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Transcription and translation mechanisms in a «permeabilized E. coli system
Transcription and translation mechanisms in a ttpermeabilized>> E. Colt system by F a k h e r BEN-HAMIDAand F r a n c o i s Gt~os.
Seroice de Phgsiologie Cellulaire, Institttt de Biologie Mol~'cttlaire, Facnltd des Sciences, Paris 2'.
Sumraarg. An E. colt ¢ permeabilized >> cell system (PCS) is described, resulting trmn 2 M-sucrose plasmolysis in the presence of 10-4 M EDTA. This treatment induces some interesting properties in the cells: permeability to various phosphorylated derivatives without osmotical fragility, depletion from endogenous metabolites and lack of growth on rich medium. This system appears well suited for the study of uncoupled maeromoleeular synthesis : (1) in the presence of the usual RNA polymerase snbstrates (ATP, CTP, GTP and UTP), PCS prominently synthesizes messenger RNA of exceptional great stability (half-life greater than 60 minutes). The reasons of this stability will be discussed ; (2') the mRNAs thus formed are active in protein synthesis as it can be shown by a stepwise induction experiment: RNA synthesized in the presence of the four ribonucleoside triphosphates plus IPTG can direct the synthesis of fJ-galaetosidase, after PCS Js transferred in an amino acid medium plus aetinomycin D ; (3) when PCS is supplemented with a complete amino mixture in addition to the four ribonueleoside triphosphates, it synthesizes proteins and also accumulates large amounts of ribosomal and transfer RNA's.
During the last decade, the study of gene expression m e c h a n i s m s in bacteria has l a r g e l ) benefited from the use of cell free systems or h i g h l y purified enzyme p r e p a r a t i o n s . Yet, certain basic problems are not fully understood, e.g. DNA replication and its control, the RC gene mediated regulation of RNA synthesis, and m a n y features of negative and positive control m e c h a n i s m s d u r i n g bacterial o,r p h a g e development: These p r o b l e m s could be looked at in more detail if more integrated subcellular systems w e r e available or if bacterial cells could be m a d e p e r m e a b l e to certain molecules. Numerous t e c h n i q u e s have been described to modify the p e r m e a b i l i t y of bacterial cells either by c h e m i c a l [1, 2, 3] or p h y s i c a l means [4, 5]. A few years ago, w e d e v e l o p e d a t e c h n i q u e based upon the p r o p e r t y of E. colt cells to u n d e r g o plasmolysis in the p r e s e n c e of 2 M sucrose at a slightly alkaline pH [fi]. This p r o c e d u r e was used by CASHEL and GALLANT [7] for e x p l o r i n g the amino acid control of RNA synthesis in RC stringent and relaxed strains. An important, albeit s o m e w h a t long-range outcome of their study was the identification of ppGpp as a regulatory cofactor for ribosomal RNA synthesis [8, 9]. Another application of the plasmolyzed cell system is found in the
w o r k of Roy BURMAN and VISSER [1Ol w h o have s h o w n that r a d i o a c t i v e 5-6 d i h y d r o - u r i d i n e 5'-trip h o s p h a t e can be d i r e c t l y and specifically incorporated into 4 S E. colt RNA. In the present paper, we shall describe an imp r o v e d t e c h n i q u e of ¢ p e r m e a b i l i z a t i o n >~ of E. colt cells, associating 2 M sucrose plasmolysis and EDTA (disodium ethylene d i a m i n o tretraacetare) treatment. The <~p e r m e a b i l i z e d >> cell system (PCS) obtained has great versatility in the study of u n c o u p l e d m a c r o m o l e c u l a r synthesis. Attention will be p a r t i c u l a r l y focused on the RNA and prot':in synthesizing capacities of the system. The main conclusions to be d r a w n are the fo,tlowing : (1) When supplemented with the usual RNA p o l y m e r a s e substrates (a m i x t u r e of the four ribonucleoside triphosphates) at the exclusion of other metabolites, PCS p r e d o m i n a n t l y synthesizes messenger RNA with little ribosomal of transfer RNA, if any. Messenger RNA synthesis is completely p r e v e n t e d by a c t i n o m y c i n D. (2) That the messengers thus f o r m e d are active in p r o t e i n synthesis can be shown by a stepwise i n d u c t i o n e x p e r i m e n t : RNA synthesized in the p r e s e n c e of the four r i b o n u c l e o s i d e t r i p h o s p h a t e s plus i s o p r o p y l ~-D thiogalactoside (IPTG) can
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d i r e c t the s y n t h e s i s of ~-galactosidase, a f t e r PCS is t r a n s f e r r e d into an a m i n o a c i d c o n t a i n i n g m e d i u m p l u s a c t i n o m y c i n D. (3) T h e m e s s e n g e r RNA f o r m e d u n d e r the a b o v e c o n d i t i o n s is m o r e stable t h a n t i n d e r o t h e r cond i t i o n s of s y n t h e s i s , s u g g e s t i n g that m e s s e n g e r d e g r a d a t i o n is n o r m a l l y e n t a n g l e d w i t h at least s o m e steps in p r o t e i n synthesis.
and Frangois
Gros.
F i g u r e 1 s h o w s the e l e c t r o n m i c r o g r a p h s of E. colt D 10 cells ¢ p e r m e a b i l i z e d ~ as d e s c r i b e d a n d n o r m a l cells. T h e s t r u c t u r a l p a t t e r n of plasm o t y z e d cells s h o w s a s h r i n k a g e of the p r o t o p l a s t d u e to loss of w a t e r . T h e p r o t o p l a s m i c m e m b r a n e is r e t r a c t e d f r o m the w a l l and the v a c u o l e are c l e a r l y visible.
(4) W h e n PCS is s u p p l e m e n t e d w i t h a c o m p l e t e a m i n o a c i d m i x t u r e in a d d i t i o n to the f o u r r i b o n u c l e o s i d e t r i p h o s p h a t e s it s y n t h e s i z e s p r o t e i n s a n d a c c u m u l a t e s large a m o u n t s of r i b o s o m a l and t r a n s f e r RNA's. MATERIALS AND METHODS.
Strains :
T h e b a c t e r i a l s t r a i n used m a i n l y in this w o r k w a s E. colt D 10, a r i b o n u c l e a s e I less m u t a n t [11]. E. c o l t M O a lac ÷ i n d u c i b l e a n d E. colt C 600 )~ t U 32 a l y s o g e n c a r r y i n g a h e a t i n d u c i b l e p r o p h a g e w e r e used o c c a s i o n a l l y . M e d i a a n d bu[[ers :
T h e cells used in t h e s e e x p e r i m e n t s w e r e g r o w n in m e d i u m 63 [12] s u p p l e m e n t e d w i t h 0.4 p. 100 g l y c e r o l and 0.4 p. 100 v i t a m i n free c a s a m i n o acids. T h e f o l l o w i n g buffers h a v e b e e n u s e d extensively : --TME m e d i u m : 0.01 M, p H 8.0, T r i s - H C l ; 0.01 M Mg SO 4 ; 0.0001 M E D T A or C o m p l e x o n III (Siegfried). -SO l, -SO4,
T M 2 b u f f e r : 0.01 M T r i s - H C l ; 0.01 M Mg p H 8.0. TM 3 b u f f e r : 0.01 M T r i s - H C l ; 0.001 M Mg p H 7.5. T M E s u c r o s e s o l u t i o n : s o l u t i o n of 2 M suc r o s e in T M E m e d i u m . -
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Fro. 1. - - (1) Electron micrograph (thin section) of E. colt D 10 plasmolyzed in 2 M sucrose and 10-4 ]~'l EDTA (.see Materials and Methods). (2) Normal E. colt D 10 (by Dr. Philippe G~AN~OULAN).
¢ P e r m e a b i l i z a t i o n ~> t e c h n i q u e :
100 ml of e x p o t e n t i a l l y g r o w n cells w e r e h a r vested in the cold at a c o n c e n t r a t i o n of 5 × 108 b a c t e r i a l p e r inl. T h e cells w e r e p e l l e t e d by cent r i f u g a t i o n for 10 m i n u t e s at 7000 r p m . T h e y w e r e w a s h e d t w i c e w i t h T M E m e d i u m and r e s u s p e n d e d in 5 ml of T M E s u c r o s e solution. After m o d e r a t e s h a k i n g at 30°C for 30 m i n u t e s , the s u s p e n s i o n w a s d i l u t e d by 100 ml of c o l d T M E a n d filtered in the c o l d on H A W P (0.45 ~) l a r g e m i l l i p o r c membranes. Absorbed bacteria were washed with 100 ml of TME and r e s u s p e n d e d in 5 ml of TM 2 buffer to a c o n c e n t r a t i o n of 5 × 109 p e r ml (PCS). O n l y 1 to 2 p. 100 of these ¢ permeabili.zed >> cells are able to give c o l o n i e s on r i c h medium. BIOCHIMIE, 1971, 53, n" 1.
SH-GTP i n c o r p o r a t i o n i n t o m e s s e n g e r R N A :
An a l i q u o t of the PCS (109 b a c t e r i a p e r ml, final c o n c e n t r a t i o n ) w a s s h a k e n at 30°C in flask cont a i n i n g 10 -2 M Tris-HC1, p H 8.0, 10 -4 M 3H-GTP (specific a c t i v i t y 100 ~cL~mole) ; 5.10 "4 M ATP, CTP and UT1a (1) ; 2.10 -4 M MnC12; 4.10-a M MgSO 4 ; 10 -4 M, KC1 and 3.10 -4 M 2 - m e r c a p t o - 6 t h a nol. To d e t e r m i n e the e x t e n t of n u c l e o s i d e incorp o r a t i o n , 50 .ul s a m p l e s w e r e m i x e d w i t h 3 ml cold 5 p. 100 TEA, after t w o h o u r s the m i x t u r e was a b s o r b e d on 0.45 ~ m i l l i p o r e or S a r t o r i u s filters. After r e p e a t e d w a s h i n g s w i t h 3 ml of c o l d TCA, (1) GTP = guanosine 5'-triphosphate ; ATP : adenosine 5'-triphosphate CTP : cytidine 5'-triphosphate ; UTP : uridine 5'-triphosphate.
T r a n s c r i p t i o n a n d t r a n s l a t i o n in a ¢ p e r m e a b i l i z e d ~ E. Colt s y s t e m . filters w e r e d r i e d a n d t r a n s f e r r e d to vials containing 10 ml of s c i n t i l l a t i o n m i x t u r e (2-5 d i p h e nyl-oxozole (PPO) 5 g ; 1-4 bis 2 (4-methyl-5-phenyl oxazolyl) b e n z e n e ( d i m e t h y l POPOP) 0.3 g in one litre of toluene. T h e y w e r e c o u n t e d in a ~ Tri-Carb P a c k a r d ~> s c i n t i l l a t i o n c o u n t e r . Determination of p r o t e i n synthesis : An a l i q u o t of PCS (109 b a c t e r i a p e r ml) w a s incubated in the p r e s e n c e of 10 -2 M, p H 8.0, T r i s HC1 ; 5.10 -4 M GTP, ATP, C T P a n d U T P ; 10-n M 14C-valine, specific a c t i v i t y 1 .uc/20 m~tm; 2.5 × 10-4 M of e a c h of the 18 o t h e r a m i n o a c i d s ; 10 .-4 M Mn C l e ; 10 -2 M MgSO4 ; 10 -2 M KC1; 3.10 -~ M 2-mercaptoethanol, 1 m g / m l c r e a t i n e p h o s p h a t e ; 20 ~ g / m l c r e a t i n e p h o s p h o k i n a s e . I n c u b a t i o n w a s carried out at 30°C w i t h shaking. To m e a s u r e the amount of 14C-valine i n c o r p o r a t i o n , 0.5 ml s a m p l e s were m i x e d w i t h 1 ml c o l d 10 p. 100 TCA. A f t e r one h o u r in the c o l d the p r e c i p i t a t e w a s p e l l e t e d by 5 m i n u t e s c e n t r i f u g a t i o n at 3000 r p m a n d the supernatant d i s c a r d e d . T h e r e s u l t i n g p e l l e t w a s dispersed in 1.5 ml 5 p. 100 TCA and the s u s p e n sion p l a c e d in b o i l i n g w a t e r for 15 m i n u t e s . T h e supernatant w a s d i s c a r d e d a f t e r c e n t r i f u g a t i o n . The n u c l e i c a c i d free p e l l e t w a s r e s u s p e n d e d in TCA and filtered on M i l l i p o r e m e m b r a n e s , w a s h e d and c o u n t e d as d e s c r i b e d in the p r e c e d i n g section. ~-galactosidase induction and assay : The << p e r m e a b i t i z e d >> cells (109 b a c t e r i a / r o D were i n c u b a t e d at 30°C w i t h s h a k i n g in the medium p r e v i o u s l y d e s c r i b e d in the s e c t i o n on p r o tein synthesis. In a d d i t i o n , h o w e v e r , 10 -8 M isopropyl ~ - D - t h i o g a l a c t o s i d e (IPTG) w a s a d d e d . F o r ~-galactosidase assays, 0.5 m l s a m p l e s w e r e vigorously s h a k e n (Vortex) for one m i n u t e in the p r e sence of 0.05 ml t o l u e n e plus 0.03 ml p. 100 sodium d e o x y c h o l a t e , f~-galactosidase a c t i v i t y w a s d e t e r m i n e d by the o r t h o - n i t r o - p h e n y l g a l a c t o s i d e (ONPG) t e c h n i q u e [13, 1,1]. RNA extraction and hybridization : Cells to be e x a m i n e d w e r e p e l l e t e d a n d resuspended in 2.5 t i m e s t h e i r w e i g h t of p H 7.5 TM3 buffer c o n t a i n i n g l y s o z y m e (200 t~g/ml) and RNAase f r e e d e o x y r i b o n u c l e a s e (50 ~ g / m l ) . A f t e r three s u c c e s s i v e f r e e z i n g a n d t h a w i n g cycles, 9.2 p. 100 s o d i u m l a u r y l sulfate w a s a d d e d to complete lysis. T h e RNA w a s p r e p a r e d by p h e n o l extraction a c c o r d i n g to KIRnY [15]. DNA-RNA annealing tests w e r e m a d e as d e s c r i b e d by GILLESPIE and SPIEGELMAN [16]. T h e filters (Sartorius MF 50) w e r e l o a d e d w i t h alkali d e n a t u r e d DNA, w a s h e d w i t h a f e w ml of 2 × SSC b u f f e r 300 mM s o d i u m c h l o r i d e a n d 30 mM s o d i u m citrate, (pH 7.5), d r i e d at r o o m t e m p e r a t u r e and BIOCHIMIE, 1971, 53, n ° 1.
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k e p t o v e r n i g h t in a v a c u u m e x c i c c a t o r . T h e c o n d i t i o n s used for h y b r i d i z a t i o n f o l l o w : E. colt DNA, 50 ~g of l a m b d a DNA 20 .ug p e r filter, 1 ml of 5 × SSC buffer p l u s the a p p r o p r i a t e RNA sample. A f t e r 48 h o u r s , at 66°C, t h e filters w e r e w a s h e d by i m m e r s i o n in 2 × SSC and r i b o n u clease t r e a t e d (10 t~g/ml ; 3 ml of 2 × SSC ; 20°C for one h o u r ) . A f t e r a n o t h e r w a s h i n g in 2 × SSC, t h e y w e r e d r i e d a n d the r a d i o a c t i v i t y d e t e r m i n e d . RESULTS. I. - - Messenger R N A synthesis by e permeabilized >>E. colt cells in the presence of the four ribonucleoside triphosphates. W h e n PCS is i n c u b a t e d in T r i s m e d i u m cont a i n i n g thc f o u r r i b o n u c l e o s i d e t r i p h o s p h a t e s (one of w h i c h is labeled, 3H-UTP) p l u s Mn ÷÷ ions, I
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50 I00 /20 Dine /'mlnu~sJ FI3. 9. 3H-UTP incorporation kinetics into messenger type RNA. Dependence on presence of the [our ribonucleoside triphosphates and e[fect of actinomllcin D. At time zero, permeabtlized >> cells (of. Materials and Methods) of E. coil D 10 (109 bacteria per mi, final concentration) were shaken at 30°C in flask containing 10-2 M Tris-HC1, pH 8.0 ; 10-4 M :~H-UTP (specific activity 100 tte/llxmole) ; 5.10-4 M ATP, CTP and G T P ; '2.10-4 M Mn C12; 4.10-3 M MgSO4; 10-4 M KCI and 3.10-~ M 2-mercaptoethanol. The incorporation was stopped by adding of 5 p. 100 TCA 3 ml to the 50 ul samples. Further precipitation and radioactivity measurement were performed as described in ¢ Materials and Methods >>. --ocomplete system with the four nucleoside triphosphates. A--- complete system without GTP. ---ocomplete system without GTP, CTP, A T P ; aH-UTP only present. V - - - complete system in the presence of actinomycin (100 ixg/ml). o ...... complete system with chloramphenieol (300 ixg/ml). - -
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a l i n e a r i n c o r p o r a t i o n of r a d i o a c t i v e U T P i n t o a TCA p r e c i p i t a b l e m a t e r i a l is o b s e r v e d ( F i g u r e 2). T h a t t h e l a b e l e d m a t e r i a l t h u s f o r m e d is R N A w i l l be subsequently shown by zone sedimentation analysis.
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(two antibiotics which do not block transcription i n i n t a c t cells). As e x p e c t e d a c t i n o m y c i n D has n o effect o n 3 H - U D P i n c o r p o r a t i o n ( F i g u r e 3). A c e r t a i n n u m b e r of a r g m n e n t s c a n b e a d v a n c e d to s h o w t h a t t h e RNA f o r m e d o n l y in t h e p r e s e n c e of t h e f o u r r i b o n u c l e o s i d e t r i p h o s p h a t e s is of the messenger type : (1) t h e e x t e n t of R N A s y n t h e s i s is n o t affected b y t h e p r e s e n c e of p r o t e i n s y n t h e s i s i n h i b i t o r s s u c h as c h l o r a m p h e n i e o l (30.0 t t g / m l ) ( F i g u r e 2) or p u r o r n y c i n (1 m g / m l ) . (2) L e v a l l o r p h a n , a s u b s t a n c e k n o w n to specifically i n h i b i t r i b o s o m a l R N A s y n t h e s i s i n E. co/i [171 s h o w s n o effect o n n u c l e o s i d e t r i p h o s p h a t e i n c o r p o r a t i o n u n d e r t h e c o n d i t i o n s d e s c r i b e d (Fig u r e 4). C o n v e r s e l y , t h e d r u g a b o l i s h e s R N A synthesis under conditions where PCS accumulates r i b o s o m a l R N A (cf. s e c t i o n III).
B 50 "~roe
T 100 (minute,y)
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Fro. 3. --- s H - U D P incorporation k i n e t i c s by E. coli K12-3000 cells p r e v i o u s l y ¢ p e r m e a b i l i z e d ~>.
Conditions of the incorporation : 109 bacteria per ml, llnal concentration, were shaken at 30°C in 10-e M Tris, pH 7.7 with :~H-UDP (Schwartz, 10 ,ttc/5 ~ m / m l and 0.01 M MgSO, - To ~top the incorporation 3 ml of 5 p. 100 TCA were added to each of the 50 ttl ~ample,~. To d e t e r m i n e the extent of incorporation, see <
A s i m i l a r r e s u l t is o b t a i n e d if 3 H - U T P is r e p l a c e d b y ~H-GTP o r ~H-CTP. T h e r a t e of i n c o r p o r a t i o n g r a d u a l l y d e c r e a s e s a f t e r 40 m i n u t e s to bec o m e s t a t i o n a r y at 60-70 m i n u t e s . C e s s a t i o n of R N A s y n t h e s i s is n o t d u e to s u b s t r a t e l i m i t a t i o n s i n c e a s e c o n d a d d i t i o n of t h e n u c l e o s i d e t r i p h o s p h a t e s m i x t u r e d o e s n o t e l i c i t a n e w r o u n d of R N A s y n t h e s i s , w h e r e a s a d d i t i o n of f r e s h cell suspension does (data not shown). RNA synthesis d e p e n d s o n t h e p r e s e n c e of all f o u r r i b o n u e l e o s i d e t r i p h o s p h a t e s , c o n f i r m i n g a n e a r l i e r res u l t I'6]. I n d e e d , o m i s s i o n of o n l y o n e of t h e s e RNA p r e c u r s o r s , G T P f o r i n s t a n c e , a l m o s t c o m pletely prevents zH-UTP incorporation. The same is t r u e , a fortio.ri, i f :~H-UTP is u s e d as t h e o n l y p r e c u r s o r ( F i g u r e 2). In c o n t r a s t a d d i t i o n of •~H-UDP ( u r i d i n e - 5 ' - d i p h o s p h a t e ) as t h e o n l y subs t r a t e is a c c o m p a n i e d b y a m a r k e d i n c o r p o r a t i o n presumably reflecting a polynueleotide phosphor y l a s e m e d i a t e d p o l y U s y n t h e s i s ( F i g u r e 3). Rib o n u c l e o s i d e t r i p h o s p h a t e i n c o r p o r a t i o n is s e n s i t i v e to a c t i n o m y c i n D ( F i g u r e 2) o r r i f a m p y c i n BIOCHIMIE, 1971, 53, n o 1.
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FIG. 4. - - s H - G T P incorporalion kinetics into messenger type R N A - E f f e c t of leuallorphan on ineorpor,lion - Action of a c l i n o m y c i n D and unlabeled GTP on p r e f o r m e d R N A stability. The i n c u b a t i o n conditions were t h a t of Figure 2 except t h a t 3H-UTP was replaced by :~H-GTP. --*-complete system. - - ~ 7 - - at 20 m i n u t e s l e v a l l o r p h a n (tartrate) 10-:¢ M was added to one part of the reaction medium. --A--at 80 minutes, non-labeled GTP (1 u m / m l ) was added to an aliquot of incorporation medium. --O--at 80 minutes, adding of actinomyein I) (90 jxg/ml) to a n o t h e r aliquot of reaction medium.
(3) S u c r o s e g r a d i e n t a n a l y s i s of <> f r o m <
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>> E . C o l t s y s t e m .
75
s t r a i n u s e d w a s E. c o l t C 600 kl U .s-°. T h e r e s i d e n t p r o p h a g e b e a r s a C~ ts m u t a t i o n of t y p e A [18] : h e a t i n g t h i s l y s o g e n a b o v e 40°C i n a c t i v e s t h e C I p r o d u c t and the r e p r e s s o r activity very slowly r e s u m e s u p o n c o o l i n g to s u b c r i t i c a l t e m p e r a t u r e s . T w o i d e n t i c a l cell s a m p l e s f r o m t h i s l y s o g e n i c s t r a i n w e r e i n c u b a t e d ; o n e ( c o n t r o l ) at 35°C f o r 10 m i n u t e s ; t h e o t h e r at 43°C f o r 10 m i n u t e s . T h e t w o s a m p l e s w e r e t h e n << p e r m e a b i l i z e d >> as d e s c r i b e d b e f o r e anti i n c u b a t e d at 30°C in t h e p r e s e n c e of t h e r a d i o a c t i v e n u c l e o s i d e t r i p h o s phate mixture. As s h o w n in T a b l e 1, t h e p e r c e n t of ). s p e c i f i c RNA p r e s e n t a m o n g t h e p o l y n u c l e o t i d e c h a i n s s y n t h e s i z e d b y t h e p u l s e - h e a t e d s a m p l e is a b o u t s i x t i m e s g r e a t e r t h a n b y t h e n o n - i n d u c e d one. T h e d i f f e r e n c e of 0.75 p. 100 c o r r e s p o n d s w e l l w i t h t h e figure u s u a l l y o b s e r v e d f o r t h e <~i m m e d i a t e e a r l y >> s p e c i e s t h a t a r e s y n t h e s i z e d f r o n t t h e r m a l l y i n d u c e d p r o p h a g e s in t h e a b s e n c e of p r o t e i n s y n t h e s i s [19~.
4,000 %
TABLE I.
3000
_P"
~21
1
I\
-3.2000
lOOO
6
I0
20 30 f,'act/onx Fro. 5. - - Sucrose density-gradient centrifugation o[ ,a <
(4) T h a t n u c l e o s i d e t r i p h o s p h a t e s u p p l e m e n t e d PCS s y n t h e s i z e s a m e s s e n g e r - l i k e RNA is also s u p ported b y e x p e r i m e n t s u s i n g l y s o g e n i c E. colt cells, c a r r y i n g a t h e r m o i n d u c i b i e p r o p h a g e . T h e
BIOCHIMIE, 1971, 53, n ° 1.
H y b r i d i z a t i o n t o k D N A c o m p a r e d rates o f R N A ' s f r o m E. colt C 600 ),t U ~ cells h e a l e d at 35"C or 43"C a n d a f t e r <
> i n c u b a ted w i t h A T P , CTP, U T P a n d .~H-GTP. Preheated cells tot 10 minutes > cells in a m e d i u m c o n t a i n i n g RNA p o l y m e r a s e p r e c u r s o r s but no amino acids p e r m i t s an u n c o u p l e d s y n t h e s i s o f m e s s e n g e r RNA. It w a s i n t e r e s t i n g to e x a m i n e w h e t h e r t h e s e cells c o u l d b e i n d u c e d to t r a n s c r i b e L a c - s p e c i f i c RNA w i t h o u t f o r m i n g O-galactosidase. E. colt M O, a Lac+ i n d u c i b l e s t r a i n , w a s s u b m i t t e d to t h e a l k a l i n e EDTA sucrose t r e a t m e n t and the cells thus obtai- > cells, some of w h i c h w i l l b e b r i e f l y d i s c u s s e d . ) cells s y n t h e s i z e large amounts of r i b o s o m a l and t r a n s f e r RNA in a d d i tion to m e s s e n g e r RNA. T h i s a m i n o a c i d d e p e n dent r i b o s o m a l RNA s y n t h e s i s seems to i n d i c a t e flint s y n t h e s i s of r i b o s o m a l p r o t e i n s is n e e d e d to stabilize n e w l y t r a n s c r i b e d r i b o s o m a l g e n e p r o ducts or that the s y s t e m m u s t be s o m e w h a t released f r o m the s t r i n g e n t c o n t r o l w h i c h is o p e r a ting w h e n no e x o g e n o u s a m i n o a c i d is p r e s e n t . Support in f a v o r of the s t a b i l i z a t i o n m e c h a n i s m comes f r o m the fact that c h l o r a m p h e n i c o l greatly diminishes - - w i t h o u t c o m p l e t e l y p r e v e n t i n g - amino acid i n d u c e d s t i m u l a t i o n of RNA synthesis. Moreover e x p e r i m e n t s to be d e s c r i b e d e l s e w h e r e [23] h a v e s h o w n that a d d i t i o n of a c o m p l e t e 70S ribosomal p r o t e i n m i x t u r e or of b a s i c p r o t e i n tractions (histories, p r o t a m i n e s ) c a u s e d a p p r e ciable s t i m u l a t i o n of r i b o s o m a l RNA s y n t h e s i s by PCS. T h i s s t i m u l a t i o n was o b s e r v e d e v e n in the presence of c h l o r a m p h e n i c o l a n d in the a b s e n c e
Counts/min hybridized t,~; ), DNA i
Total input (cpm)
i Percent lhybridized ]
35oC . . . . . . . . . . . . .
'
310
~ 208,000 ~
0.15
43oC . . . . . . . . . . . . .
!
614
69,000 !
0.89
Culture of E. colt C 600 )~t U32 was grown in casaminoacid 63 medium plus glycerol, t h y m i n e (50 ttg/ml) as usual. When the OD at 420 mtt was equal to 1.0, the cells were divided into two :amples of OD 10 in TMEbuffer : one (control) was incubated for 10 minutes at 35°C, the other was t h e r m a l l y induced by a 10 minute:s heat at 35°C, lhe other was thermally indnced by a 10 minutes heat at 43°C. Then both saniples were submitted to the same procedure : cooling in ice and after <
M e s s e n g e r R N A s t a b i l i t y in the a b s e n c e of amino acids. T h e m e s s e n g e r f r a c t i o n w h i c h is s y n t h e s i z e d under the afore-mentioned conditions -- namely i n t h e p r e s e n c e of t h e f o u r n u c l e o s i d e t r i p h o s p h a t e s a n d a b s e n c e of a n t i n o a c i d s - - is of e x c e p t i o n a l m e t a b o l i c s t a b i l i t y . A c c o r d i n g l y , if, a f t e r t h e r a t e of n u c l e o s i d e i n c o r p o r a t i o n h a s b e c o m e c o n s t a n t , f u r t h e r i n c o r p o r a t i o n is s t o p p e d b y iso-
76
Fakher
Ben-Hamida
topic dilution or by a c t i n o m y c i n D (90 ~g/ml), a slow d e g r a d a t i o n of p r e f o r m e d RNA occurs, w i t h a half-life greater than 60 m i n u t e s (Figure 4). Reasons for this very m a r k e d stability will be c o n s i d e r e d afterwards. II.
Protein synthesis by ¢ permeabilized
--
>>
and Francois
The rate is linear with time. The amounts of 6-galactosidase are 20-30 p. 100 that of normal cells.
150-
cells.
When E. c o l t is submitted to a p l a s m o l y t i c shock u n d e r the conditions already described, not only does it b e c o m e p e r m e a b l e to metabolites or drugs that usually do not cross the cell b a r r i e r (nucleotides or nucleoside triphosphates, actinomyein, etc...) but endogenous metabolites apparently leak out from the plasmolysed cell bodies. Hence p r o t e i n synthesis will not take place unless the system is supplemented with RNA precursors, an A'FP r e g e n e r a t i n g system, specific cations and a complete m i x t u r e of a m i n o acids (see Materials and Methods). F i g u r e 6 illustrates the effect of a d d i n g a complete a m i n o acid m i x t u r e on the rate of 14C valine i n c o r p o r a t i o n . In this complete a m i n o acid medium, protein synthesis goes on linearly for several hours. A c t i n o m y c i n D almost completely p r e v e n t s protein synthesis s h o w i n g the nucleoside t r i p h o s p h a t e d e p e n d e n t RNA synthesis generates active messenger templates.
Gros.
/
.£ u
/
go,loo-
~ 50-
,,7
o
~
Ido
~;o Time
mn
Fie. 7. - - i•-galactosidase induced synthesis by E. colt D 10 cells ¢ permeabilized >>. For experimental details see ¢ Materials and Methods >>. - - V - - complete system. --o-without IPTG. - - A - - c o m p l e t e system with actinomycin D (100 ~g/ml).
III. - - R i b o s o m a l a n d t r a n s f e r R N A s y n t h e s i s .
.3a~
•~
•
~
•
•
50
I00
150
200
Time (m,;~utes)
Fire 6, - - ll, C-valine incorporation kinetics into proteins by PCS (E. colt D 10). For incorporation eomlitions see <
When in the p r e s e n c e of a complete amino acid mixture, PCS, o t h e r w i s e supplemented w i t h ribonueleoside t r i p h o s p h a t e precursors, actively synthesizes at a l i n e a r rate r i b o s o m a l and transfer RNA's. This rate c o r r e s p o n d s to a total RNA increase of 12 p. 100 per hour. F i g u r e 8 shows the effect of a d d i n g the complete a m i n o acid mixture w h e n nucleotide i n c o r p o r a t i o n has ceased (the same c o n d i t i o n s as in Figure 2) : total nueleotide i n c o r p o r a t i o n resumes at a linear rate for several hours. Adding c h l o r a m p h e n i c o l (340 vtg/ml), some time after the amino acids, c o n s i d e r a b l y reduces the rate of RNA synthesis. Existence of an <
Transcription
and
translation
i n a <~p e r m e a b i l i z e d
crose g r a d i e n t s t u d i e s : F i g u r e 9 s h o w s t h e r a d i o . activity p a t t e r n o f a n e x t r a c t f r o m ¢ p e r m e a b i l i zed~ cells t h a t w e r e p r e i n c u b a t e d f o r 220 minutes in a n u c l e o s i d e t r i p h o s p h a t e - a m i n o acid
>> E . C~>li s y s t e m .
77
T h e p r e s e n c e of p o l l y d i s p e r s e d RNA m a t e r i a l s e d i m e n t i n g b e t w e e n 5 S antt 27 S also i n d i c a t e s t h a t s t a b l e m e s s e n g e r RNA c o n t i n n e s to be f o r m e d .
lO 2,~000 I
I
I
50 5
_0.8
20000
305
~,pm
6000
ISO00
~000 ~/0.000
a v
2000 SO00
6
I00
200
300
Fla. 8. - - . S H - G T P incorporation kinetics into ribosomal and transfer RNA's by PCS (E. eali D 10). Effect of the omission of a single amino acid. E f f e c t of chloramphenicol and levallorphan. --e--incorporation of ~H-GTP into messenger type RNA (conditions of Figures 2 and 4) : (A). --O-at 90 minutes, adding to an A aliquot of a complete amino acid mixture (the incorporation medium is the same as that described in ¢ Materials and Methods ~ section : ¢ Det e r m i n a t i o n of protein synthesis , ) : (B). --A-at 90 minutes, adding to an A aliquot of a complete amino acid mixture, excep~ valine. ......~7...... at 120 minutes, levallorphan (tartrate) 10-3 M was added to a B aliquot. ..... O...... at 150 minutes, chloramphenicol 340 ttg/ml added to a B aliquot.
m e d i u m c o n t a i n i n g aH-GTP. In profile of F i g u r e 5, 50 S a n d 30 m a r k e d l y l a b e l e d ; a s h o u l d e r is sible in t h e 4 S r e g i o n s u g g e s t i n g
BIOCHIMIE, 1971, 53, n ° 1.
c o n t r a s t to t h e S subunits are also c l e a r l y viiRNA s y n t h e s i s .
[O
20 30 /-tact~arts FIfi. 9. - - Sucrose density-gradient centrifugation of an <
IV.Uncoupling between transcription a n d t r a n s l a t i o n o f L a c - s p e c i [ i c R N A in PCS. As i l l u s t r a t e d b e f o r e , i n c u b a t i o n of <
78
F a k h e r B e n - H a m i d a and Francois Gros.
n e d i n c u b a t e d in a n u c l e o s i d e t r i p h o s p h a t e m e d i u m c o n t a i n i n g I P T G (no ~ - g a l a c t o s i d a s e i n d u c t i o n c o u l d be d e t e c t e d at t h e e n d of t h i s stage). I P T G t r e a t e d as w e l l as an u n i n d u c e d s a m p l e w e r e f i l t e r e d a n d t h e c e l l s r e s u s p e n d c d in a p r e warmed mediuin containing a complete amino m i x t u r e . As s h o w n in F i g u r e 10 t h i s s t e p w i s e t r a n s f e r e l i c i t e d ~ - g a t a c t o s i d a s e s y n t h e s i s in t h e I P T G p r e i n c u b a t e d s a m p l e b u t n o t in t h e c o n t r o l . !
A c t i n o n l y c i n D h a d v i r t u a l l y n o e f f e c t w h e n pres e n t in t h e m e d i u m a l l o w i n g RNA e x p r e s s i o n but c o m p l e t e l y p r e v e n t e d i n d u c t i o n of L a c specific RNA w h e n a d d e d t o g e t h e r w i t h IPTG. As s h o w n in F i g u r e 11, ~ - g a l a c t o s i d a s e f o r m a t i o n d u r i n g the s e c o n d s t e p Was p r o p o r t i o n a l to t h e l e n g t h of the induction period.
_
I
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-u o
o
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100
50 "]'1 r'N e
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I
50 100 T/me (mlnutea) Fro. 10. - - Kinetics of .~-galactosidase f o r m a t i o n in the presence of a complete amino acid mixture, by PCS of E. colt MO, preinduced (IPTG) in the presence of the nucleoside lriphosphates m i x t u r e and then filtered. E. colt MO <
10-4 M 2 - m e r c a p t o e t h a n o l
and
5 X
10-4 M I P T G .
After 60 minutes induction, the samples were adsorbed on 0.45 U Sartorius filters and washed with TM 2 buffer. The filters then are transferred in a second step to flasks containing the pre-warmed following mixture : 10-2 M Tris-HC1 pH 7.5, 10g M KC1, 10 -'~ M Mg SO~, 5 X 10-4 M Mn Cl.~, creatine phosphate (1 m g / m l ) , creatine phosphokinase (20 p.g/ml). The cells were easily resuspended with some rotary motions and the m e m b r a n e s w e r e removed from the flasks. At time zero of Figure 10, a complete amino acid mixture (2.5 × 10-4 M) was added into the flasks, shaken at 30°C. fl-galactosidase assays were performed as described in the <
BIOCIIlMIE, 1971, 53, n" 1.
r~th
Fla. 11. - - Compared kinetics of i~-galactosidase formarion in the presence of a complete amino acid mixture, by PCS of E. colt MO, preinduced (IPTG) for various periods and then filtered. The experimental conditions are those of Figure 10 except for the IPTG-induetion period. --e-no IPTG in the first step. --©-IPTG-indueed cells for 6 minutes. --A-IPTG-indueed cells for 25 minutes --V-IPTG-induced cells .for 40 minutes.
DISCUSSION. As s h o w n in t h i s w o r k a l k a l i n e E D T A - s u c r o s e t r e a t m e n t of E. colt c e l l s i n d u c e s i n t e r e s t i n g alter a t i o n s in t h e i r p e r m e a b i l i t y to v a r i o u s p h o s p h o r y l a t e d d e r i v a t i v e s w i t h o u t m a k i n g t h e m osmotic a l l y f r a g i l e . T h e s e ceils b e h a v e in m a n y r e s p e c t s like an o r d i n a r y cell f r e e s y s t e m s i n c e t h e i r p e r m e a b i l i t y b a r r i e r is lost o r d r a s t i c a l l y a l t e r e d , while their synthesizing machinery remains i n t a c t . S u c h a s i t u a t i o n a p p e a r s p a r t i c u l a r l y well s u i t e d f o r t h e s t u d y of u n c o u p l e d m a c r o m o l e c u l a r synthesis ~ the present paper illustrates this point by describing the transcription and translation p r o p e r t i e s of E. colt <
Transcription
and translation
i n a <~p e r m e a b i l i z e d
I n c u b a t i o n of PCS w i t h the f o u r RNA p o l y m e rose substrates but in the a b s e n c e of a m i n o a c i d s causes a c t i v e RNA synthesis. T h e p o l y n u c l e o t i d e fraction t h u s f o r m e d e x h i b i t s so far all the p r o perties of m e s s a g e r RNA. T h i s is p a r t i c u l a r l y evidenced by the e x p e r i m e n t s i n v o l v i n g t h e r m a l l y induced ~ l y s o g e n s o r cells t r e a t e d w i t h a gratuitous 3-galactosidase i n d u c e r . Interestingly, newly synthesized messenger RNA is of e x c e p t i o n a l l y great stability (half-life greater t h a n 60 m i n u t e s ) . T h i s m i g h t be due in part to the h i g h l y r e s t r i c t i v e c o n d i t i o n s that are realized d u r i n g the i n c u b a t i o n , c o n d i t i o n s w h i c h almost c o m p l e t e l y p r e c l u d e p r o t e i n s y n t h e s i s (Fig. 6 a n d 8). A n o t h e r e x p l a n a t i o n m i g h t be related to the p o s s i b l e r e m o v a l of p e r i p l a s t i c nucleases [20, 21]. T h i s is suggested by the results of Figure 8 in w h i c h a stable m e s s e n g e r like profile can be d e t e c t e d e v e n a m o n g <~ p e r m e a b i l i z e d ~ cells s y n t h e s i z i n g both RNA and p r o t e i n s . T h i s point is p r e s e n t l y u n d e r study. When i n c u b a t e d w i t h the f o u r n u c l e o s i d e triphosphates as the o n l y substrates, PCS s y n t h e s i z e s no a p p r e c i a b l e a m o u n t of r i b o s o m a l or t r a n s f e r RNA, a situation w h i c h f o r m a l l y r e c a l l s the behavior of RC str b a c t e r i a w h e n s t a r v e d of t h e i r essential a m i n o a c i d [221. Yet the t r a n s c r i p t i o n p r o duets m a d e u n d e r s i m i l a r c o n d i t i o n s by a suspension of (~ p e r m e a b i l i z e d ~ b a c t e r i a w i t h a RCr~ genotype h a v e not yet been a n a l y z e d . RNA t r a n s l a t i o n in PCS r e q u i r e s the p r e s e n c e of a c o m p l e t e a m i n o a c i d m i x t u r e (cf. F i g u r e 8). This i n d i c a t e s that s u c r o s e - i n d u c e d p l a s m o t y s i s presumably d e p l e t e s cells f r o m t h e i r e n d o g e n o u s metabolites. U n d e r c o m p l e t e a m i n o a c i d s u p p l e mentation, <
BIOCHIMIE, 1 9 7 1 ,
53, n ° 1.
~> E. C o l t s y s t e m .
79
of e x o g e n o u s a m i n o acids, s u g g e s t i n g that s t r u c t u ral p r o t e i n s m i g h t h a v e free a c c e s s to the cell interior. A n o t h e r i l l u s t r a t i o n of the u n c o u p l e d s y n t h e s i s that can be s t u d i e d w i t h the <( p e r m e a b i l i z e d ~ cell s y s t e m is p r o v i d e d by the s t e p - w i s e ~-galact o s i d a s e i n d u c t i o n e x p e r i m e n t d e s c r i b e d in t h i s w o r k . I n c u b a t i o n of the s y s t e m w i t h t h e RNA pol y m e r a s e substrates p l u s I P T G elicits t r a n s c r i p tion of the ~( z ~> gene w i t h o u t c o n c o m i t a n t e n z y m e synthesis. Accordingly such a preinduced system can s y n t h e s i z e ~-galactosidase u p o n t r a n s f e r to an a m i n o a c i d c o n t a i n i n g I P T G free m e d i u m , e v e n if a c t i n o m y c i n D is p r e s e n t . Use of PCS m i g h t thus be of great i n t e r e s t to dissect the var i o u s steps a c c o m p a n y i n g gene e x p r e s s i o n and its control. In the p r e c e d i n g , e m p h a s i s has b e e n p l a c e d on the m e c h a n i s m s of gene e x p r e s s i o n . W e h a v e seen that (~ p e r m e a b i l i z e d >~ cells a r e u n a b l e to m a k e c o l o n i e s on a g a r plates e v e n w h e n s u p p l e m e n t e d w i t h a r i c h m e d i u m . Lack of c o l o n y f o r m i n g c a p a city is not due, h o w e v e r , to an i n a c t i v a t i o n of the DNA r e p l i e a s e . PC,S not o n l y c o n t a i n s an a c t i v e DNA po.lymerase as e v i d e n c e by its a b i l i t y to incorporate deoxyribonucleoside triphosphates but it also e x h i b i t s a m e m b r a n e b o u n d A T P d e p e n d e n t DNA p o l y m e r a s e a c t i v i t y . T h i s c o n c l u s i o n stems f r o m use of pol A- m u t a n t s and w i l l be r e p o r t e d in a s e p a r a t e p a p e r [2,4]. A ckno~wledfem e n ts.
We wish to express our thanks to Dr. Philippe GRAN1~OULAN who has performed the electron mierographs. This work was supported by grants from the Fonds de D6veloppement de la Recherche Scientifique et Technique, the Commissariat h l'Energie Atomique, the Centre National de la Recherche Scientifique, the Ligue Nationale Frangaise co ntre le Cancer, and the Fondation pour la Recherche M6dicale Franqaise. R~su,~. Dans cet article, nous d~crivons les propri6tds biosynth~tiques d'un syst6me de cellules d'E. colt <
80
Fakher
Ben-Hamida
(2) Les ARN messagers ainsi form6s sont fonctionnels dans la synth6se des prot6ines, eomme le montre une experience d'induction en deux t e m p s : I'ARN, synth6tis~ en pr6sence des quatre ribonucl6oside triphosphates et de I'IPTG, peut diriger la synth6se de [~-galactosidase quand le syst6me PCS est transf6r6 dans un m61ange complet d'acides amines en pr6senee d'actinomycine. (3) Quand ce syst6me PCS est mis en pr6sence des quatre ribonucl6oside triphosphates et de tous les acides amin6s, il synth6tise des prot6ines et il y a une forte acumulation d'ARN de t r a n s f e r t et d'ARN ribosomal. ZUSAMMENFASSUNG.
In dieser Arbeit beschreiben w i r die biosynthetischen Eigenschaften eines Systems yon • permeabilisierten >> E. c o l i - Z e l l e n (PCS) das durch Behandlung mittels Plasmolyse der Bakterien (2 M - S a c c h a r o s e ) und Wirkung des EDTA (10-4 M) erhalten wird. Die so behandelten Zellen weisen interessante Eigenschaften a u f : Permeabilit~it gegeniiber gewissen Phosphorylderivaten ohne Empfindlichkeit gegentiber osmotischer Wirkung, Verlust der Reserven and endogenen Metaholiten und Anhalten der Zellvermehrung auf reichem Medium Dieses System ist besonders interessant fiir die Untersuchung der entkoppelten Synthesen der Makromolekiile. 1 - - In Gegenwart der gew6hnlichen Substrate der RNS-Polymerase (ATP, CTP, GTP und UTP), synthetisiert das System hauptsfichlich Messenger-RNS, die ausserordentlich stahil ist (Halbleben von m e h r als 60 Minutenl. Die Griinde dieser Stabilitiit werden besprochen. 2 - Die so gebildeten Messenger-RNS sind in der P r o t c i n s y n t h e s e funktionnell, wie ein I n d u k t | o n s v e r :;uch in zwei Etappen es zeigt : die in Gegenwart der 5 Ribonukleosidtriphosphate und des IPTG gebildete RNS kann die Synthese der ~J-Galaktosidase steuern wenn das PCS-System in die vollstfindige Mischung der Aminosaiircn in Gegen~vart yon Aetinomycin fiberge. ffihrt wird. 3 --- Wenn dieses PCS-System in Gegenwart der vier Ribonukleosidtriphosphate und aller Aminosaiiren
BIOCHIMIE, 1971, 53, n ° 1.
and Francois
Gros.
gesetzt wird, so synthetisiert es Proteine und es gibt eine starke Anhiiufung von Transfer-RNS und Rihosom-RNS.
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