
1 . 5 0 m i n labeling
1 2 3
1.3 1.5 1.2
1 .S 2.0 1.6
441 l a b e l i n g
1 2 3
4.0 4.7 5.2
4.2 5.0 5.4
Conformational changes in a macromolecule exhibiting a t w o - c o m p o n e n t EPR spectrum can be detected b y estimating the variation in the proportion of the mobile and the less mobile part of the spectrum. This is c o m m o n l y estimated b y measuring the ratio o f the heights of the low field lines of the mobile and less mobile components, hi and h2, respectively (Fig. 1A: insert) (e.g. see refs. 28, 29). An increase in the index of mobility, the h~/h2 ratio, indicates a loosening in the molecular structure in the environment of the binding site of the label. Direct examination o f experimental data reveals that EPR spectra of ribosomes labeled for 1.50 min are sensitive to the presence of streptomycin in that the mobile c o m p o n e n t of the spectrum increases at the expense of the less mobile c o m p o n e n t (Fig. 1A). Examples of the variation in the h~/h2 ratio o f EPR spectra after addition of streptomycin are presented in Table II. Irrespective o f fluctuations in the h~/h2 ratio values, an increase of 35 +5% was always detected in the presence of streptomycin. This result demonstrates that u p o n binding of streptomycin structural changes occur in the ribosome in the vicinity o f labeled sulfhydryl groups, and the increase in the mobility of the labels indicates that these changes involve a loosening in the structure of some ribosomal proteins. After a 4 h labeling period, the two components o f the EPR spectra of labeled ribosomes can still be distinguished (Fig. 1B) b u t these spectra are much more mobile that after a labeling o f 1.50 min since the intensity of the signal due to the constrained part is less important and the hi~h2 ratio is higher (Table II). In contrast to the situation observed after a labeling period of 1.50 min, addition o f streptomycin to 4 h-labeled ribosomes did n o t significantly alter the spectrum * nor m o d i f y the h~/h2 ratio (Table II). • A s m a l l a n d r e p r o d u c i b l e d e c r e a s e in t h e r a t i o of t h e h e i g h t o f t h e c e n t r a l b a n d t o t h e h i g h field b a n d o f t h e m o b i l e p a r t o f t h e s p e c t r u m is n o t i c e a b l e h o w e v e r . S u c h a d e c r e a s e w h i c h i n d i c a t e s a slight i n c r e a s e in t h e m o b i l i t y o f t h e spin labels h a d led u s to suggest p r e v i o u s l y t h a t t h e b i n d i n g o f s t r e p t o m y c i n l o o s e n s t h e r i b o s o m a l s t r u c t u r e [ 5].
420
Radioactive labeling (Table I) showed that after a labeling of 1.50 min, two 30-S proteins, $1 and $18, and two 50-S proteins, L10 and L17, were preferentially labeled with [14C]MalNEt. That these data are also applicable to the spin label was demonstrated by exposing the ribosomes to a mixture of radioactive and spin labels in a 1 : 1 molar ratio. It was found that radioactive labeling decreased by about 50%, which indicates that the radioactive label and the spin label compete for the same sites. Following a treatment of 4 h with MalNEt derivatives, a larger number of ribosomal sites are labeled. A comparison of the influence of streptomycin on spin-labeled ribosomes after a treatment of 1.50 min and 4 h thus reveals two interesting points: first, the binding of streptomycin loosens the structure of ribosomal proteins in the environment of some derivatized sulfhydryl groups, and second, this loosening is probably located in a restricted area of the ribosome, since it is readily observed only when a limited number of ribosomal sites are labeled.
10
Z <
E c O
>.
5
z_ u Z
~
I~ MOLAR
RATIO OF ETHIDIUM BROMIDE
200 TO
RIBOSOME
Fig. 2. E f f e c t o f t h e 7 0 - S r i b o s o m e s o n t h e f l u o r e s c e n c e o f e t h i d i u m b r o m i d e in t h e a b s e n c e (-=), or in t h e p r e s e n c e ( o o) of streptomycin. ~ A, i n t e n s i t y o f f l u o r e s c e n c e o f e t h i d i u m b r o m i d e alone. No correction to the fluorescence intensity of ethidium bromide bound to ribosomes was made f o r t h e c o n t r i b u t i o n o f u n b o u n d d y e . T h e v e r t i c a l b a z s r e p r e s e n t t h e s t a n d a r d v a r i a t i o n o v e r six e x p e r i ments.
421
Binding of ethidium bromide The fluorescence intensity of ribosomes as a function of the molar ratio of ethidium bromide to ribosome is shown in Fig. 2. In the absence or presence of streptomycin, there is a rapid initial increase in fluorescence intensity, followed by a more gradual increase. This type of fluorescence enhancement is known to be characteristic of an intercalation of ethidium bromide molecules into ordered regions of RNA [10]. At the ionic strength of buffer C' in which labeling is carried out, electrostatic interactions between ethidium bromide and RNA would be minimal [9]. The fluorescence intensity of ribosomes labeled with ethidium bromide in the presence of streptomycin was found to be slightly less than the fluorescence observed in the absence of the antibiotic. The differences are small but larger than those due to experimental variations. No change in the fluorescence intensity was seen when ribosomes from streptomycin-resistant cells were labeled with ethidium bromide in the presence of streptomycin (data not shown). The fluorescence intensity o f ethidium bromide alone was unaffected by the presence of streptomycin. These results suggest that the binding of streptomycin to the ribosome perturbs the secondary structure of ribosomal RNA; the lower fluorescence intensity could be due to a reduced number of ethidium bromide binding sites, resulting from conformational changes in ribosomal RNA. r x 10 - 6 C
0.:
0.2
00
~
2o
r
Fig. 3. S c a t c h a ~ ! P l o t s o f e t h i d i u m b r o m i d e b i n d i n g t o r i b o s o m e s i n the absence ( e ) o r i n the presence (A) o f s t r e p t o m y c i n ; r represents the n u m b e r o f b o u n d e t h i d i u m b r o m i d e molecules p e r r i b o s o m e and c is the m o l a r c o n c e n t r a t i o n o f free d y e ; r a n d c w e r e d e t e r m i n e d f r o m a b s o r p t i o n m e a s u r e m e n t s at 4 6 5 n m . Each e x p e r i m e n t a l p o i n t is t h e m e a n o f six e x p e r i m e n t s . Lines d r a w n t h r o u g h data p o i n t s w e r e c a l c u l a t e d from the parameters given in Table III.
422
TABLE III C H A R A C T E R I Z A T I O N O F E T H I D I U M B R O M I D E B I N D I N G T O 70-S R I B O S O M E S I N T H E A B S E N C E O R IN T H E P R E S E N C E O F S T R E P T O M Y C I N T h e n u m b e r o f e t h i d i u m b r o m i d e b i n d i n g sites a n d t h e b i n d i n g c o n s t a n t s are c a l c u l a t e d f r o m t h e Scatc h a r d p l o t s p r e s e n t e d in Fig. 3. B i n d i n g p a r a m e t e r s are d e t e r m i n e d f r o m t h e e s t i m a t e d i n t e r c e p t s o f the S e a t c h a r d p l o t s and f r o m data p o i n t s a c c o r d i n g to H a l f m a n a n d Nishida [ 3 0 ] . Conditions
Without streptomycin With s t r e p t o m y c i n
N u m b e r o f sites
Binding c o n s t a n t s (×10--4 M - l )
Total number
1st t y p e
95 80
8 8
2nd type
87 72
1st t y p e
2nd type
2.5 2.5
0.18 O. 18
Direct evidence for this hypothesis was sought by absorption measurements of the a m o u n t of free and b o u n d dye at different molar ratios of ethidium bromide to ribosome. The binding isotherms of ethidium bromide to ribosomes in the absence or in the presence of streptomycin are shown in Fig. 3 in terms of Scatchard plots (r/c vs. r, where r is the number of dye molecules bound per ribosome and c is the molar concentration of free dye}. It can be observed from Fig. 3 that the a m o u n t of ethidium bromide bound per ribosome is lower in the presence of streptomycin since, at equal values of r, the ratio r/c is lower in the presence of streptomycin. The Scatchard plots are not linear, which indicates more than one type of binding site. They can be successfully resolved in terms of a two-site binding process according to the m e t h o d of Halfman and Nishida [30]. The number of binding sites of each type and their association constants are presented in Table III. The total number of sites is 95 in the absence of streptomycin. It decreases to 80 in its presence and this decrease appears to affect the sites with the lower association constant. As suggested by fluorescence measurements and demonstrated by absorption studies, the number of ethidium bromide binding sites is reduced in the presence of streptomycin and since ethidium bromide binds specifically to doublestranded regions of RNA, this reduction indicates a decrease in the a m o u n t of ordered structure in ribosomal RNA. Ballesta et al. [20] observed that treatment of ribosomes with 10-4M ethidium bromide causes partial loss of two 50-S proteins, L7 and L12. No significant loss of proteins could be detected under our labeling conditions with concentrations of ethidium bromide up to 6 • 10 -s M * Discussion
EPR spectroscopy revealed that the binding of streptomycin to the 70-S ribosome induces a loosening of the structure. This loosening is particularly evident when the 30-S proteins, S1 and S18, and the 50-S proteins, L10 and * T h e p r e s e n c e o f p r o t e i n s in t h e s u p e r n a t a n t after c e n t r i f u g a t i o n o f e t h i d i u m b r o m i d e - l a b e l e d r i b o s o m e s w a s b e l o w the level o f s e n s i t i v i t y o f t h e assay. We c a n t h u s a s s u m e t h e loss in L 1 7 / L 1 2 r i b o s o m a l c o n t e n t t o b e less t h a n or e q u a l to 3%.
423 L17, are derivatized. Streptomycin may affect the structure of both the 30-S and the 50-S subunit [6,7]; b u t since the binding site of this antibiotic is located on the 30-S subunit and it interferes mainly with ribosomal functions controlled b y the 30-S subunit, it seems reasonable to assume that the structural alteration induced b y streptomycin occurs predominantly in the 30-S subunit, in the region containing the sulfhydryl groups o f proteins $1 and $18. It should be mentioned that the structural changes induced by streptomycin need not be restricted solely to proteins $1 and S18, but other unlabeled proteins which are located in the same region are probably also affected. Studies on the binding of ethidium bromide indicated that streptomycin also decreases the degree of organization of ribosomal RNA. This result is in agreement with the data of Delihas et al. [6], who reported that the binding o f streptomycin to ribosomes reduces the amount of base-paired regions in ribosomal R N A while enhancing the number of unpaired guanine residues. Although labeling of the ribosomes with ethidium bromide did not provide any information concerning the localization of the changes induced in RNA b y streptomycin, they might also occur in the area containing the sulfhydryl groups of proteins S1 and $18. These studies on the binding of ethidium bromide would be more easily interpreted if one knew what influence ribosomal proteins play on the process of intercalation of the dye into helical regions of ribosomal RNA. It can be mentioned that interaction between ribosomal proteins and 5-S or 16-S R N A resulted in a decrease in the number of binding sites for ethidium bromide [31,32]. By analogy, since streptomycin decreases the number of binding sites for ethidium bromide, it might also enhance interaction between R N A and proteins. While purely speculative, this might satisfactorily explain why streptomycin confers to ribosomes an increased stability against denaturation [33--35] and a decreased ability to undergo conformational changes [36]. The precise location of the binding site of streptomycin on the 30-S ribosomal subunit is still unknown. Some workers favour a location of this site on .16-S I~NA with a possible involvement of the 3' end of 16-S R N A [37,38]. Furthermore, several ribosomal proteins have been found to influence the binding of streptomycin [39--45,22]. Localization by immunoelectron microscopy of the proteins involved in the binding of streptomycin [46,47] and of the 3' end o f 16-S R N A [48] suggests that the antibiotic binds at a site close to the neck region o f the 30-S subunit, at the interface b e t w e e n the subunits. Proteins $1 and S18 are among the proteins involved in the binding of strept o m y c i n [44,45]. These t w o proteins are known to be implicated in important ribosomal functions, since they are part of the decoding site (refs. 49--51, reviewed in ref. 52), and their sulfhydryl groups have been shown to be involved in the positioning of aminoacyl- and initiator-tRNAs [22,53] and in the binding o f natural messengers [54]. The functions in which proteins S1 and S18 are involved are controlled by a set of ribosomal components located in the head of the 30-S subunit, near the binding site of streptomycin, as deduced from immunoelectron microscopy [46--48,55]. In addition to the functions enumerated above, this region also contains the binding site of the initiation factor IF-3 [56--59], which promotes the dissociation of ribosomes into subunits. It can, therefore, be concluded that streptomycin induces structural rear-
424
rangements in a ribosomal area, in the vicinity of its site of attachment. The induction of structural changes in such a key-region for the control of protein synthesis may obviously explain the multiplicity of effects characterizing the action of streptomycin, such as a decrease in translational fidelity, a destabilization of the initiation complex and an interference with ribosome dissociation mediated by factor IF-3.
Acknowledgments We express our appreciation to Drs. A. Bollen, G. Drapeau, H. Dugas, M. Herrington, G. Willick and R. Zimmermann for a number of valuable discussions and criticisms. We thank Dr. F. Capet for the use of her Cary ll8-Spectrophotometer and M. Lacaille for expert assistance. This investigation was supported by grants from the Medical Research Council of Canada and the Minist~re de l'Education du Quebec. G.B., S.G., and N.B. acknowledge support of a studentship from the Conseil des Recherches en Sant~ du Quebec, from the Medical Research Council of Canada and from the National Research Council of Canada respectively. References 1 Gale, E.F., Cundliffe, E., Reynolds, P.E., Richmond, M.H. and Waring, M.J. (1972) The Molecular Basis of An tibiotic Action, pp. 290--306, J o h n Wiley and Sons, New Y ork 2 Schlessinger, O. and Medoff, G. (1975) in Antibiotics I n . Mechanism of A c t i on of Antimicrobial and A n t i t u m o r Agents (Corcoran, J.W. and Hahn, F.E., eds.), pp. 535--550, Springer-Verlag, New York 3 Sherman, M.I. and Simpson, M.V. (1969) Proc. Natl. Acad. Sci. U.S. 64, 1388--1395 4 Sherman, M.I. (1972) Ettr. J. Biochem. 25, 291--300 5 Brakier-Gingras, L., Provost, L. and Dugas, H. (1974) Biochem. Biophys. Res. C ommun. 60, 1238-1244 6 Delihas, N., Topol, E. and Larrinda, I. (1975) FEBS Lett. 53, 170--175 7 Martinez, O., Vazquez, D. and Modolell, J. (1978) FEBS Lett. 87, 21--25 8 Chang, F.N. and Flaks, J.G. (1972) Antimicrob. Agents Chemother. 2, 308--319 9 LePecq, J.B. and Paoletti, C. (1967) J. Mol. Biol. 27, 87--106 10 Gatti, C., Houssier, C. and Fredericq, E. (1975) Biochim. Biophys. Acta 4 0 7 , 3 0 8 - - 3 1 9 11 Moore, P.B. (1971) J. Mol. Biol. 60, 169--184 12 Iwasaki, K., Sabol, S., Wahba, A.J. and Ochoa, S. (1968) Arch. Biochem. Biophys. 125, 542--547 13 Eikenberry, E.F., Bickle, T.A., Traut, R.R. and Price, C.A. (1970) Ettr. J. Biochem. 12, 113--116 14 Tissi~res, A., Watson, J.D., Schlessinger, D. and Hollingworth, B ~ . (1959) J. Mol. Biol. 1, 221--233 15 Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2897--2905 16 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 36, 401--412 17 Dwek, R.A. (1975) Nuclear Magnetic Resonance in Biochemistry, pp. 285--326, Clarendon Press, Oxford 18 Douthart, R.J., Burnett, J.P., Beasley, F.W. and Frank, B.H. (1973) Biochemistry 12, 214--220 19 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 5 1 , 6 6 0 - - 6 7 2 20 Ballesta, J.P.G., Waling, M.J. and Vazquez, D. (1976) Nucleic Acids Res. 3, 1307--1322 21 Ginzburg, I., Miskin, R. and Zamir, A. (1973) J. Mol. Biol. 79, 481--494 22 Ginzburg, I. and Zamir, A. (1976) J. Mol. Biol. 100, 387--398 23 Acharya, A.S. and Moore, P.B. (1973) J. MoL Biol. 76, 207--221 24 Rosenberg, M., Berman, D. and Chang, F.N. (1973) J. Baeteriol. 116, 497--499 25 McConnell, H.M., Deal, W. and Ogata, R.T. (1969) Biochemistry 8, 2580--2585 26 Ebashi, S., Ohnishi, S., Abe, S. and Maruyama, K. (1974) J. Biochem. ( T o k y o ) 75, 211--213 27 Arai, K., Kawakita, M., Kaziro, V., Maeda, T. and Ohnishi, S. (1974) J. Biol. Chem. 249, 3311--3313 28 Stone, D., Prevost, S.C. and Botts, J. (1970) Biochemistry 9, 3937--3947 29 Hemminga, M.A., Van den Boomgaard, T. and De Wit, J.L. (1978) FEBS Lett. 85, 171--174 30 Halfman, C.J. and Nishida, T. (1972) Biochemistry 11, 3 4 9 3 - - 3 4 9 8 31 Feunteun, J., Monier, R., Garrett, R., Le Bret, M. and LePecq, J.B. (1975) J. Mol. Biol. 93, 535--541 32 Bollen, A., Herzog, A., Favre, A., Thibault, J. and Gros, F . ( 1 9 7 0 ) FEBS Lett. 11, 49--54
425 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Leon, S.A. and Brock, T . D . ( 1 9 6 7 ) J. Mol. Biol. 24, 391- -404 Wolfe, A.D. and Hahn, F.E. (1968) Biochem. Biophys. Res. Commun. 3 1 , 9 4 5 - - 9 4 9 Zamir, A., Miskin, R. and Elson, D. (1971) J. MoL Biol. 60, 347--364 Miskin, R. and Zamir, A. ( 1 9 7 2 ) Nat. New Biol. 2 3 8 . 7 8 - - 8 0 Garvin, R.T., Biswas, D.K. and Gorini, L. (1974) Proc. Natl. Aead. Sci. U.S. 71, 3 8 1 4 - - 3 8 1 8 Tai, P.C. and Davis, B.D. (1974) Proc. Natl. Aead. Sci. U.S. 71, 1021--1025 Nomura, M., Mizushima, S., Ozaki, M., Traub, P. and Lowry, C.V. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 49--61 Schreiner, G. and Nierhaus, K.H. (1973) J. Mol. Biol. 81, 71--82 Pongs, O. and Erdmann, V.A. (1973) FEBS Lett. 37, 47--50 Girshovieh, A.S.. Boehkareva, E.S. and Ovehinnikov, Y.A. (1976) MoL Gen. Genet. 144, 205---212 Chang, F.N. and Flaks, J.G. (1970) Proc. Natl. Acad. Scl- U.S. 67, 1321--1328 Lelong, J.C., Gros, D., Gros, F., Bollen, A., Maschler, R. and Stoffler, G. (1974) Proc. Natl. Acad. Sci. U.S. 71, 248--252 Cantor, C.R., Huang, K.H. and Fairclough, R. (1974) in Ribosomes (Nomura, M., Tissi~res, A. and Lengyel, P., eds.), pp. 587--599, Cold Spring Harbor Laboratory, New Y ork Lake, J.A. (1976) J. Mol. Biol. 105, 131--159 Tischendorf, G.W., Zeichhardt, H. and StSffler, G. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4 8 2 0 - - 4 8 2 4 Politz, S.M. and Glitz, D.G. (1977) Proe. Natl. Acad. Sci. U.S. 74, 1468--1472 Pongs, O. and Lanka, E. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1505---1509 Fiser, I., Scheit, K.H., StSffler, G. and Kuechler, E. (1975) FEBS Lett. 5 6 , 2 2 6 - - 2 2 9 Li~hrmann, R., Gassen, H.G. and StSffler, G. (1976) Eur. J. Bioehem. 66, 1--9 Kurland, C.G. (1977) Annu. Rev. Bioehem. 4 6 , 1 7 3 - - 2 0 0 Ginzburg, I. and Zamir, A. (1975) J. Mol. Biol. 93,465---476 Kolb, A., Hermoso, J.M., Thomas, J.O. and Szer, W. (1977) Proc. Natl. Acad. Sei. U.S. 74, 2379-2383 Lake, J.A. (1977) Proc. Natl. Aead. Sci. U.S. 74, 1903--1907 Hawley, D.A., Slobin, L.I. and Wahba, A.J. (1974) Biochem. Biophys. Res. Commun. 61, 544--550 Van Duin, J., Kurland, C.G., Dondon, J. and Grunberg-Manago, M. (1975) FEBS Lett. 59, 287--290 Van Duin, J., Kurland, C.G., Dondon, J., Grunberg-Manago, M., Branlant, C. and Ebel, J. (1976) FEBS Lett. 62, 111--114 Heimark, R.L., Kahan, L., J o h n s t o n , K., Hershey, J.W.B. and Traut, R.R. (1976) J. Mol. Biol. 105, 21 9--230
Biochimica et Biophysica Acta, 521 (1978) 413--425 © Elsevier/North-Holland Biomedical Press
BBA 99332
STREPTOMYCIN-INDUCED CONFORMATIONAL CHANGES IN THE 70-S BACTERIAL RIBOSOME LI~A BRAKIER-GINGRAS a, GUY BOILEAU a, SOPHIE G L O R I E U X a and NORMAND BRISSON b
Ddpartements de a Biochimie et de b Chimie, Universitd de Montreal, Montreal, Quebec (Canada) (Received December 8th, 1977) (Revised manuscript received June 27th, 1978)
Summary Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and $18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.
Introduction
Streptomycin binds to the 30-S ribosomal subunit and inhibits several steps of protein biosynthesis [1,2]. The known effects of this antibiotic are induction of misreading, phenotypic suppression, destabilization of the initiation complex, interference with the dissociation of ribosome into subu~its, and a breakdown of polysomes. Several studies have demonstrated that the structure of the ribosome is altered upon binding of streptomycin. These included hydrogen-tritium exchange [3,4], spin labeling [5], reaction with kethoxal [6] and iodination [7]. Structural changes in the ribosome were also suggested by an examination MalNEt, N-ethylmaleimide; NO-MalNEt, nitxoxide analog of MalNEt; EPR, electron pazarnagnetie resonance. Abbreviations:
414
of the thermodynamic parameters which characterize the binding of streptomycin [8]. This study investigated the changes induced by streptomycin in the ribosome, using fluorescent, radioactive and spin-labeled probes. The fluorescent probe, ethidium bromide, was used since it intercalates preferentially into double-stranded regions of nucleic acids [9,10] and is therefore expected to reveal structural changes in ribosomal RNA. The radioactive marker, N-[14C]ethylmaleimide and the spin-labeled probe, a nitroxide analog of N-ethylmaleimide, which react selectively with sulfhydryl groups of ribosomal proteins [ 11 ] were chosen to monitor conformational changes at the protein level. Preliminary studies in which many ribosomal proteins were spin-labeled suggested that the binding of streptomycin loosens the structure of the ribosome [5]. In the present study, more specific labeling conditions have been used and the derivatized proteins identified with a radioactive label. These data, combined with the results on fluorescence, allow a more conclusive description of the structural changes in the ribosome induced by streptomycin. Materials and Methods Materials Unlabeled MalNEt was purchased from Eastman Corp., [14C]MalNEt (specific activity: 8.4 Ci/mol) was from New England Nuclear and NO-MalNEt, N-(l~xyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide, was obtained from Syva Associates. Ethidium bromide (3,8-diamino-5-ethyl-6-phenyl phenanthridinium bromide) was purchased from Sigma Chemical Co. Streptomycin was obtained from General Biochemicals and [3H]dihydrostreptomycin (3 Ci/mmol) was a product from Amersham Radiochemical Centre. Scintillation counting fluids for extracting 14C-labeled materials from acrylamide gels (Protosol-Econofluor) were purchased from New England Nuclear. Reagents for two
poly(U)~iirected polyphenylalanine synthesis. Their sedimentation constant and purity were checked by analytical ultracentrifugation at 30 000 rev./min
415 with a Beckman-Spinco ultracentrifuge model E, equipped with schlieren and ultraviolet optics.
Binding of streptomycin ethidium bromide
to ribosomes labeled with N-ethylmaleimide or
The influence of the labeling of ribosomes on [3H]dihydrostreptomycin binding was determined b y filtration on Millipore filters according to Chang and Flaks [8]. The concentration of ribosomes was 30 A 2 6 0 n m units/ml (0.72 • 10 -6 M) *. Ethidium bromide and [3H]dihydrostreptomycin were added to give a molar ratio relative to ribosomes of 250 and 100, respectively, and the labeling time was 15 min. Two types of labeling with MalNEt were performed: a limited labeling, in which the ratio of label to ribosome was 125 and the labeling time was 1.50 min and a longer labeling with a ratio of label to ribosome of 250 and an incubation period of 4 h. The antibiotic was added before or after labeling.
Radioactive labeling Labeling of ribosomes with [14C]MalNEt was performed in the absence or presence of streptomycin at 37°C in buffer C. The reaction volume was 0.1 ml, the concentration of ribosomes was 300 A2eonm units/ml; [14C]MalNEt (0.42 Ci/mol) was at a molar ratio of label to ribosome of 125 or 250, corresponding to a labeling of 1.50 min and 4 h respectively. Streptomycin, when present, was added at a molar ratio of antibiotic to ribosome of 100. Ribosomes were first preincubated for 15 min in the presence or absence of streptomycin at 37°C, before adding [14C]MalNEt. After an incubation period of 1.50 min or 4 h, the reaction was stopped b y gel filtration through Sephadex G-25. Ribosomes were precipitated with a cold solution of 5% trichloroacetic acid, filtered on Reeve Angel glass fiber filters, dried and counted in a Packard Tri-Carb Scintillation Counter using a toluene-based scintillation fluid. To identify which ribosomal proteins were labeled, labeling was carried o u t under the same conditions except that the specific activity of [14C]MalNEt was 8.4 Ci/mol. Ribosomal proteins were extracted by the acetic acid m e t h o d of Hardy et al. [ 15] and their separation effected by two-dimensional polyacrylamide gel electrophoresis according to Kaltschmidt and Wittmann [16]. Proteins were stained with 0.2% Coomassie Blue, the spots were cut o u t from the gels, crushed, incubated overnight at 37°C in 10 ml of a 5% Protosol solution in Econofluor, and then counted.
Spin labeling Labeling of the ribosomes with NO-MalNEt was performed under conditions similar to those described above for the radioactive label. Ribosomes were labeled either for 1.50 min or 4 h at 37°C. U n b o u n d NO-MalNEt was removed b y gel filtration through Sephadex G-25. Ribosomes were then incubated at 37°C for 15 min, with or without streptomycin. The EPR spectra were * O n e A 2 6 0 n m u n i t is t h e q u a n t i t y o f m a t e r i a l c o n t a i n e d i n 1 m l o f a s o l u t i o n w h i c h has an a b s o r b a n c e o f 1 a t 2 6 0 n m , w h e n m e a s u r e d i n a c e l l w i t h a 1 - c m p a t h length. One A 2 6 0 n m u n i t c o r r e s p o n d s t o 24 pmol of 70-S ribosomes [14].
416 recorded at 37°C _+ I°C on a Brucker 414S spectrometer operating at 9.33 GHz and equipped with a temperature control unit. The samples were examined in a small aqueous flat cell (Scanlon Co.). Changes in the EPR spectra arise from modifications in the mobility of the labels (for a discussion of the factors involved in EPR line shape analysis, see ref. 17). The labeling conditions were slightly different from those used previously [ 5], in t h a t a lower ratio of label to ribosome and a shorter period of treatment with NO-MalNEt (1.50 min or 4 h instead of 24 h) were used. Under these conditions, less dissociation was found to occur and ribosome degradation was avoided.
Fluorescence labeling Fluorescence labeling of ribosomes in the presence or in the absence of streptomycin with different concentrations of ethidium bromide was performed at 37°C in buffer C'. The reaction volume (3 ml) contained ribosomes (10 A260nm units/ml) and ethidium bromide at a molar ratio of label to ribosome of 50 to 250. The molar ratio of streptomycin to ribosome was 100. When appropriate, ribosomes were preincubated with streptomycin for 15 min before adding ethidium bromide. The fluorescence intensity of labeled ribosomes was measured at 37°C with a PMQ II Zeiss spectrophotometer equipped with a fluorescent attachment and a temperature control unit. The excitation wavelength was 540 nm, the emission was measured at 590 nm. Absorption studies were performed in order to determine the difference in the number o f ethidium bromide molecules bound to ribosomes in the presence or in the absence of streptomycin. The conditions for labeling were similar to those used in fluorescence labeling except for the amounts of ribosomes and ethidium bromide used: the reaction volume was 2 ml, the ribosome concentration was 100 A2e0nm units/ml, the concentration of ethidium bromide was in a molar ratio of label to ribosome varying from 5 to 100. The molar ratio of streptomycin to ribosome was 100. The absorption of ribosome-bound ethidium bromide was measured at 465 nm at 37°C with a C a r y - l l 8 recording spectrophotometer using a l~cm light path cell in a thermostated cell holder. The a m o u n t o f free and bound ethidium bromide was determined following the procedure o f Douthart et al. [18], which is based on the difference in the extinction coefficient of free and bound form. The association constant and the number o f binding sites for ethidium bromide were estimated using the Scatchard relationship [19]. Possible release of ribosomal proteins after treatment with ethidium bromide [20] was checked by two-dimensional polyacrylamide gel electrophoresis on the 150 000 X g supernatant of labeled ribosomes. Results
Effect o f labeling with N-ethylmaleimide or ethidium bromide on the binding o f streptomycin to ribosomes Labeling with MalNEt or with ethidium bromide did not interfere with the binding of [3H]dihydrostreptomycin to ribosomes as assayed by Millipore
417 TABLE I I D E N T I F I C A T I O N O F T H E 70-S R I B O S O M A L P R O T E I N S L A B E L E D W I T H N - [ 1 4 C ] E T H Y L M A L E I MIDE A F T E R A T R E A T M E N T OF 1.50 MIN P r o t e i n s w e r e e x t r a c t e d f r o m r i b o s o m e s l a b e l e d w i t h [ 1 4 C ] M a l N E t f o r 1 . 5 0 m i n as d e s c r i b e d in Materials and M e t h o d s . T h e y w e r e f r a c t i o n a t e d 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 c t r o p h o r e s i s and t h e a m o u n t o f r a d i o a c t i v i t y b o u n d t o e a c h p r o t e i n s p o t w a s d e t e r m i n e d . R e s u l t s are a v e r a g e s o f five d i f f e r e n t e x p e r i m e n t s . C o u n t s are u n c o r r e c t e d f o r t h e p e r c e n t a g e o f m a t e r i a l r e m a i n i n g at t h e origin o f p o l y a c r y l a m i d e gels. T o a c c o u n t for d i f f e r e n c e s in t h e a m o u n t o f p r o t e i n s s u b m i t t e d t o e l e e t r o p h o r e s i s , results are n o r m a l i z e d t o a t o t a l o f 1 6 0 0 c o u n t s m i n -x p e r gel. S t a n d a r d d e v i a t i o n o n i n d i v i d u a l c o u n t s p e r p r o t e i n is 10%. P r o t e i n s l a b e l e d w i t h less t h a n 5% o f t h e r a d i o a c t i v i t y f o u n d in t h e p r o t e i n w i t h m a x i m u m l a b e l i n g h a v e n o t b e e n listed. 30-S p r o t e i n s ( c o u n t m i n - 1 )
50-S p r o t e i n s ( c o u n t r a i n - 1 )
Withoutstreptomycin
With s t r e p t o m y c i n
Withoutstreptomycin
With s t r e p t o m y c i n
S1 S18
295 885
L10 LI7
156 264
285 880
167 268
filtration (data not shown) *. It is known that labeling with a higher concentration o f MalNEt does interfere with the binding of streptomycin [ 5,21], because of the blocking of the sulfhydryl group of protein S14 [22]. However, as will be shown later, labeling of protein S14 did not occur under the experimental conditions used in this study.
Binding of N-[14C]ethylmaleimide Depending on whether the incubation with MalNEt lasted 1.50 min or 4 h, an average of one or six sulfhydryl groups per ribosome were found to react with MalNEt. The presence of streptomycin did not modify the amount o f binding. To identify the proteins which are derivatized under our conditions, ribosomes were labeled with [14C]MalNEt, ribosomal proteins were extracted and fractionated b y two
418
labeled. This reflects the variability in the reactivity of the different sulfhydryl groups [23,24] and indicates that all the ribosomes are not equally labeled.
Spin labeling The EPR spectra of spin-labeled ribosomes obtained after a 1.50 min or a 4 h labeling period are presented in Fig. 1 (A--B). These spectra are typical for spin labels bound to proteins. They are t w o - c o m p o n e n t spectra consisting of a constrained (I) and a more mobile (M) part. The existence of the two components in the EPR spectra of spin-labeled ribosomes can be explained if there are two types of sulfhydryl binding sites corresponding to the mobile and the less mobile part of the spectra, or if the label, at each of the binding sites, can alternatively occupy t w o isomeric states, one mobile and one constrained, a situation frequently encountered with spin-labeled proteins [25--27].
iM
I
w
I+M Z 0
m
~M
f
~M
10G
MAGNETIC FIELD (G) Fig. I . A, E P R s p e c t r u m o f 70-S r i b o s o m e s spin-labeled f o r 1 . 5 0 m i n w i t h N O - M a l N E t in t h e a b s e n c e (solid line) o r in t h e p r e s e n c e o f s t r e p t o m y c i n ( d a s h e d line). T h e a r r o w s M a n d I r e f e r t o t h e m o b i l e a n d less m o b i l e p a r t o f t h e s p e c t r u m , r e s p e c t i v e l y . T h e i n s e r t is a n e n l a r g e d s e c t i o n o f t h e l o w field p a r t of t h e s p e c t n a m w i t h t h e s y m b o l s h I a n d h 2 r e p r e s e n t i n g t h e h e i g h t s o f t h e l o w field b a n d s c o r r e s p o n d i n g t o t h e m o b i l e a n d less m o b i l e p a r t o f t h e s p e c t r u m . E P R s p e c t r a w e r e m e a s u r e d w i t h m i c r o w a v e f r e q u e n c y , 9 . 3 3 G H z ; m o d u l a t i o n a m p l i t u d e , I G; m i c r o w a v e p o w e r , 1 0 m W a n d a m p l i f i c a t i o n 5 • 104. B, E P R spect r u m o f 70-S r i b o s o m e s spin-labeled f o r 4 h w i t h N O - M a l N E t . C o n d i t i o n s o f m e a s u r e m e n t are t h e s a m e as a b o v e e x c e p t f o r m o d u l a t i o n a m p l i t u d e , 0.6 G; m i c r o w a v e p o w e r , 5 m W a n d a m p l i f i c a t i o n , 4 • 104. T h e s p e c t r u m is n o t s i g n i f i c a n t l y a l t e r e d in t h e p r e s e n c e o f s t r e p t o m y c i n ( n o t s h o w n ) .
419 T A B L E II EFFECT OF STREPTOMYCIN ON THE EPR SPECTRA OF SPIN-LABELED RIBOSOMES T h e i n d e x o f m o b i l i t y , t h e h 1/h 2 r a t i o , is d e f i n e d as t h e r a t i o o f t h e h e i g h t s o f t h e l o w field p e a k s corresp o n d i n g t o t h e m o b i l e a n d less m o b i l e p a r t o f t h e s p e c t r a (Fig. 1). T h e h l / h 2 r a t i o v a r i e s slightly w i t h t h e d i f f e r e n t p r e p a r a t i o n s or b e c a u s e o f t h e d e l a y o c c u r r i n g b e f o r e r e c o r d i n g t h e s p e c t r a . T h e m o s t p r e v a l e n t eases are p r e s e n t e d h e r e . F o r e a c h e x p e r i m e n t , t h r e e s a m p l e s f r o m t h e s a m e p r e p a r a t i o n w e r e l a b e l e d und e r s i m i l a r c o n d i t i o n s a n d t h e E P R s p e c t r a w e r e r e c o r d e d in t h e a b s e n c e a n d t h e p r e s e n c e o f s t r e p t o m y c i n . T o i n c r e a s e t h e a c c u r a c y i n t h e m e a s u r e m e n t o f h l / h 2, t h e p a r t o f t h e s p e c t r u m c o r r e s p o n d i n g t o t h e l o w field lines w a s s c a n n e d w i t h a n i n c r e a s e d gain s e t t i n g . F o r a n y given set o f e x p e r i m e n t s , t h e v a l u e s o f h l / h 2 w e r e r e p r o d u c i b l e w i t h i n a s t a n d a r d d e v i a t i o n o f -+5%. Conditions
Experiment
I n d e x o f m o b i l i t y , hl/h 2 Without streptomycin
With s t r e p t o m y c i n
1 . 5 0 m i n labeling
1 2 3
1.3 1.5 1.2
1 .S 2.0 1.6
441 l a b e l i n g
1 2 3
4.0 4.7 5.2
4.2 5.0 5.4
Conformational changes in a macromolecule exhibiting a t w o - c o m p o n e n t EPR spectrum can be detected b y estimating the variation in the proportion of the mobile and the less mobile part of the spectrum. This is c o m m o n l y estimated b y measuring the ratio o f the heights of the low field lines of the mobile and less mobile components, hi and h2, respectively (Fig. 1A: insert) (e.g. see refs. 28, 29). An increase in the index of mobility, the h~/h2 ratio, indicates a loosening in the molecular structure in the environment of the binding site of the label. Direct examination o f experimental data reveals that EPR spectra of ribosomes labeled for 1.50 min are sensitive to the presence of streptomycin in that the mobile c o m p o n e n t of the spectrum increases at the expense of the less mobile c o m p o n e n t (Fig. 1A). Examples of the variation in the h~/h2 ratio o f EPR spectra after addition of streptomycin are presented in Table II. Irrespective o f fluctuations in the h~/h2 ratio values, an increase of 35 +5% was always detected in the presence of streptomycin. This result demonstrates that u p o n binding of streptomycin structural changes occur in the ribosome in the vicinity o f labeled sulfhydryl groups, and the increase in the mobility of the labels indicates that these changes involve a loosening in the structure of some ribosomal proteins. After a 4 h labeling period, the two components o f the EPR spectra of labeled ribosomes can still be distinguished (Fig. 1B) b u t these spectra are much more mobile that after a labeling o f 1.50 min since the intensity of the signal due to the constrained part is less important and the hi~h2 ratio is higher (Table II). In contrast to the situation observed after a labeling period of 1.50 min, addition o f streptomycin to 4 h-labeled ribosomes did n o t significantly alter the spectrum * nor m o d i f y the h~/h2 ratio (Table II). • A s m a l l a n d r e p r o d u c i b l e d e c r e a s e in t h e r a t i o of t h e h e i g h t o f t h e c e n t r a l b a n d t o t h e h i g h field b a n d o f t h e m o b i l e p a r t o f t h e s p e c t r u m is n o t i c e a b l e h o w e v e r . S u c h a d e c r e a s e w h i c h i n d i c a t e s a slight i n c r e a s e in t h e m o b i l i t y o f t h e spin labels h a d led u s to suggest p r e v i o u s l y t h a t t h e b i n d i n g o f s t r e p t o m y c i n l o o s e n s t h e r i b o s o m a l s t r u c t u r e [ 5].
420
Radioactive labeling (Table I) showed that after a labeling of 1.50 min, two 30-S proteins, $1 and $18, and two 50-S proteins, L10 and L17, were preferentially labeled with [14C]MalNEt. That these data are also applicable to the spin label was demonstrated by exposing the ribosomes to a mixture of radioactive and spin labels in a 1 : 1 molar ratio. It was found that radioactive labeling decreased by about 50%, which indicates that the radioactive label and the spin label compete for the same sites. Following a treatment of 4 h with MalNEt derivatives, a larger number of ribosomal sites are labeled. A comparison of the influence of streptomycin on spin-labeled ribosomes after a treatment of 1.50 min and 4 h thus reveals two interesting points: first, the binding of streptomycin loosens the structure of ribosomal proteins in the environment of some derivatized sulfhydryl groups, and second, this loosening is probably located in a restricted area of the ribosome, since it is readily observed only when a limited number of ribosomal sites are labeled.
10
Z <
E c O
>.
5
z_ u Z
~
I~ MOLAR
RATIO OF ETHIDIUM BROMIDE
200 TO
RIBOSOME
Fig. 2. E f f e c t o f t h e 7 0 - S r i b o s o m e s o n t h e f l u o r e s c e n c e o f e t h i d i u m b r o m i d e in t h e a b s e n c e (-=), or in t h e p r e s e n c e ( o o) of streptomycin. ~ A, i n t e n s i t y o f f l u o r e s c e n c e o f e t h i d i u m b r o m i d e alone. No correction to the fluorescence intensity of ethidium bromide bound to ribosomes was made f o r t h e c o n t r i b u t i o n o f u n b o u n d d y e . T h e v e r t i c a l b a z s r e p r e s e n t t h e s t a n d a r d v a r i a t i o n o v e r six e x p e r i ments.
421
Binding of ethidium bromide The fluorescence intensity of ribosomes as a function of the molar ratio of ethidium bromide to ribosome is shown in Fig. 2. In the absence or presence of streptomycin, there is a rapid initial increase in fluorescence intensity, followed by a more gradual increase. This type of fluorescence enhancement is known to be characteristic of an intercalation of ethidium bromide molecules into ordered regions of RNA [10]. At the ionic strength of buffer C' in which labeling is carried out, electrostatic interactions between ethidium bromide and RNA would be minimal [9]. The fluorescence intensity of ribosomes labeled with ethidium bromide in the presence of streptomycin was found to be slightly less than the fluorescence observed in the absence of the antibiotic. The differences are small but larger than those due to experimental variations. No change in the fluorescence intensity was seen when ribosomes from streptomycin-resistant cells were labeled with ethidium bromide in the presence of streptomycin (data not shown). The fluorescence intensity o f ethidium bromide alone was unaffected by the presence of streptomycin. These results suggest that the binding of streptomycin to the ribosome perturbs the secondary structure of ribosomal RNA; the lower fluorescence intensity could be due to a reduced number of ethidium bromide binding sites, resulting from conformational changes in ribosomal RNA. r x 10 - 6 C
0.:
0.2
00
~
2o
r
Fig. 3. S c a t c h a ~ ! P l o t s o f e t h i d i u m b r o m i d e b i n d i n g t o r i b o s o m e s i n the absence ( e ) o r i n the presence (A) o f s t r e p t o m y c i n ; r represents the n u m b e r o f b o u n d e t h i d i u m b r o m i d e molecules p e r r i b o s o m e and c is the m o l a r c o n c e n t r a t i o n o f free d y e ; r a n d c w e r e d e t e r m i n e d f r o m a b s o r p t i o n m e a s u r e m e n t s at 4 6 5 n m . Each e x p e r i m e n t a l p o i n t is t h e m e a n o f six e x p e r i m e n t s . Lines d r a w n t h r o u g h data p o i n t s w e r e c a l c u l a t e d from the parameters given in Table III.
422
TABLE III C H A R A C T E R I Z A T I O N O F E T H I D I U M B R O M I D E B I N D I N G T O 70-S R I B O S O M E S I N T H E A B S E N C E O R IN T H E P R E S E N C E O F S T R E P T O M Y C I N T h e n u m b e r o f e t h i d i u m b r o m i d e b i n d i n g sites a n d t h e b i n d i n g c o n s t a n t s are c a l c u l a t e d f r o m t h e Scatc h a r d p l o t s p r e s e n t e d in Fig. 3. B i n d i n g p a r a m e t e r s are d e t e r m i n e d f r o m t h e e s t i m a t e d i n t e r c e p t s o f the S e a t c h a r d p l o t s and f r o m data p o i n t s a c c o r d i n g to H a l f m a n a n d Nishida [ 3 0 ] . Conditions
Without streptomycin With s t r e p t o m y c i n
N u m b e r o f sites
Binding c o n s t a n t s (×10--4 M - l )
Total number
1st t y p e
95 80
8 8
2nd type
87 72
1st t y p e
2nd type
2.5 2.5
0.18 O. 18
Direct evidence for this hypothesis was sought by absorption measurements of the a m o u n t of free and b o u n d dye at different molar ratios of ethidium bromide to ribosome. The binding isotherms of ethidium bromide to ribosomes in the absence or in the presence of streptomycin are shown in Fig. 3 in terms of Scatchard plots (r/c vs. r, where r is the number of dye molecules bound per ribosome and c is the molar concentration of free dye}. It can be observed from Fig. 3 that the a m o u n t of ethidium bromide bound per ribosome is lower in the presence of streptomycin since, at equal values of r, the ratio r/c is lower in the presence of streptomycin. The Scatchard plots are not linear, which indicates more than one type of binding site. They can be successfully resolved in terms of a two-site binding process according to the m e t h o d of Halfman and Nishida [30]. The number of binding sites of each type and their association constants are presented in Table III. The total number of sites is 95 in the absence of streptomycin. It decreases to 80 in its presence and this decrease appears to affect the sites with the lower association constant. As suggested by fluorescence measurements and demonstrated by absorption studies, the number of ethidium bromide binding sites is reduced in the presence of streptomycin and since ethidium bromide binds specifically to doublestranded regions of RNA, this reduction indicates a decrease in the a m o u n t of ordered structure in ribosomal RNA. Ballesta et al. [20] observed that treatment of ribosomes with 10-4M ethidium bromide causes partial loss of two 50-S proteins, L7 and L12. No significant loss of proteins could be detected under our labeling conditions with concentrations of ethidium bromide up to 6 • 10 -s M * Discussion
EPR spectroscopy revealed that the binding of streptomycin to the 70-S ribosome induces a loosening of the structure. This loosening is particularly evident when the 30-S proteins, S1 and S18, and the 50-S proteins, L10 and * T h e p r e s e n c e o f p r o t e i n s in t h e s u p e r n a t a n t after c e n t r i f u g a t i o n o f e t h i d i u m b r o m i d e - l a b e l e d r i b o s o m e s w a s b e l o w the level o f s e n s i t i v i t y o f t h e assay. We c a n t h u s a s s u m e t h e loss in L 1 7 / L 1 2 r i b o s o m a l c o n t e n t t o b e less t h a n or e q u a l to 3%.
423 L17, are derivatized. Streptomycin may affect the structure of both the 30-S and the 50-S subunit [6,7]; b u t since the binding site of this antibiotic is located on the 30-S subunit and it interferes mainly with ribosomal functions controlled b y the 30-S subunit, it seems reasonable to assume that the structural alteration induced b y streptomycin occurs predominantly in the 30-S subunit, in the region containing the sulfhydryl groups o f proteins $1 and $18. It should be mentioned that the structural changes induced by streptomycin need not be restricted solely to proteins $1 and S18, but other unlabeled proteins which are located in the same region are probably also affected. Studies on the binding of ethidium bromide indicated that streptomycin also decreases the degree of organization of ribosomal RNA. This result is in agreement with the data of Delihas et al. [6], who reported that the binding o f streptomycin to ribosomes reduces the amount of base-paired regions in ribosomal R N A while enhancing the number of unpaired guanine residues. Although labeling of the ribosomes with ethidium bromide did not provide any information concerning the localization of the changes induced in RNA b y streptomycin, they might also occur in the area containing the sulfhydryl groups of proteins S1 and $18. These studies on the binding of ethidium bromide would be more easily interpreted if one knew what influence ribosomal proteins play on the process of intercalation of the dye into helical regions of ribosomal RNA. It can be mentioned that interaction between ribosomal proteins and 5-S or 16-S R N A resulted in a decrease in the number of binding sites for ethidium bromide [31,32]. By analogy, since streptomycin decreases the number of binding sites for ethidium bromide, it might also enhance interaction between R N A and proteins. While purely speculative, this might satisfactorily explain why streptomycin confers to ribosomes an increased stability against denaturation [33--35] and a decreased ability to undergo conformational changes [36]. The precise location of the binding site of streptomycin on the 30-S ribosomal subunit is still unknown. Some workers favour a location of this site on .16-S I~NA with a possible involvement of the 3' end of 16-S R N A [37,38]. Furthermore, several ribosomal proteins have been found to influence the binding of streptomycin [39--45,22]. Localization by immunoelectron microscopy of the proteins involved in the binding of streptomycin [46,47] and of the 3' end o f 16-S R N A [48] suggests that the antibiotic binds at a site close to the neck region o f the 30-S subunit, at the interface b e t w e e n the subunits. Proteins $1 and S18 are among the proteins involved in the binding of strept o m y c i n [44,45]. These t w o proteins are known to be implicated in important ribosomal functions, since they are part of the decoding site (refs. 49--51, reviewed in ref. 52), and their sulfhydryl groups have been shown to be involved in the positioning of aminoacyl- and initiator-tRNAs [22,53] and in the binding o f natural messengers [54]. The functions in which proteins S1 and S18 are involved are controlled by a set of ribosomal components located in the head of the 30-S subunit, near the binding site of streptomycin, as deduced from immunoelectron microscopy [46--48,55]. In addition to the functions enumerated above, this region also contains the binding site of the initiation factor IF-3 [56--59], which promotes the dissociation of ribosomes into subunits. It can, therefore, be concluded that streptomycin induces structural rear-
424
rangements in a ribosomal area, in the vicinity of its site of attachment. The induction of structural changes in such a key-region for the control of protein synthesis may obviously explain the multiplicity of effects characterizing the action of streptomycin, such as a decrease in translational fidelity, a destabilization of the initiation complex and an interference with ribosome dissociation mediated by factor IF-3.
Acknowledgments We express our appreciation to Drs. A. Bollen, G. Drapeau, H. Dugas, M. Herrington, G. Willick and R. Zimmermann for a number of valuable discussions and criticisms. We thank Dr. F. Capet for the use of her Cary ll8-Spectrophotometer and M. Lacaille for expert assistance. This investigation was supported by grants from the Medical Research Council of Canada and the Minist~re de l'Education du Quebec. G.B., S.G., and N.B. acknowledge support of a studentship from the Conseil des Recherches en Sant~ du Quebec, from the Medical Research Council of Canada and from the National Research Council of Canada respectively. References 1 Gale, E.F., Cundliffe, E., Reynolds, P.E., Richmond, M.H. and Waring, M.J. (1972) The Molecular Basis of An tibiotic Action, pp. 290--306, J o h n Wiley and Sons, New Y ork 2 Schlessinger, O. and Medoff, G. (1975) in Antibiotics I n . Mechanism of A c t i on of Antimicrobial and A n t i t u m o r Agents (Corcoran, J.W. and Hahn, F.E., eds.), pp. 535--550, Springer-Verlag, New York 3 Sherman, M.I. and Simpson, M.V. (1969) Proc. Natl. Acad. Sci. U.S. 64, 1388--1395 4 Sherman, M.I. (1972) Ettr. J. Biochem. 25, 291--300 5 Brakier-Gingras, L., Provost, L. and Dugas, H. (1974) Biochem. Biophys. Res. C ommun. 60, 1238-1244 6 Delihas, N., Topol, E. and Larrinda, I. (1975) FEBS Lett. 53, 170--175 7 Martinez, O., Vazquez, D. and Modolell, J. (1978) FEBS Lett. 87, 21--25 8 Chang, F.N. and Flaks, J.G. (1972) Antimicrob. Agents Chemother. 2, 308--319 9 LePecq, J.B. and Paoletti, C. (1967) J. Mol. Biol. 27, 87--106 10 Gatti, C., Houssier, C. and Fredericq, E. (1975) Biochim. Biophys. Acta 4 0 7 , 3 0 8 - - 3 1 9 11 Moore, P.B. (1971) J. Mol. Biol. 60, 169--184 12 Iwasaki, K., Sabol, S., Wahba, A.J. and Ochoa, S. (1968) Arch. Biochem. Biophys. 125, 542--547 13 Eikenberry, E.F., Bickle, T.A., Traut, R.R. and Price, C.A. (1970) Ettr. J. Biochem. 12, 113--116 14 Tissi~res, A., Watson, J.D., Schlessinger, D. and Hollingworth, B ~ . (1959) J. Mol. Biol. 1, 221--233 15 Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2897--2905 16 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 36, 401--412 17 Dwek, R.A. (1975) Nuclear Magnetic Resonance in Biochemistry, pp. 285--326, Clarendon Press, Oxford 18 Douthart, R.J., Burnett, J.P., Beasley, F.W. and Frank, B.H. (1973) Biochemistry 12, 214--220 19 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 5 1 , 6 6 0 - - 6 7 2 20 Ballesta, J.P.G., Waling, M.J. and Vazquez, D. (1976) Nucleic Acids Res. 3, 1307--1322 21 Ginzburg, I., Miskin, R. and Zamir, A. (1973) J. Mol. Biol. 79, 481--494 22 Ginzburg, I. and Zamir, A. (1976) J. Mol. Biol. 100, 387--398 23 Acharya, A.S. and Moore, P.B. (1973) J. MoL Biol. 76, 207--221 24 Rosenberg, M., Berman, D. and Chang, F.N. (1973) J. Baeteriol. 116, 497--499 25 McConnell, H.M., Deal, W. and Ogata, R.T. (1969) Biochemistry 8, 2580--2585 26 Ebashi, S., Ohnishi, S., Abe, S. and Maruyama, K. (1974) J. Biochem. ( T o k y o ) 75, 211--213 27 Arai, K., Kawakita, M., Kaziro, V., Maeda, T. and Ohnishi, S. (1974) J. Biol. Chem. 249, 3311--3313 28 Stone, D., Prevost, S.C. and Botts, J. (1970) Biochemistry 9, 3937--3947 29 Hemminga, M.A., Van den Boomgaard, T. and De Wit, J.L. (1978) FEBS Lett. 85, 171--174 30 Halfman, C.J. and Nishida, T. (1972) Biochemistry 11, 3 4 9 3 - - 3 4 9 8 31 Feunteun, J., Monier, R., Garrett, R., Le Bret, M. and LePecq, J.B. (1975) J. Mol. Biol. 93, 535--541 32 Bollen, A., Herzog, A., Favre, A., Thibault, J. and Gros, F . ( 1 9 7 0 ) FEBS Lett. 11, 49--54
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