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Inhibition of peptide chain initiation in Escherichia coli by thermorubin
310
Biochimica et Biophysica Acta, 366 (1974) 310--318 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98114
INHIBITION OF PEPTIDE CHAIN INITIATION IN ESCHERICHIA COLI BY THERMORUBIN
G. PIRALI a, S. SOMMA a, G.C. LANCINI a and F. SALA b
aLaboratori Ricerche, Gruppo Lepetit S.p.A., via Durando, 38, Milano and bLaboratorio di Genetica biochimica ed evoluzionistica del Consiglio Nazionale delle Ricerche, Pavia (Italy) (Received April 22rid, 1974)
Summary The antibiotic thermorubin specifically inhibits protein synthesis when added to growing cultures of Escherichia coli, while RNA and DNA synthesis are not affected. In vitro studies show that thermorubin is a specific inhibitor of initiation of protein synthesis directed by natural mRNA. The drug inhibits protein synthesis directed by MS2 RNA but not poly(U)-dependent polyphenylalanine synthesis. Furthermore, during protein synthesis directed by endogenous messenger RNA, only the fraction of polypeptide synthesis dependent on ex novo-synthesized mRNA is sensitive to the drug. Thermorubin inhibits the binding of fMet--tRNA to ribosomes in the presence of AUG, GTP and initiation factors whereas the synthesis of the first peptide bond, assayed as fMet-puromycin synthesis, is not impaired by the antibiotic. The implications of these findings, with respect to the hypothesis that RNA synthesis in E. coli is controlled by initiation of protein synthesis, are discussed.
Introduction Thermorubin is an antibiotic produced by Thermoactinomyces antibioticus, a thermophilic actinomycete, active against Gram positive and many Gram negative bacteria, but ineffective on fungi and Protozoa [1]. It is an unusual example of a metabolite composed of a xanthonic and a substituted anthraquinonic moiety [2]. The product shows a low degree of toxicity in mice (LDs0~-300 mg/kg intraperitoneally) but is poorly effective in curing experimental infections when administered orally or subcutaneously (Arioli, V., unpublished results). In this paper we report evidence, obtained from growing cells and cell-free systems, that thermorubin inhibits peptide synthesis at the level of translation. The primary target of the antibiotic seems to be a step of the initiation process of protein synthesis prior to the formation of the first peptide bond. The elongation of polypeptide chains is not affected by thermorubin.
311
Materials and Methods
Escherichia coli K12 (761, his, try- and 690S T h y , Met-, L.L. Cavalli Sforza) was used. Cell free extracts ($30) were prepared from cells collected during early exponential growth in nutrient broth. Growth conditions and extraction procedures were essentially as described by Niremberg and Matthei [3] but in some cases preincubation and DNAase treatment were omitted as specified in the text. MS2 bacteriophage was prepared according to Sargeant [4] and its RNA was extracted following the method of Gesteland and Boedtker [5], without CsCl2 gradient centrifugation. ['4C]phenylalanyl-tRNA ([14 C] phe--tRNA) was prepared according to Kaji et al. [6]. The mixture of [' 4 C] aminoacyl-tRNAs was prepared in the same manner using stripped tRNA from E. coli and 14 C-labelled amino acids from Chlorella protein acid hydrolysate, supplemented with the amino acids not contained in this mixture (Try, CySH, GluNH~ and AspNH2 ). Polypeptide synthesis was assayed as described by Ciferri et al. [7]. Ribosomes, initiation factors, synthesis of N-formyl[' 4 C] methionyl-tRNA (f[l 4 C] Met-tRNA), and the assays of initiation reactions, binding f[14 C] Met-tRNA to the ribosomes and fMet-puromycin synthesis were performed as described by Sala and Kiintzel [ 8]. Thermorubin was dissolved in dimethylformamide: the solvent did not impair the assays at concentrations lower than 2% (v/v). Radioactivity was determined by a Philips Liquid Scintillation Analyzer. Stripped tRNA from E. coli was obtained from General Biochemical, Chargin, Ohio, [1-'4C]phenylalanine (57 Ci/mole), the mixture of (1-14 C)-labelled amino acid Chlorella hydrolysate (25 Ci/atom of carbon labelled) and [1-14C]methionine (233 pCi/mmole) from the Radiochemical Centre, Amersham, England; Polyuridilic acid was from Sigma, St. Louis, Mo., U.S.A., AUG from Miles Laboratories, Kankakee, Illinois, U.S.A., and puromycin from Serva, Heidelberg, Germany. Results
In vivo studies The minimal inhibitory concentration of thermorubin on the strain of E. coli used by us is 2pg]ml. The action of the antibiotic appears to be bacteriostatic; a culture in exponential growth, containing 108 cells/ml, was treated with 200 pg/ml of thermorubin and at different time intervals the number of viable cells was counted by plating after suitable dilution: the number of colonies formed did not decrease even after 7 h contact with the drug {data not shown). The effect of thermorubin on macromolecular synthesis in intact cells was studied by adding 10 pg/ml of the drug to a culture growing in the presence of radioactive thymine, uracil and phenylalanine. As shown in Fig. 1, within 2--3 min of the addition of the drug, the incorporation of phenylalanine is blocked, whereas incorporation of thymidine is only partially depressed and no effect is evident on uracil incorporation for about 40 min. The overall picture suggests that the antibiotic specifically interferes with the translation process. This is also supported by the experiment reported in
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Fig. 1. E f f e c t o f t h e r m o r u b i n o n t h e i n c o r p o r a t i o n o f [ 14 C] t h y m i n e ( A ) [ 14 C] u r a c i l (B) a n d [ 14 C] p h e n y l a l a n i n e (C) b y i n t a c t cells o f E. coll. T i m e o f a d d i t i o n o f t h e r m o r u b i n is i n d i c a t e d b y a n a r r o w . E. coli K 1 2 ( 6 9 0 S T h y , M e t - R C s i r ) f o r [ 1 4 C ] t h y m i n e i n c o r p o r a t i o n a n d E. coil K 1 2 ( 7 6 1 , h i s , t r y - R C s t r ) f o r [ 1 4 C ] u r a c i l a n d [ 14 C] p h e n y l a l a n i n e i n c o r p o r a t i o n , w e r e g r o w n in Davis M i n i m a l m e d i u m s u p p l e m e n t e d with 0.2% Difco casarnino acids and, respectively, 10 #g/ml of thymine, 7 #g/ml of uracil and 10 #g/mi of p h e n y l a l a n i n e . W h e n all c o n c e n t r a t i o n s r e a c h e d 1 0 8 c e l l / m i , [ 2 - 1 4 C ] t h y m i n e ( 0 . 0 5 # C i / m l , final) [ 2 - 1 4 C ] u r a c i l ( 0 . 0 2 5 # C i / m l , final) a n d [U° 1 4 C ] p h e n y l a l a r d n e ( 0 . 0 2 5 # C i / m l ) w e r e a d d e d . T h e r m o r u b i n was added after 10 min at a 10 #g/mi final concentration. Aliquots of 2 ml were withdrawn at intervals, d i l u t e d w i t h 3 m l o f 8% c o l d t r i c h l o r o a c e t i c a c i d a n d t h e r a d i o a c t i v i t y o f i n s o l u b l e m a t e r i a l c o l l e c t e d o n glass-fiber d i s c s d e t e r m i n e d .
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Fig. 2. R e s u m p t i o n o f R N A s y n t h e s i s is s t r i n g e n t E. coli cells s t a r v e d f o r a m i n o a c i d s f o l l o w i n g a d d i t i o n o f t h e r m o r u b i n . E. coil K 1 2 ( 6 9 0 S , thy-, m e t - , R C s t r ) w a s g r o w n in Davis m i n i m a l m e d i u m s u p p l e m e n t e d w i t h 0 . 2 % c a s a m i n o a c i d s a n d 1 0 # g / m l o f t h y m i n e u p t o a c o n c e n t r a t i o n o f 5 • 1 0 7 cells/ml. 2 0 0 m l o f t h i s c u l t u r e w e r e f i l t e r e d o n a m f l l i p o r e m e m b r a n e filter ( 0 . 4 5 #), w a s h e d a n d r e s u s p e n d e d i n a n e q u a l v o l u m e o f t h e s a m e m e d i u m b u t w i t h o u t a m i n o acids. A f t e r 15 m i n i n c u b a t i o n at 3 7 ° C [ 2 - 1 4 C ] u r a c i l w a s a d d e d a t 5 ~ g / m l (spec. a c t . 3.1 # C i / m m o l e ) a n d a f t e r a n a d d i t i o n a l 1 0 r a i n t h e c u l t u r e w a s s u b d i v i d e d in f o u r p a r t s a n d t o t h r e e o f t h e s e w e r e a d d e d : c h l o r a m p h e n i c o l , 2 0 # g / m 1 a n d t h e r m o r u b i n , 5 a n d 2 0 # g / m 1 , r e s p e c t i v e l y . A l i q u o t s o f 2 m l w e r e w i t h d r a w n a t i n t e r v a l s a n d r a d i o a c t i v i t y d e t e r m i n e d as d e s c r i b e d in Fig. 1.
313
Fig. 2 in which it is shown that stringent (RCStr)E. coli cells starved of methionine resume synthesizing RNA after addition of thermorubin. This effect is characteristic of agents inhibiting ribosomal functions [9]. Activity in cell free systems The activity of thermorubin on cell-free polypeptide-synthesizing systems was first tested on polyuridilic acid-directed polyphenylalanine synthesis. As shown in Fig. 3 no significant inhibition was observed up to a drug concentration of 16 pg/ml. This results, when compared with the observations made with intact-cells experiments, could only be interpreted as an indication that some aspect of the protein synthesis process, not adequately represented by the poly(U)-directed model, was the target of antibiotic action. Thus, inhibition of other systems in which the synthesis is directed by natural messenger RNAs was tested. In one Such system, RNA of Phage MS2 was used as the messenger. This was incubated with a mixture of 20 aminoacyl-tRNAs and the 30 000 × g supernatant fraction of an extract of E. Coli previously treated with DNAase and preincubated to exhaust the endogenous synthesis as described by Niremberg and Matthei [3]. Polypeptide synthesis in this system, which is strongly dependent on added m R N A , was inhibited at antibiotic concentrations of 0.5--1 pg/ml, providing support that the antibiotic interferes with the protein synthesis
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Therrnoru bin JJg/ml Fig. 3. E f f e c t o f t h e r m o r u b i n o n ( A ) p o l y ( U ) - d i r e c t e d p o l y p h e n y l a l a n i n e s y n t h e s i s and (B) MS2 R N A d e p e n d e n t p o l y p e p t i d e synthesis, in cell-free e x t r a c t s prepared f r o m E. coli K 1 2 . R e a c t i o n m i x t u r e s ( 0 . 2 mi) c o n t a i n e d : 5 0 m M Tris--rnaleatc b u f f e r (pH 6.8), 9 m M m a g n e s i u m a c e t a t e , 6 0 m M KCI, 5 m M ~ - m e r c a p t o e t h a n o l , 0 . 5 m M GTP, 5 rnM creatine p h o s p h a t e , 2 5 / ~ g / m i creatine p h o s p h o k i n a s e , 1 m M r e d u c e d g l u t a t h i o n e , 5 0 ~ g / m i s p e r m i n e and r e s p e c t i v e l y : in ( A ) 1 0 0 ~ g / m i p o l y u r i d i l i c acid and 3 0 p m o l e s / m i [ 1 4 C ] p h e n y l a l a n l l t R N A ( 5 0 0 C i / m o l e ) or in (B) 2 5 0 / ~ g / m i MS2 R N A and 5 5 0 ~ g / m i of a m i x t u r e o f [ 14C] a m i n o a c y l - - t R N A s ( 8 5 cpm//~g). Assay t u b e s c o n t a i n e d also $ 3 0 , ( 3 5 0 m g / m l o f protein) a n d t h c r m o r u b i n at variable c o n c e n t r a t i o n s . I n c u b a t i o n was at 3 5 ° C for 3 0 rain. R e a c t i o n w a s s t o p p e d b y a d d i n g 1 m i o f 10% t r i c h l o r o a c e t i c acid a n d the r a d i o a c t i v i t y o f insoluble m a t e r i a l was c o l l e c t e d o n glass-fiber discs and m e a s u r e d in a Philips liquid s c i n t i l l a t i o n analyzer. T h e assays w i t h o u t a n t i b i o t i c in the p o l y ( U ) - d i r e c t e d s y s t e m and in the MS2 R N A - d i r e c t e d s y s t e m i n c o r p o r a t e d 4 . 5 p m o l e s of [ 14C] p h c n y l a i a n i n e a n d 1 2 5 0 c p m o f the m i x t u r e o f [ 14C]-labelled a m i n o acids per 0 . 2 ral o f r e a c t i o n mixture.
314 machinery. Moreover, since charged tRNAs were present in the reaction mixture, we could rule o u t amino acid activation and t R N A aminoacilation as the site of inhibitory action. We then tested the effect of the antibiotic on a cell-free system dependent on endogenous mRNA. In such a system, obtained by using a cell extract not DNAase-treated or preincubated, protein synthesis is supported both by RNA chains already engaged with ribosomes and by newly synthesized messenger chains. In this system, the antibiotic, as shown in the first column of Table I, reduced the synthesizing activity to a b o u t 50% of the control when added at concentrations between 0.25--2.5 pg/ml b u t no further inhibition was observed by increasing the concentration up to 25 pg/ml. This result, together with that obtained using MS2 RNA, suggested that peptide synthesis is sensitive to the action of thermorubin only when depending on exogenous or newly formed messenger chains. This was confirmed by experiments in which streptolydigin, a known inhibitor of RNA synthesis [ 10] was added to the system described above. As shown in the second column of Table I, in the presence of streptolydigin about 50% of protein synthesizing activity is still detected b u t this residual activity is not affected by the addition of thermorubin. In contrast, this residual activity is completely inhibited by sporangiomycin [11], an inhibitor of peptide chain elongation [ 12]. Furthermore, the above assumption implies that the effect of thermorubin on the endogenous mRNA-dependent system is weaker in the first minutes when translation of pre-existing RNA chains is more relevant. The inhibitory effect would increase when, as a consequence of completion of initiated chains, polypeptide synthesis became more dependent on newly synthesized m R N A molecules. The time course in Fig. 4 confirms the expectations and shows a similar behaviour to streptolydigin which prevents initiation of new peptide chains by inhibiting the synthesis of new mRNA. To exclude the possibility that thermorubin had an effect on R N A synthesis, we have tested the effect of the drug on RNA synthesis in a system utilizing purified DNA-dependent RNA TABLE I 14C-LABELLED AMINO ACID INCORPORATION BY E N D O G E N O U S m R N A
I N C R U D E E. C O L 1 E X T R A C T S ( $ 3 0 ) D I R E C T E D
R e a c t i o n m i x t u r e s ( 0 . 2 m l ) w e r e as d e s c r i b e d in t h e l e g e n d to F i g . 3 e x c e p t t h a t t h e a s s a y t u b e s c o n t a i n e d u n t r e a t e d $ 3 0 ( 3 5 0 m g / m l ) , 5.5 m g / m l o f a m i x t u r e o f 14C-labelled a m i n o a c y l - - t R N A s ( 8 5 c p m / p g ) , t h e r m o r u b i n at v a r i a b l e c o n c e n t r a t i o n s a n d , w h e r e i n d i c a t e d , s t r e p t o l i d y g i n at 25 ~ug/ml. I n c u b a t i o n w a s f o r 30 r a i n at 3 6 ° C . T h e c p m axe e x p r e s s e d p e r 0 . 2 m l o f r e a c t i o n m i x t u r e . Thermorubin (pg/ml)
C r u d e E. coli e x t r a c t s ( $ 3 0 ) Complete
0 0.025 0.25 2.5 2.5
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F i g . 4. T i m e c o u r s e o f p o l y p e p t i d e synthesis directed b y e n d o g e n o u s m R N A i n presence of v a r i o u s antibiotics. R e a c t i o n m i x t u r e s and assay p r o c e d u r e w e r e the s a m e a s described under the T a b l e I. T h e r morubin was present at 10 #g/m], streptolidygin at 25 ~g/ml and sporangiomycin at 10 ~g/ml. Incubation was at 35°C for variable times.
polymerase; no inhibition was observed at the drug concentrations affecting protein synthesis (data n o t shown). Altogether, these results are consistent with the hypothesis that thermorubin inhibits a step in the initiation of protein synthesis b u t has no effect on elongation of polypeptide chains. To test this assumption and to gain a better understanding of the precise mechanism by which thermorubin interferes with protein synthesis, we assayed the effect of the drug on the reactions of peptide chain initiation. The binding of fMet--tRNA to ribosomes was assayed using a Mg 2÷ concentration at which binding is dependent on initiation factors, GTP and on the initiation trinucleotide AUG. The data reported in Fig. 5 show that the formation of such a complex is prevented b y thermorubin at the same concenixations affecting MS2 RNAdirected protein synthesis. In order to check if the thermorubin interferes also with further steps of peptide chain initiation, we have also tested the puromycin reaction. As shown in Fig. 5 the inhibition of the synthesis of fMet--puromycin parallels the inhibition of binding of fMet--tRNA~ to ribosomes. The results of Table II show that thermorubin does n o t inhibit the synthesis of fMet-puromycin, i.e. the synthesis of the first peptide bond. Its addition to the reaction mixture interferes with the synthesis of fMet-puromycin only if added before, not after, the binding reaction has occurred. The partial inhibition of synthesis recorded even when the antibiotic is added at 30 min has to be expected; indeed, thermorubin inhibits any further binding of fMet--tRNA
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Thermorubin pg/rnl Fig. 5. E f f e c t o f t h e r m o r u b i n o n t h e b i n d i n g o f N - f o r m y l [ 1 4 C ] M e t - - t R N A f to ribosomes and on the synthesis of N - f o r m y l [ t 4C] m e t h i o n y l - - p t t r o m y c i n . R e a c t i o n m i x t u r e s (0.2 m l ) contained: 100 m M Tris-HCl, p H 7.2, 50 m M KCl, 5 m M Mg a c e t a t e , 1 m M G T P , 10 m M ~ - m e r c a p t o e t h a n o l , 0 . 8 u n i t s / m l A U G a n d 4 2 2 p m o l e s / m l f[ ! 4 C ] M e t - - t R N A f ( 2 3 3 p C / p M ) 0 . 9 m g / m l r i b o s o m e s a n d 0 . 5 m g / m l i n i t i a t i o n factors. A f t e r 15 r a i n i n c u b a t i o n at 2 4 ° C t h e s a m p l e s w e r e d i v i d e d in t w o p a r t s ; t o 0.1 ml w e r e a d d e d 3 m l o f a n i c e - c o l d b u f f e r (0.1 M T r i s - - H C l , p H 7.2, 0 . 0 5 KC1, 5 m M Mg a c e t a t e ) t o s t o p t h e r e a c t i o n a n d t h e b i n d i n g o f f[ 1 4 C ] M e t - - t R N A f t o r i b o s o m e s w a s m e a s u r e d ; t o t h e o t h e r s 0.1 ml 1 p m o l e / m l o f p u r o m y c i n w a s a d d e d a n d , a f t e r a d d i t i o n a l 20 r a i n i n c u b a t i o n , f o r m a t i o n o f f[ ! 4 C ] M e t - p u r o m y c i n w a s d e t e r m i n e d . R e f e r e n c e s c o n c e r n i n g t h e d e t a i l s o f t h e s e a s s a y m e t h o d s are r e p o r t e d u n d e r M a t e r i a l s a n d M e t h o d s . T h e assay w i t h o u t a n t i b i o t i c i n c o r p o r a t e d 2.2 p m o l e s o f f[ 14 C ] - M e t - - t R N A f p e r m g o f r i b o s o m e s .
to ribosomes during the second incubation period, when puromycin reacting with fMet--tRNA makes new binding sites on ribosomes free for the fMet-tRNA still present in the reaction mixture. In Table II we can observe that the amount of fMet--puromycin synthesized in the presence 2 or 5 pg/ml of thermorubin added at the end of the first incubation period (2.45 and 2.16 pmoles/ mg ribosome) is very close to the amount of fMet--tRNA bound to ribosomes in the absence of the antibiotic (2.36 pmoles/mg of ribosome). T A B L E II EFFECT OF THERMORUBIN
ON THE SYNTHESIS OF fMet--PUROMYCIN
R e a c t i o n m i x t u r e s (0.1 m l ) w e r e as d e s c r i b e d in t h e l e g e n d t o Fig. 5. T h e a s s a y t u b e s w e r e i n c u b a t e d a t 2 4 ° C f o r 3 0 rain, a f t e r w h i c h t i m e , in s o m e t u b e s , t h e r e a c t i o n w a s s t o p p e d a n d t h e f [ 1 4 C ] M e t - - t R N A f b o u n d t o r i b o s o m e s w a s m e a s u r e d ; in o t h e r t u b e s p u r o m y c i n (1 /zMole/ml) w a s a d d e d a n d t h e i n c u b a t i o n c o n t i n u e d f o r 20 rain. T h e r m o r u b i n w a s a d d e d e i t h e r b e f o r e t h e r e a c t i o n s t a r t e d ( t i m e 0) or j u s t b e f o r e t h e a d d i t i o n o f p u r o m y c i n ( t i m e 30 r a i n ) , as i n d i c a t e d . Addition of thermorubin (ug/ml) Time 0 - -
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317 Discussion
The data reported here show that thermorubin specifically inhibits in vivo and in vitro protein synthesis in E. coli. As a target of the antibiotic action the reactions related to amino acid activation and the aminoacilation of t R N A can be excluded because; (a) in vivo a stringent E. coli strain starved of methionine resumes uracil incorporation on addition of thermorubin (Fig. 2). Inhibition of t R N A aminoacylation would give the opposite effect; (b) in vitro protein synthesis is inhibited in a system to which charged tRNAs have been added (Table I, first column). If, as usual, the events taking place on ribosomes during protein synthesis are divided into a "chain initiation process" and a "chain elongation process", our data provide evidence that a step in the former is inhibited whereas the latter is most probably unaffected. This conclusion is supported by the following consideration; a low concentration of the antibiotic reduces polypeptide synthesis to a b o u t 50% in a cell-free system in which amino acid polymerization is almost equally dependent on pre-existing R N A chains and RNA molecules synthesized ex novo. The residual synthesis is not reduced even by a 10-fold concentration of the drug. The fraction of polypeptide synthesis inhibited by thermorubin is clearly the one supported by newly formed RNA chains; in fact, when RNA synthesis is prevented by addition of streptolydigin, total polypeptide synthesis is reduced and the residual activity is insensitive to thermorubin (Table I). Moreover, the time course of inhibition by thermorubin substantially parallels that of streptolydigin; the inhibitory effect appears to increase with the time of incubation {Fig. 4). These data, together with the inhibition observed in the system suggested by exogenous natural RNA, are consistent with the assumption that the initiation process is inhibited. On the other hand, the lack of inhibition of the fraction of endogenous synthesis not supported by newly formed R N A makes an effect of the antibiotic on the elongation process highly improbable. More direct evidence that initiation is involved is provided by the effect on formation of the initiation complex (70 S ribosome--AUG--fMet--tRNA) which is inhibited by concentrations comparable to those inhibiting the phage RNA-directed synthesis (Fig. 5). Once this complex is formed, the " p u r o m y c i n reaction", i.e. the formation of a peptide bond between formyl methionine and puromycin, is no longer sensitive to the presence of the antibiotic {Table II) providing additional evidence that the c o m p o u n d acts specifically on a step prior to the first peptide bond. In contrast to thermorubin, several agents impairing initiation of protein synthesis, i.e. hydroxylamine [ 1 4 ] , trimethoprim [ 1 3 ] , aurintricarboxilic acid [9,15] and negamycin [ 1 6 ] , also have an inhibitory effect on RNA synthesis in intact cells of E. coli. In the case of aurintricarboxilic acid this is not surprising since recently it has been shown that low concentrations of this drug also inhibit in vitro RNA synthesis, probably by binding to RNA polymerase [17]. With negamycin, depression of RNA synthesis and protein-synthesis inhibition are not necessarily linked because the concentrations at which the two effects can be observed are different. The inhibition of RNA synthesis in RC 'tr strains of E. coli. by hydroxy-
318 lamine and trimethoprim is a more complex phenomenon. When E. coli R c s t r cells are deprived of amino acids, the synthesis of stable RNA species is decreased (stringent response) whereas in the same conditions RC fez mutants continue to accumulate RNA {relaxed response). In the RC re~ mutant RNA synthesis is insensitive to trimethoprim and NH2 OH, if the culture medium is supplied with purinic bases. On this basis it has been proposed that there is a coupling between the initiation of protein synthesis and the control of RNA synthesis in the RC ~tr and this coupling is abolished in RC tel strains [13,14,9]. Hydroxylamine and trimethoprim, however, are not inhibitors of ribosome functions b u t cause depletion of the formyltetrahydrofolate pool, thus preventing the formation of fMet-tRNA M e t, the initiator of peptide chains. During stringent control the arrest of RNA synthesis is associated with the restricted supply of any aminoacyl--tRNA. Moreover, it has been shown that the stringent response imposed by trimethoprim may arise primarily from a decrease in the intracellular concentration of glycine and methionine, rather than from an inhibition of the initiation of new protein chains [ 1 8 - - 2 0 ] . Furthermore, in stringent E. coli cells in which RNA synthesis is blocked by amino acid starvation, addition of thermorubin results in resumption of RNA synthesis, as described with several agents inhibiting protein synthesis at ribosomal level such as chloramphenicol, puromycin, erythromycin, tetracycline and fusidic acid. This resumption of R N A synthesis does n o t take place with hydroxylamine and trimethoprim [9]. On the other hand, kasugamycin, an inhibitor of protein synthesis which acts by blocking the formation of initiation complex among AUG, fMet--tRNA and the 30 S ribosomal subunit [ 2 1 ] , does not reduce RNA synthesis rate in intact cells [22]. So , we conclude that, if any coupling exists between the control of RNA synthesis and the initiation of protein synthesis, it must lay in a step preceding those affected by thermorubin and kasugamycin. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Craveri, R., Coronelli, C., Pagani, H. and Sensi, P. (1964) Clin. Med. 7 1 , 5 1 1 - - 5 2 2 Moppett, C., Dix, D., Johnson, F. and Coronelli, C. (1972) J. Am. Chem. Soc. 94, 3269--3272 Niremberg, M.W. and Matthei, J.M. (1961) Proc. Natl. Acad. Sci. U.S. 47, 1558--1602 Sargeant, K. (1969) Methods in Microbiology (Norris, J.R. and Ribbons, D.W., eds), Vol. 1, p p . 517--518, Academic Press, L o n d o n Gesteland, R.F. and Boetdker, S. (1964) J. Mol. Biol. 8, 496--507 Kaji, A., Kaji, M. and Novelli, G.D. (1965) J. Biol. Chem. 240, 1885--1891 Ciferri, O., Parisi, B., Perani, A. and Grandi, M. (1968) J. Mol. Biol. 37, 529--533 Sala, F. and K/intzel, M. (1970) Eur. J. Biochem. 15, 280--286 Lund, E. and Kjeldegaard, N.O. (1972) J. Biochem. (Tokyo) 2 8 , 3 1 6 - - 3 2 6 Siddhikol, C., ErbstSszer, J.W. and Weisblum, B. (1969) J. Bacteriol. 99, 151--155 Thiemann, J.E., Coronelli, C., Pagani, H., Beretta, G., Tamoni, G. and Arioli, V. (1968) J. Antibiot. XXI, 525--531 Pirali, G., Lancini, G.C., Parisi, B. and Sala, F. (1972) J. Antibiot. XXV, 561--567 Shih, A . , E i s e n s t a d t , J. and Sengyel, P. (1966) Proc. Natl. Acad. Sci. U.S. 56, 1599--1605 Klein, A., Eisenstadt, A., Eisenstadt, J. and Sengagel, P. (1970) Biochemistry 9, 4542--4548 Stewart, M.L., Grollman, A.P. and Huang, M. (1971) Proc. Natl. Acad. Sci. U.S. 68, 97--101 Mizuno, S., Nitta, K. and Umezawa, H. (1970) J. Antibiot. XXIII, 581--588 Blumenthal, T. and Landers, T.A. (1973) Bioehem. B i o p h y s . R e s . C ommun. 55, 680--688 Moivic, M. and Pizer, L.I. (1971) J. Bacteriol. 1 0 6 , 8 5 6 - - 8 6 2 Dale, B.A. and Greenberg, G.R. (1972) J. Bacteriol. 110, 905--916 Smith, R.J. and Midgley, J.E.M. (1973) Biochem. J. 1 3 6 , 2 2 5 - - 2 3 4 Okuyama, A., Machyama, N., Kinoshita, T. and Tanaka, N. (1971) Biochem. Biophys. Res. C ommun. 43, 196--199 Tanaka, N., Nishimura, T., Yamaguchi, M., Y a m a m o t o , C., Yoshida, Y., Sashikata, K. and Umezawa, H. (1965) J. Antibiot. XVIII. 139--144
Biochimica et Biophysica Acta, 366 (1974) 310--318 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98114
INHIBITION OF PEPTIDE CHAIN INITIATION IN ESCHERICHIA COLI BY THERMORUBIN
G. PIRALI a, S. SOMMA a, G.C. LANCINI a and F. SALA b
aLaboratori Ricerche, Gruppo Lepetit S.p.A., via Durando, 38, Milano and bLaboratorio di Genetica biochimica ed evoluzionistica del Consiglio Nazionale delle Ricerche, Pavia (Italy) (Received April 22rid, 1974)
Summary The antibiotic thermorubin specifically inhibits protein synthesis when added to growing cultures of Escherichia coli, while RNA and DNA synthesis are not affected. In vitro studies show that thermorubin is a specific inhibitor of initiation of protein synthesis directed by natural mRNA. The drug inhibits protein synthesis directed by MS2 RNA but not poly(U)-dependent polyphenylalanine synthesis. Furthermore, during protein synthesis directed by endogenous messenger RNA, only the fraction of polypeptide synthesis dependent on ex novo-synthesized mRNA is sensitive to the drug. Thermorubin inhibits the binding of fMet--tRNA to ribosomes in the presence of AUG, GTP and initiation factors whereas the synthesis of the first peptide bond, assayed as fMet-puromycin synthesis, is not impaired by the antibiotic. The implications of these findings, with respect to the hypothesis that RNA synthesis in E. coli is controlled by initiation of protein synthesis, are discussed.
Introduction Thermorubin is an antibiotic produced by Thermoactinomyces antibioticus, a thermophilic actinomycete, active against Gram positive and many Gram negative bacteria, but ineffective on fungi and Protozoa [1]. It is an unusual example of a metabolite composed of a xanthonic and a substituted anthraquinonic moiety [2]. The product shows a low degree of toxicity in mice (LDs0~-300 mg/kg intraperitoneally) but is poorly effective in curing experimental infections when administered orally or subcutaneously (Arioli, V., unpublished results). In this paper we report evidence, obtained from growing cells and cell-free systems, that thermorubin inhibits peptide synthesis at the level of translation. The primary target of the antibiotic seems to be a step of the initiation process of protein synthesis prior to the formation of the first peptide bond. The elongation of polypeptide chains is not affected by thermorubin.
311
Materials and Methods
Escherichia coli K12 (761, his, try- and 690S T h y , Met-, L.L. Cavalli Sforza) was used. Cell free extracts ($30) were prepared from cells collected during early exponential growth in nutrient broth. Growth conditions and extraction procedures were essentially as described by Niremberg and Matthei [3] but in some cases preincubation and DNAase treatment were omitted as specified in the text. MS2 bacteriophage was prepared according to Sargeant [4] and its RNA was extracted following the method of Gesteland and Boedtker [5], without CsCl2 gradient centrifugation. ['4C]phenylalanyl-tRNA ([14 C] phe--tRNA) was prepared according to Kaji et al. [6]. The mixture of [' 4 C] aminoacyl-tRNAs was prepared in the same manner using stripped tRNA from E. coli and 14 C-labelled amino acids from Chlorella protein acid hydrolysate, supplemented with the amino acids not contained in this mixture (Try, CySH, GluNH~ and AspNH2 ). Polypeptide synthesis was assayed as described by Ciferri et al. [7]. Ribosomes, initiation factors, synthesis of N-formyl[' 4 C] methionyl-tRNA (f[l 4 C] Met-tRNA), and the assays of initiation reactions, binding f[14 C] Met-tRNA to the ribosomes and fMet-puromycin synthesis were performed as described by Sala and Kiintzel [ 8]. Thermorubin was dissolved in dimethylformamide: the solvent did not impair the assays at concentrations lower than 2% (v/v). Radioactivity was determined by a Philips Liquid Scintillation Analyzer. Stripped tRNA from E. coli was obtained from General Biochemical, Chargin, Ohio, [1-'4C]phenylalanine (57 Ci/mole), the mixture of (1-14 C)-labelled amino acid Chlorella hydrolysate (25 Ci/atom of carbon labelled) and [1-14C]methionine (233 pCi/mmole) from the Radiochemical Centre, Amersham, England; Polyuridilic acid was from Sigma, St. Louis, Mo., U.S.A., AUG from Miles Laboratories, Kankakee, Illinois, U.S.A., and puromycin from Serva, Heidelberg, Germany. Results
In vivo studies The minimal inhibitory concentration of thermorubin on the strain of E. coli used by us is 2pg]ml. The action of the antibiotic appears to be bacteriostatic; a culture in exponential growth, containing 108 cells/ml, was treated with 200 pg/ml of thermorubin and at different time intervals the number of viable cells was counted by plating after suitable dilution: the number of colonies formed did not decrease even after 7 h contact with the drug {data not shown). The effect of thermorubin on macromolecular synthesis in intact cells was studied by adding 10 pg/ml of the drug to a culture growing in the presence of radioactive thymine, uracil and phenylalanine. As shown in Fig. 1, within 2--3 min of the addition of the drug, the incorporation of phenylalanine is blocked, whereas incorporation of thymidine is only partially depressed and no effect is evident on uracil incorporation for about 40 min. The overall picture suggests that the antibiotic specifically interferes with the translation process. This is also supported by the experiment reported in
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Fig. 1. E f f e c t o f t h e r m o r u b i n o n t h e i n c o r p o r a t i o n o f [ 14 C] t h y m i n e ( A ) [ 14 C] u r a c i l (B) a n d [ 14 C] p h e n y l a l a n i n e (C) b y i n t a c t cells o f E. coll. T i m e o f a d d i t i o n o f t h e r m o r u b i n is i n d i c a t e d b y a n a r r o w . E. coli K 1 2 ( 6 9 0 S T h y , M e t - R C s i r ) f o r [ 1 4 C ] t h y m i n e i n c o r p o r a t i o n a n d E. coil K 1 2 ( 7 6 1 , h i s , t r y - R C s t r ) f o r [ 1 4 C ] u r a c i l a n d [ 14 C] p h e n y l a l a n i n e i n c o r p o r a t i o n , w e r e g r o w n in Davis M i n i m a l m e d i u m s u p p l e m e n t e d with 0.2% Difco casarnino acids and, respectively, 10 #g/ml of thymine, 7 #g/ml of uracil and 10 #g/mi of p h e n y l a l a n i n e . W h e n all c o n c e n t r a t i o n s r e a c h e d 1 0 8 c e l l / m i , [ 2 - 1 4 C ] t h y m i n e ( 0 . 0 5 # C i / m l , final) [ 2 - 1 4 C ] u r a c i l ( 0 . 0 2 5 # C i / m l , final) a n d [U° 1 4 C ] p h e n y l a l a r d n e ( 0 . 0 2 5 # C i / m l ) w e r e a d d e d . T h e r m o r u b i n was added after 10 min at a 10 #g/mi final concentration. Aliquots of 2 ml were withdrawn at intervals, d i l u t e d w i t h 3 m l o f 8% c o l d t r i c h l o r o a c e t i c a c i d a n d t h e r a d i o a c t i v i t y o f i n s o l u b l e m a t e r i a l c o l l e c t e d o n glass-fiber d i s c s d e t e r m i n e d .
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Fig. 2. R e s u m p t i o n o f R N A s y n t h e s i s is s t r i n g e n t E. coli cells s t a r v e d f o r a m i n o a c i d s f o l l o w i n g a d d i t i o n o f t h e r m o r u b i n . E. coil K 1 2 ( 6 9 0 S , thy-, m e t - , R C s t r ) w a s g r o w n in Davis m i n i m a l m e d i u m s u p p l e m e n t e d w i t h 0 . 2 % c a s a m i n o a c i d s a n d 1 0 # g / m l o f t h y m i n e u p t o a c o n c e n t r a t i o n o f 5 • 1 0 7 cells/ml. 2 0 0 m l o f t h i s c u l t u r e w e r e f i l t e r e d o n a m f l l i p o r e m e m b r a n e filter ( 0 . 4 5 #), w a s h e d a n d r e s u s p e n d e d i n a n e q u a l v o l u m e o f t h e s a m e m e d i u m b u t w i t h o u t a m i n o acids. A f t e r 15 m i n i n c u b a t i o n at 3 7 ° C [ 2 - 1 4 C ] u r a c i l w a s a d d e d a t 5 ~ g / m l (spec. a c t . 3.1 # C i / m m o l e ) a n d a f t e r a n a d d i t i o n a l 1 0 r a i n t h e c u l t u r e w a s s u b d i v i d e d in f o u r p a r t s a n d t o t h r e e o f t h e s e w e r e a d d e d : c h l o r a m p h e n i c o l , 2 0 # g / m 1 a n d t h e r m o r u b i n , 5 a n d 2 0 # g / m 1 , r e s p e c t i v e l y . A l i q u o t s o f 2 m l w e r e w i t h d r a w n a t i n t e r v a l s a n d r a d i o a c t i v i t y d e t e r m i n e d as d e s c r i b e d in Fig. 1.
313
Fig. 2 in which it is shown that stringent (RCStr)E. coli cells starved of methionine resume synthesizing RNA after addition of thermorubin. This effect is characteristic of agents inhibiting ribosomal functions [9]. Activity in cell free systems The activity of thermorubin on cell-free polypeptide-synthesizing systems was first tested on polyuridilic acid-directed polyphenylalanine synthesis. As shown in Fig. 3 no significant inhibition was observed up to a drug concentration of 16 pg/ml. This results, when compared with the observations made with intact-cells experiments, could only be interpreted as an indication that some aspect of the protein synthesis process, not adequately represented by the poly(U)-directed model, was the target of antibiotic action. Thus, inhibition of other systems in which the synthesis is directed by natural messenger RNAs was tested. In one Such system, RNA of Phage MS2 was used as the messenger. This was incubated with a mixture of 20 aminoacyl-tRNAs and the 30 000 × g supernatant fraction of an extract of E. Coli previously treated with DNAase and preincubated to exhaust the endogenous synthesis as described by Niremberg and Matthei [3]. Polypeptide synthesis in this system, which is strongly dependent on added m R N A , was inhibited at antibiotic concentrations of 0.5--1 pg/ml, providing support that the antibiotic interferes with the protein synthesis
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Therrnoru bin JJg/ml Fig. 3. E f f e c t o f t h e r m o r u b i n o n ( A ) p o l y ( U ) - d i r e c t e d p o l y p h e n y l a l a n i n e s y n t h e s i s and (B) MS2 R N A d e p e n d e n t p o l y p e p t i d e synthesis, in cell-free e x t r a c t s prepared f r o m E. coli K 1 2 . R e a c t i o n m i x t u r e s ( 0 . 2 mi) c o n t a i n e d : 5 0 m M Tris--rnaleatc b u f f e r (pH 6.8), 9 m M m a g n e s i u m a c e t a t e , 6 0 m M KCI, 5 m M ~ - m e r c a p t o e t h a n o l , 0 . 5 m M GTP, 5 rnM creatine p h o s p h a t e , 2 5 / ~ g / m i creatine p h o s p h o k i n a s e , 1 m M r e d u c e d g l u t a t h i o n e , 5 0 ~ g / m i s p e r m i n e and r e s p e c t i v e l y : in ( A ) 1 0 0 ~ g / m i p o l y u r i d i l i c acid and 3 0 p m o l e s / m i [ 1 4 C ] p h e n y l a l a n l l t R N A ( 5 0 0 C i / m o l e ) or in (B) 2 5 0 / ~ g / m i MS2 R N A and 5 5 0 ~ g / m i of a m i x t u r e o f [ 14C] a m i n o a c y l - - t R N A s ( 8 5 cpm//~g). Assay t u b e s c o n t a i n e d also $ 3 0 , ( 3 5 0 m g / m l o f protein) a n d t h c r m o r u b i n at variable c o n c e n t r a t i o n s . I n c u b a t i o n was at 3 5 ° C for 3 0 rain. R e a c t i o n w a s s t o p p e d b y a d d i n g 1 m i o f 10% t r i c h l o r o a c e t i c acid a n d the r a d i o a c t i v i t y o f insoluble m a t e r i a l was c o l l e c t e d o n glass-fiber discs and m e a s u r e d in a Philips liquid s c i n t i l l a t i o n analyzer. T h e assays w i t h o u t a n t i b i o t i c in the p o l y ( U ) - d i r e c t e d s y s t e m and in the MS2 R N A - d i r e c t e d s y s t e m i n c o r p o r a t e d 4 . 5 p m o l e s of [ 14C] p h c n y l a i a n i n e a n d 1 2 5 0 c p m o f the m i x t u r e o f [ 14C]-labelled a m i n o acids per 0 . 2 ral o f r e a c t i o n mixture.
314 machinery. Moreover, since charged tRNAs were present in the reaction mixture, we could rule o u t amino acid activation and t R N A aminoacilation as the site of inhibitory action. We then tested the effect of the antibiotic on a cell-free system dependent on endogenous mRNA. In such a system, obtained by using a cell extract not DNAase-treated or preincubated, protein synthesis is supported both by RNA chains already engaged with ribosomes and by newly synthesized messenger chains. In this system, the antibiotic, as shown in the first column of Table I, reduced the synthesizing activity to a b o u t 50% of the control when added at concentrations between 0.25--2.5 pg/ml b u t no further inhibition was observed by increasing the concentration up to 25 pg/ml. This result, together with that obtained using MS2 RNA, suggested that peptide synthesis is sensitive to the action of thermorubin only when depending on exogenous or newly formed messenger chains. This was confirmed by experiments in which streptolydigin, a known inhibitor of RNA synthesis [ 10] was added to the system described above. As shown in the second column of Table I, in the presence of streptolydigin about 50% of protein synthesizing activity is still detected b u t this residual activity is not affected by the addition of thermorubin. In contrast, this residual activity is completely inhibited by sporangiomycin [11], an inhibitor of peptide chain elongation [ 12]. Furthermore, the above assumption implies that the effect of thermorubin on the endogenous mRNA-dependent system is weaker in the first minutes when translation of pre-existing RNA chains is more relevant. The inhibitory effect would increase when, as a consequence of completion of initiated chains, polypeptide synthesis became more dependent on newly synthesized m R N A molecules. The time course in Fig. 4 confirms the expectations and shows a similar behaviour to streptolydigin which prevents initiation of new peptide chains by inhibiting the synthesis of new mRNA. To exclude the possibility that thermorubin had an effect on R N A synthesis, we have tested the effect of the drug on RNA synthesis in a system utilizing purified DNA-dependent RNA TABLE I 14C-LABELLED AMINO ACID INCORPORATION BY E N D O G E N O U S m R N A
I N C R U D E E. C O L 1 E X T R A C T S ( $ 3 0 ) D I R E C T E D
R e a c t i o n m i x t u r e s ( 0 . 2 m l ) w e r e as d e s c r i b e d in t h e l e g e n d to F i g . 3 e x c e p t t h a t t h e a s s a y t u b e s c o n t a i n e d u n t r e a t e d $ 3 0 ( 3 5 0 m g / m l ) , 5.5 m g / m l o f a m i x t u r e o f 14C-labelled a m i n o a c y l - - t R N A s ( 8 5 c p m / p g ) , t h e r m o r u b i n at v a r i a b l e c o n c e n t r a t i o n s a n d , w h e r e i n d i c a t e d , s t r e p t o l i d y g i n at 25 ~ug/ml. I n c u b a t i o n w a s f o r 30 r a i n at 3 6 ° C . T h e c p m axe e x p r e s s e d p e r 0 . 2 m l o f r e a c t i o n m i x t u r e . Thermorubin (pg/ml)
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polymerase; no inhibition was observed at the drug concentrations affecting protein synthesis (data n o t shown). Altogether, these results are consistent with the hypothesis that thermorubin inhibits a step in the initiation of protein synthesis b u t has no effect on elongation of polypeptide chains. To test this assumption and to gain a better understanding of the precise mechanism by which thermorubin interferes with protein synthesis, we assayed the effect of the drug on the reactions of peptide chain initiation. The binding of fMet--tRNA to ribosomes was assayed using a Mg 2÷ concentration at which binding is dependent on initiation factors, GTP and on the initiation trinucleotide AUG. The data reported in Fig. 5 show that the formation of such a complex is prevented b y thermorubin at the same concenixations affecting MS2 RNAdirected protein synthesis. In order to check if the thermorubin interferes also with further steps of peptide chain initiation, we have also tested the puromycin reaction. As shown in Fig. 5 the inhibition of the synthesis of fMet--puromycin parallels the inhibition of binding of fMet--tRNA~ to ribosomes. The results of Table II show that thermorubin does n o t inhibit the synthesis of fMet-puromycin, i.e. the synthesis of the first peptide bond. Its addition to the reaction mixture interferes with the synthesis of fMet-puromycin only if added before, not after, the binding reaction has occurred. The partial inhibition of synthesis recorded even when the antibiotic is added at 30 min has to be expected; indeed, thermorubin inhibits any further binding of fMet--tRNA
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1 5
I 10
Thermorubin pg/rnl Fig. 5. E f f e c t o f t h e r m o r u b i n o n t h e b i n d i n g o f N - f o r m y l [ 1 4 C ] M e t - - t R N A f to ribosomes and on the synthesis of N - f o r m y l [ t 4C] m e t h i o n y l - - p t t r o m y c i n . R e a c t i o n m i x t u r e s (0.2 m l ) contained: 100 m M Tris-HCl, p H 7.2, 50 m M KCl, 5 m M Mg a c e t a t e , 1 m M G T P , 10 m M ~ - m e r c a p t o e t h a n o l , 0 . 8 u n i t s / m l A U G a n d 4 2 2 p m o l e s / m l f[ ! 4 C ] M e t - - t R N A f ( 2 3 3 p C / p M ) 0 . 9 m g / m l r i b o s o m e s a n d 0 . 5 m g / m l i n i t i a t i o n factors. A f t e r 15 r a i n i n c u b a t i o n at 2 4 ° C t h e s a m p l e s w e r e d i v i d e d in t w o p a r t s ; t o 0.1 ml w e r e a d d e d 3 m l o f a n i c e - c o l d b u f f e r (0.1 M T r i s - - H C l , p H 7.2, 0 . 0 5 KC1, 5 m M Mg a c e t a t e ) t o s t o p t h e r e a c t i o n a n d t h e b i n d i n g o f f[ 1 4 C ] M e t - - t R N A f t o r i b o s o m e s w a s m e a s u r e d ; t o t h e o t h e r s 0.1 ml 1 p m o l e / m l o f p u r o m y c i n w a s a d d e d a n d , a f t e r a d d i t i o n a l 20 r a i n i n c u b a t i o n , f o r m a t i o n o f f[ ! 4 C ] M e t - p u r o m y c i n w a s d e t e r m i n e d . R e f e r e n c e s c o n c e r n i n g t h e d e t a i l s o f t h e s e a s s a y m e t h o d s are r e p o r t e d u n d e r M a t e r i a l s a n d M e t h o d s . T h e assay w i t h o u t a n t i b i o t i c i n c o r p o r a t e d 2.2 p m o l e s o f f[ 14 C ] - M e t - - t R N A f p e r m g o f r i b o s o m e s .
to ribosomes during the second incubation period, when puromycin reacting with fMet--tRNA makes new binding sites on ribosomes free for the fMet-tRNA still present in the reaction mixture. In Table II we can observe that the amount of fMet--puromycin synthesized in the presence 2 or 5 pg/ml of thermorubin added at the end of the first incubation period (2.45 and 2.16 pmoles/ mg ribosome) is very close to the amount of fMet--tRNA bound to ribosomes in the absence of the antibiotic (2.36 pmoles/mg of ribosome). T A B L E II EFFECT OF THERMORUBIN
ON THE SYNTHESIS OF fMet--PUROMYCIN
R e a c t i o n m i x t u r e s (0.1 m l ) w e r e as d e s c r i b e d in t h e l e g e n d t o Fig. 5. T h e a s s a y t u b e s w e r e i n c u b a t e d a t 2 4 ° C f o r 3 0 rain, a f t e r w h i c h t i m e , in s o m e t u b e s , t h e r e a c t i o n w a s s t o p p e d a n d t h e f [ 1 4 C ] M e t - - t R N A f b o u n d t o r i b o s o m e s w a s m e a s u r e d ; in o t h e r t u b e s p u r o m y c i n (1 /zMole/ml) w a s a d d e d a n d t h e i n c u b a t i o n c o n t i n u e d f o r 20 rain. T h e r m o r u b i n w a s a d d e d e i t h e r b e f o r e t h e r e a c t i o n s t a r t e d ( t i m e 0) or j u s t b e f o r e t h e a d d i t i o n o f p u r o m y c i n ( t i m e 30 r a i n ) , as i n d i c a t e d . Addition of thermorubin (ug/ml) Time 0 - -
2 5
fMet--tRNA bound to ribosomes
fMet puromycin synthesized
T i m e 30
pmoles per mg of ribosome
% of t h e control
pmoles per mg of r i b o s o m e
% of the control
----
2.36 0.73 0.64
-30.8 27.0
2.98 1.03 0.62 2.45 2.16
-34.8 20.1 82.3 72.4
- -
2
- -
- -
--
5
--
--
317 Discussion
The data reported here show that thermorubin specifically inhibits in vivo and in vitro protein synthesis in E. coli. As a target of the antibiotic action the reactions related to amino acid activation and the aminoacilation of t R N A can be excluded because; (a) in vivo a stringent E. coli strain starved of methionine resumes uracil incorporation on addition of thermorubin (Fig. 2). Inhibition of t R N A aminoacylation would give the opposite effect; (b) in vitro protein synthesis is inhibited in a system to which charged tRNAs have been added (Table I, first column). If, as usual, the events taking place on ribosomes during protein synthesis are divided into a "chain initiation process" and a "chain elongation process", our data provide evidence that a step in the former is inhibited whereas the latter is most probably unaffected. This conclusion is supported by the following consideration; a low concentration of the antibiotic reduces polypeptide synthesis to a b o u t 50% in a cell-free system in which amino acid polymerization is almost equally dependent on pre-existing R N A chains and RNA molecules synthesized ex novo. The residual synthesis is not reduced even by a 10-fold concentration of the drug. The fraction of polypeptide synthesis inhibited by thermorubin is clearly the one supported by newly formed RNA chains; in fact, when RNA synthesis is prevented by addition of streptolydigin, total polypeptide synthesis is reduced and the residual activity is insensitive to thermorubin (Table I). Moreover, the time course of inhibition by thermorubin substantially parallels that of streptolydigin; the inhibitory effect appears to increase with the time of incubation {Fig. 4). These data, together with the inhibition observed in the system suggested by exogenous natural RNA, are consistent with the assumption that the initiation process is inhibited. On the other hand, the lack of inhibition of the fraction of endogenous synthesis not supported by newly formed R N A makes an effect of the antibiotic on the elongation process highly improbable. More direct evidence that initiation is involved is provided by the effect on formation of the initiation complex (70 S ribosome--AUG--fMet--tRNA) which is inhibited by concentrations comparable to those inhibiting the phage RNA-directed synthesis (Fig. 5). Once this complex is formed, the " p u r o m y c i n reaction", i.e. the formation of a peptide bond between formyl methionine and puromycin, is no longer sensitive to the presence of the antibiotic {Table II) providing additional evidence that the c o m p o u n d acts specifically on a step prior to the first peptide bond. In contrast to thermorubin, several agents impairing initiation of protein synthesis, i.e. hydroxylamine [ 1 4 ] , trimethoprim [ 1 3 ] , aurintricarboxilic acid [9,15] and negamycin [ 1 6 ] , also have an inhibitory effect on RNA synthesis in intact cells of E. coli. In the case of aurintricarboxilic acid this is not surprising since recently it has been shown that low concentrations of this drug also inhibit in vitro RNA synthesis, probably by binding to RNA polymerase [17]. With negamycin, depression of RNA synthesis and protein-synthesis inhibition are not necessarily linked because the concentrations at which the two effects can be observed are different. The inhibition of RNA synthesis in RC 'tr strains of E. coli. by hydroxy-
318 lamine and trimethoprim is a more complex phenomenon. When E. coli R c s t r cells are deprived of amino acids, the synthesis of stable RNA species is decreased (stringent response) whereas in the same conditions RC fez mutants continue to accumulate RNA {relaxed response). In the RC re~ mutant RNA synthesis is insensitive to trimethoprim and NH2 OH, if the culture medium is supplied with purinic bases. On this basis it has been proposed that there is a coupling between the initiation of protein synthesis and the control of RNA synthesis in the RC ~tr and this coupling is abolished in RC tel strains [13,14,9]. Hydroxylamine and trimethoprim, however, are not inhibitors of ribosome functions b u t cause depletion of the formyltetrahydrofolate pool, thus preventing the formation of fMet-tRNA M e t, the initiator of peptide chains. During stringent control the arrest of RNA synthesis is associated with the restricted supply of any aminoacyl--tRNA. Moreover, it has been shown that the stringent response imposed by trimethoprim may arise primarily from a decrease in the intracellular concentration of glycine and methionine, rather than from an inhibition of the initiation of new protein chains [ 1 8 - - 2 0 ] . Furthermore, in stringent E. coli cells in which RNA synthesis is blocked by amino acid starvation, addition of thermorubin results in resumption of RNA synthesis, as described with several agents inhibiting protein synthesis at ribosomal level such as chloramphenicol, puromycin, erythromycin, tetracycline and fusidic acid. This resumption of R N A synthesis does n o t take place with hydroxylamine and trimethoprim [9]. On the other hand, kasugamycin, an inhibitor of protein synthesis which acts by blocking the formation of initiation complex among AUG, fMet--tRNA and the 30 S ribosomal subunit [ 2 1 ] , does not reduce RNA synthesis rate in intact cells [22]. So , we conclude that, if any coupling exists between the control of RNA synthesis and the initiation of protein synthesis, it must lay in a step preceding those affected by thermorubin and kasugamycin. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
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