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Further characterization of yeast RNA polymerases. Effect of subunits removal
BIOCHIMIE, 19.76, 58, 71-80.
Further characterization of yeast RNA polymerases. Efl ct of subunits removale). J a n i n e HUET, S y b i l l e DEZ~L~E, F r a n q o i s IBORRA, J e a n - M a r i e BUHLER, A n d r 6 SENTEN&C a n d P i e r r e FIROMAGEOT <~.
Service de Biocbimie, D~partement de Biologic, Centre d'Etudes Nucldaires de Saclay, B.P. n ° 2, 91190 Gif-sur-Yvette, France. Summary. - - Two f o r m s of yeast RNA polymerase A are resolved by phosphoeellulose c h r o m a t o g r a p h y . One of these, called RNA p o l y m e r a s e A*, is lacking two polypeptide c h a i n s of 48,0'0.0 a n d 37,000 daltons. The properties of the two enzymes are compared in t h e p r e s e n t paper. R~NA p o l y m e r a s e A* t r a n s c r i b e s dfA-T), w i t h a s i m i l a r efficiency as the complete enzyme, b u t it is c o m p a r a t i v e l y much less active w i t h native DNA. The two enzymes can also be differen,tiated on t h e basis of t h e i r ionic s t r e n g t h and divalent cation r e q u i r e m e n t s . RNA p o l y m e r a s e A* h a s a p a r t i c u ] a r l y low activity at high salt and low Mg2+ concentrations. T h e r m a l i n a c t i v a t i o n cur~'es of the two enzymes are different when residual activity is assayed w i t h native DNA. I n c o n t r a s t w i t h d(A-T), as t e m pl,~te the a p p a r e n t i n a c t i v a t i o n curves of the t w o enzymes are identical. The data sugges,t t h a t the two dissociable polypcptide chains play an i m p o r t a n t role in transcri,ption. The t e m p l a t e specificity of yeast RNA .polymerase B was f u r t h e r investigated using SV40 DNA-FI as template. RNA polym,erase B is able to ~etain [3H]SV40 DNA-FI on nitrocellulose filters b u t the enzyme-DNA complex is very unstable. The o b s e r v a t i o n t h a t RNA p o l y m e r a s e B can t r a n s c r i b e to some extent a supercoiled DNA b u t not a l i n e a r double s t r a n d e d t e m p l a t e supports the h y p o t h e s i s t h a t the enzyme needs some u n p a i r e d DNA s t r u c t u r e to i n i t i a t e t r a n s c r i p t i o n .
INTRODUCTION. T h e c o m p l e x i t y of g e n e t r a n s c r i p t i o n i n h i g h e r cells is r e f l e c t e d b y t h e s t r u c t u r a l f e a t u r e s of t h e m u l t i p l e f o r m s of R N A p o l y m e r a s e s . T h r e e c l a s s e s of e n z y m e s , w h i c h c a n b e c o n v e n i e n t l y d i s t i n g u i s h e d o n t h e b a s i s of t h e i r t t - a m a n i t i n s e n s i t i v i t y , a p p e a r to b e r e s p o n s i b l e f o r all n u c l e a r R N A s y n t h e s i s It, 2]. Nu,clear R N A p o l y m e r a s e s , p u r i fied fro~m v a r i o u s o r g a n i s m s , a r e c o m p l e x m u l t i m e r l e p r o t e i n s w h i c h c o n s i s t of t w o 1,arge s u b u n i t s i n e q u i m o l a r a m o u n t , a s s o c i a t e d w i t h a s e r i e s of s m a l l e r p o l y p e p t i d e c h a i n s [3]. A n u m b e r of q u e s tion,s a r i s e w h e n c o n s i d e r i n g t h e s e c o m p l e x p r o tein structures. (1) A r e aH t h e p o l y p e p t i d e c o m p o n e n t s associated with the enzymes required for the basic, template-directed polynlerisation reaction ? (2) A l t e r n a t i v e l y , d o e s e a c h of t h e s e p o l y m e r a s e s c o n s i s t of a f u n d a m e n t a l e n z y m e s u r r o u n d e d w i t h regulatory components or specificity determinants having a specialized role in transcription ? (3) A r e t h e m u l t i p l e f o r m s of R N A p o l y m e r a s e A, B an,d C c o m p , l e t e l y g e n e t i c a l l y d i s t i n c t (*) Th~s. p a p e r is dedicated to the m e m o r y of Huguette de R.obi.chon-Szulmajster. To w h o m all eorrespond.ence should he addressed.
proteins or do they share common subunits which could carry a basic function in DNA transcription ? (4) A r e t h e s u b c l a s s e s of t h e s e e n z y m e s d u e to t h e loss o r a l t e r a t i o n of o n e o r m o r e p o l y p e p t i d e c o m p o n e n t s d u r i n g p u r i f i c a t i o n o r a r e -they i n d e e d i m p l i c a t e d i n s o m e r e g u l a t o r y p r o c e s s at the polymerase level ? W e h a v e d e s c r i b e d [4, 5] t h e p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n of R N A p o l y m e r a s e A a n d B f r o m y e a s t cells Saccharomyces cerevisiae. T h e s t r u c t u r a l p r o p e r t i e s of t h e t w o e n z y m e s w e r e i n v e s t i g a t e d to b r i n g s o m e l i g h t o n s.ome of t h e a b o v e q u e s t i o n s . It w a s f o u n d t h a t a l t h o u g h R N A p o l y m e r a s e s A a n d B a p p e a r lo b e d i s t i n c t p r o t e i n s o n t h e b a s i s of a n u m b e r of c r i t e r i a i n c l u d i n g s o m e i m m u n o l o g i c a l a n d s t r u c t u r a l p r o p e r t i e s , t h e s e enz y m e s s h a r e t h r e e c o m m o n s u b u n i t s of l o w m o l e c u l a r w e i g h t [6]. I n t h e c o u r s e of t h i s .~tudy y e a s t R N A p o l y l n e r a s e A w a s f o u n d to b e c o n v e r t e d u n d e r c e r t a i n c o n d i t i o n s to a n e w f o r m of e n z y m e c a l l e d R N A p o l y m e r a s e A* w h i c h is l a c k i n g t w o p o l y p e p t i d e c h a i n s [6, 7]. H e n c e t h e o p p o r t u n i t y w a s g i v e n to b e g i n to a n s ~ - e r -the f i r s t t w o q u e s t i o n s c o n c e r n i n g t h e r o l e of t h e d i f f e r e n t p o l y p e p t i d e c o m p o n e n t s of t h i s e n z y m e . I t is t h e p u r p o s e of t h i s c o m m u n i c a t i o n to d e s c r i b e a d d i -
72
J. H u r t a n d coll.
tional properties of RNA polymerase A" c o m p a r e d to that of the complete enzyme. I n a d d i t i o n , some n e w results are r e p o r t e d on the template specificity of yeast RNA polymerase B.
MATERIAL AND METHODS.
fected 3T6 cells g r o w n i n the p r e s e n c e of [3H]t h y m i d i n e by the methcvd of Hirt [9, 10]. Native calf t h y m u s DNA was p u r i f i e d on nitrocellulose c o l u m n [5~. [i¢Cl-nucleoside triphosphates were o b t a i n e d from N.E.N. Corp. [3H]UTP a n d Ea32,p]GTP was obtained from C.E.N. Saclay. Other materials were as p r e v i o u s l y described.
"YEAST CELLS.
RNA POLYMERASE A ASSAY. S a c c h a r o m y c e s cerevisiae (4094 B a Ad 2 Ur 1) was obtained from Huguette de Robichon-Szulmajster. The cells were g r o w n and collected as p r e v i o u s l y described [51.
RNA POLYMEnASE:S A, A* AN,D B. E n z y m e s A a n d B were p u r i f i e d as p r e v i o u s l y des.cribe~d [4, 51 a n d were homogeneous p r o t e i n s as seen by gel electrophoresis (Fig. 1). RN,A polyInerase A* was o b t a i n e d by phos.phocellulose c h r o m a t o g r a p h y of pure RNA po.lylne.rase A [7].
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S t a n d a r d i n c u b a t i o n m i x t u r e (0.1 ml) c o n t a i n e d : 0.07 M Tris-HC,1 (pH 8), 5 InM M.gCl~, 1 mM dithiothreitol, 1 InM each of AT!P, GT.P, a n d CT~, 0.5 mM [3H]UTP (2,0 to 30 corn per pmole), 12 ~g of native calf t h y m u s DNA a n d 1 to 2 ~g of RNA polymerase A or A*. W h e n s y n t h e t i c p o l y m e r s were used as temp'late, only the c o m p l e m e n t a r y nucleoside triphosphates were added w i t h one labelled nucleotide at 0.5 InM as i n d i c a t e d . After a 20 m i n i n c u b a t i o n at 30 °, acid insoluble radioactivity was d e t e r m i n e d Ell]. As,s'AY Fort TRANSCRIPTION OF SV40 D[N.A-FI BY RI~A POLYME.RASEB. The i n c u b a t i o n m i x t u r e (.0.1 Inl) c o n t a i n e d : 0.07 M Tris-HCl (pH 8), 4.5 InM MnC12, 5 mM dithiothreitol, 10 mM a m m o n i u m su'lfate, 0.4 InM each ATP, CTP, UTP a n d 0.2 mM [ct32PIGTP (65 epm per pmole), enzyme a n d DNA as i n d i c a ted. Reaction was initiated by a d d i t i o n of the salt a n d substrate m i x t u r e ; after 5 Inin i n c u b a t i o n at 37 ° to p e r f o r m RNA c h a i n i n i t i a t i o n , the ammon i u m sulfate concentr,ation was i n c r e a s e d to 80 m,M and i n c u b a t i o n was co n'tinued for 25 rain at 37 ° . Fort B I N D I N G [ZH1 SV49 DNA-FI.
ASSAY
FIG. 1. --Polyacrylamide gel electrophoresis of yeast RNA polymerases A and B under non denaturing conditions. Analytic electrop,h.oresis was performed as indicated under M,ethods. Left : RNA polymcrasc B (5 ~g ; phosphocellu.lose fraction [5]) ; right : RNA polymerase A (4 ~g ; glycerol gradient fraction [4]). NUCLEIC ACIDS
OF R N A
POLYME,RASE B
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B i n d i n g r e a c t i o n was c o n d u c t e d as f o l l o w s : [3H] S V 4 0 - D N A (95 per cent form I D N A ; 7500,0, cpm per u,g) an,d RNA p o l y m e r a s e B were naixed i n 50 td of b i n d i n g bu,ffer c o n t a i n i n g 0.05 M Tris-HC1 (pH 8), 1 InM dithiothreit(fl, 0.1 mM EDT~A, 3 mM MnCl,, a n d 3,0 per cenl glycerol. After 1,0 m i n at 37 ° the m i x t u r e was diluted with 1 ml of b i n d i n g buffer at 37 °, i m m e d i a t e l y filtered on nitrocellulose m e m b r a n e (Mil]ipore HAWP 02.5) and r a d i o a c t i v i t y r e t a i n e d on the filter was determ i n e d [12].
AND N U C ~ E O T I D E S .
Synthetic p o l y m e r s d(A-T)n, 4(I-C)n, (d T) n were o b t a i n e d as p r e v i o u s l y described [81. Unlabelled SV40 D,NA FI was a gift of Dr. M. Yaniv (Institut Pasteur). [3H] SV40 DNA was p u r i f i e d from in-
BIOCHIMIE, 1976, 58, n ° 1-2.
P O L Y A C R Y L A M I D E , GEL .~ECTROPHORE,SIS.
The electrophoresis of ,I~NA polylnerase u n d e r n o n .denaturing c o n d i t i o n s was p e r f o r m e d as desc r i b e d [4]. P o l y a c r y l a m i d e gel electrophoresis in
73
Yeast RNA polymerases. the p r e s e n c e of s o d i u m d o d e c y l sulfate w a s essenflatly p e r f o r m e d a c c o r d i n g to the p r o c e d u r e desc r i b e d b y L a e m m l i [13]. T h e t w o d i m e n s i o n a l m a p p i n g of s u b u n i t s w a s c a r r i e d out b y electrof o c u s s i n g in 6M urea f o l l o w e d b y s o d i u m d o d e c y l sulfate gel e l e c t r o p h o r e s i s in the s e c o n d d i m e n sion. T h e m e t h o d w i l l be d e s c r i b e d e l s e w h e r e
C o m p a r e d a c t i v i t y of RNA p o l y m e r a s e s A a n d A* w i t h various templates.
1.1. - -
A p r e l i m i n a r y s c r e e n i n g of r e m e n t s of the t w o f o r m s of that the r e m o v a l of the t w o p o n e n t s m a r k e d l y altered the
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F o l l o w i n g p h o s p h o c e l i u l o s e c h r o m a t o g r a p h y of p u r e R N A p o l y m e r a s e A, t w o e n z y m a t i c fractions are o b t a i n e d w h i c h h a v e the s a m e s p e c i f i c a c t i v i t y w i t h d(A-T)n as t e m p l a t e [7]. One fraction is l a c k i n g t w o p o l y p e p t i d e c o m p o n e n t s of 48,000 a n d 37,0.0.0 daltons. T h i s e n z y m e , calle.d R N A p o l y m e r a s e A* has a v e r y r e d u c e d a c t i v i t y on native calf t h y m u s D N A [7]. T h e r e c o v e r y
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FIG. 3. - - Kinetics of RNA synthesis with different templates by RNA polymerases A and A*. Four standard incubation mixtures described under Methods were scaled up to 1 ml. Template was either 100 ~xg of native calf t h y m u s DNA, 31 ~g d(I-C)~, 30 Ixg d(A-T)n or 30 ~g (dT),,. Incubation with RNA potymerase A (7 ;~g) or RNA polymerase A* (10 ug) was carried out at 30 °. At different times 0.1 ml aliquots were withdrawn and processed to estimate acid-insoluble radioactivity as usuaI. (G--C)), RNA polymerase A ; (Q--O)~ RNA polymerase A*.
Fx6. 2. - - Polyacrylamide gel eleclrophoresis with sodium dodecyl sulfate of RATA polymerases A and A* after phosphocellulose chromatography. Glycerol gradient RNA polymerase A was chromatograp'hed on phosphoeeilul, ose as described [7]. Protein fractions were analysed by electrophoresis with sodium dodecyl sulfate. From l,eft to right fraction I : RNA polymerase A* ; fraction 2' : RNA polym,erasc A ; 3 : the two dissociated po]ypeptides.
f r o m p h o s p h o c e l l u l o s e of the t w o e n z y m e s fractions A a n d A* as wel,1 as the t w o d i s s o c i a t e d p o l y p e p t i d e s is illustrated in figure 2.
BIOCHIMIE, 1976, 58, n ° 1-2.
of the e n z y m e ~7]. F i g u r e 3 s h o w s the k i n e t i c s of R N A s y n t h e s i s u s i n g four different t e m p l a t e s w i t h R N A p o l y m e r a s e A a n d A*. W i t h the alternated c o p o . l y m e r d(A-T) n, both e n z y m e s are e q u a l l y active. An e x t e n s i v e s y n t h e s i s of r(A-U) n o c c u r e d w i t h o u t lag p h a s e a n d lasted for m o r e than one hour. On the other h a n d , w i t h native c a l f t h y m u s D N A as t e m p l a t e , R N A p o l y m e r a s e A* w a s about 6 f o l d less active than the c o m p l e t e e n z y m e . R N A p o l y m e r a s e A* w a s also c o m p , a r a t i v e l y less active w i t h d(I-C) n as t e m p l a t e a n d a m a r k e d lag p h a s e in R N A s y n t h e s i s s u g g e s t e d that the e n z y m e h a d s o m e d i f f i c u l t y in i n i t i a t i n g the p o l y m e r i s a t i o n reaction. W i t h (dT) n as t e m p l a t e , the c o m plete e n z y m e w a s also m o r e active than e n z y m e A* a n d a p r o n o u n c e d lag p h a s e w a s s e e n w i t h
J. H u e t a n d coll.
74
this template. These results c l e a r l y i n d i c a t e s that the two m i s s i n g p o l y p e p t i d e s are not r e q u i r e d for the basic p r o c e s s o,f t r a n s c r i p t i o n . On the o t h e r han~d, since the v a r i o u s templates, e x c e p t for d(A-T)n, are t r a n s c r i b e d w i t h a different efficiency, it is likely that the t w o polype,p,tides play an i m p o r t a n t role in transcri,ption. 1.2. - - Effect of enzyme concentration. The lag p h a s e in RNA synthesis could be due to the p r e s e n c e of trace a m o u n t of a catalytic comp o n e n t in RNA p o l y m e r a s e A* p r e p a r a t i o n . If this
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T h e effect of a m m o n i u m sulfate c o n c e n t r a t i o n u p o n p o l y m e r a s e s activities is i l l u s t r a t e d in figure 6. W i t h calf t h y m u s DNA and My2+ both enzymes are m o r e active at l o w salt c o n c e n t r a tions and h a v e o p t i m a l activity at 5 mM ammon i m n sul, fate. In the p r e s e n c e of Mg 2+ plus Mn2+ ions I={NA p o l y m e r a s e A shows b i p h a s i c p r o f i l e w i t h m a x i m a at 5 mM an,d 50 mM a m m o n i u m sulfate. The i n t e r e s t i n g o b s e r v a t i o n was that RNA p o l y m e r a s e A* w a s p r a c t i c a l l y i n a c t i v e at h i g h
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i o n i c s t r e n g t h u p o n e n z y m e activity [3]. It was t h e r e f o r e of interest to c o m p a r e the d i v a l e n t cations and i o n i c strength o p t i m a of the two enzymes u n d e r o t h e r w i s e well d e f i n e d a n d s i m i l a r conditions. F i g u r e 5 s h o w s RNA p o l y m e r a s e s A and A* activities w i t h native DNA as template, in the p r e s e n c e of Mg 2+ or Mn 2+ i n d i v i d u a l l y or w i t h a c o m b i n a t i o n of the t w o d i v a l e n t cations. The same o p t i m a l c o n c e n t r a t i o n of Mg 2+, about 5 mM, a c t i v a t e d RNA p o l y m e r a s e s A and A* but, s u r p r i s i n g l y , e n z y m e A* w a s p r a c t i c a l l y i n a c t i v e at l o w c o n c e n t r a t i o n s of MgCI 2 (2 to 3 raM) w h i c h stimulated RNA p o l y m e r a s e A. W i t h Mn 2+ ions alone, the activity of b o t h enzymes w a s m u c h r e d u c e d and to the same extent. The c o m b i n a t i o n of 5 mM MgG] 2 w i t h v a r y i n g c o n c e n t r a t i o n s of MnC12 stimulated only to a l i m i t e d extent transc r i p t i o n by the two polymerases.
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FIe. 4. - - Effect of enzyme concentration. RNA synthesis was carried out as described under Nleth'ods wit'h calf thymus DN.A (O, Q) (12 l~g) or d(A-T),. ~A, A) (3 ivg)~as template. RNA polymerases A ar A* were added at varying concentration as indicated (©, Zk), RNA polymerase A ; (O, A), RNA polymerase A*.
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w e r e the case the enzyme a c t i v i t y w o u l d be e x p e c t e d to v a r y as a f u n c t i o n of e n z y m e c o n c e n tration. F i g u r e 4 s h o w s the extent of RNA synthesis w i t h the t w o f o r m s of enzymes at different e n z y m e c o n c e n t r a t i o n s . W i t h i n a large range, RNA synthesis w a s in both cases l i n e a r l y dependent on ihe amount of enzyme. No a p p a r e n t c o o p e r a t i v i t y was o b s e r v e d w i t h d(A-T)~ or native DNA as template.
Fie. 5. - - Effect of divalent cations on RNA polgmerases A and A* activity. RNA synthesis was carried out in 0.1 ml standard mixtures described under Methods using calf thymus DNA and either RNA polymerase A (1.0 !~g) or RNA polyme.rase A* (1.2 ~g). Diva,lent cations were added at varying concentrations as indicated in the figure. Left : (A, Jk); MnC12 alone (©, O) 5 mM~ lVfgCla plus varying concentrations of MInC12 ; right (O, @) MgC12 alone. Open symbols, RNA polymerase A ; closed symbols, RNA po~ymerase A.*.
Divalent cations and ionic strength optima. The v a r i o u s forms of I~NA p o l y m e r a s e s h a v e often been d i f f e r e n t i a t e d by t h e i r p r e f e r e n t i a l a c t i v a t i o n by Mg 2+ or Mn 2÷ and by the effect of
salt c o n c e n t r a t i o n s (above 50 mM a m m o n i u m sulfate w i t h Mg 2+ or w i t h Mg 2+ plus Mn2+). Thus about a 15 to 30 fold d i f f e r e n c e in s p e c i f i c acti-
1.3.-
BIOCHIMIE, 1976, 58, n ° 1-2.
Yeast RNA polymerases. vity of the two e n z y m e s w a s observed at 70 mM salt. The effect of salt concentration w a s also investigated w i t h s y n t h e t i c p o l y m e r s as templates. (fig. 6). One can see that, as p r e v i o u s l y noted by others [15], the effect of i o n i c strength d e p e n d s
m a i n l y influence the e n z y m e D N A interaction in the case of bacterial e n z y m e [16] this suggests that RNA p o l y m e r a s e A* c o u l d have s o m e difficulty for DNA b i n d i n g or c h a i n initiation.
Optimum pH and temperature.
1.4.NATIVE
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p H o p t i m u m for the two e n z y m e s is around p H 8 to 8.5. Practically the s a m e ratio of activity b e t w e e n the p o l y m e r a s e s A and A* w a s f o u n d from p H 6.5 to p H 9. Maximal activity w i t h calf t h y m u s DNA w a s obtained at 3,0 °. RNA p o l y m e rase A* w a s c o m p a r a t i v e l y less active than e n z y m e A at l o w e r and higher temperatures (fig. 7).
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The p a r t i c i p a t i o n of the t w o subunits of 48,000 and 37,000 daltons in transcription of native DNA w a s e x p e c t e d to appear in the thermal inactivation curves of the p o l y m e r a s e s . We therefore c o m pared the heat i n a c t i v a t i o n of RNA p o l y m e r a s e s A and A* u s i n g calf t h y m u s DNA to assay their residual activity. The results are s h o w n in figure 8. It is interesting to observe that first the activity of e n z y m e A d r o p p e d r a p i d l y at 53 ° then the rate of inaetivation and the total r e m a i n i n g activity f o l l o w e d those of RNA p o l y m e r a s e A*. In contrast, w i t h d(A-T) n as template the apparent inactivation curves of the t w o e n z y m e s w a s s t r i k i n g l y
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Fro. 6. - - Effect of ammonium sulfate concentration. RNA synthesis was carried out in 0.1 ml standard mixtures, described under l~ethods vri~h either RNA polymerase A (1.0 It~g) or RNA polymerase A* (1.2' t~g). Ammonium sulfate was present at varying concentratior~s as indicated. T~he templates were calf thymus DNA (6 'l~g) or d(A-T), (3 ~g) or d(I-C)=. RNA synthesis is given as nmoles of total polymerized nucleotides as cal.culated from base composition of the templates. (O), R.NA potymerase A ; (O), P~NA polymerase A*.
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Fro. 7. on the template used. W i t h d(A-T) n or d(I-C)n, m,aximal activity w a s again obtained at l o w salt concentrations. Although RNA p o l y m e r a s e s A and A* have the same activity w i t h d(A-T)= at l o w i o n i c strength, a difference is again observed at about 50 mM a n m l o n i u m sulfate. The above results s h o w that the difference in s p e c i f i c activity betw e e n the t w o forms of e n z y m e s can be m u c h enh a n c e d u n d e r c o n d i t i o n s of h i g h i o n i c strength or 1.ow Mg 2+ concentration. As the i o n i c c o n d i t i o n s
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R N A s y n t h e s i s w a s c a r r i e d o u t in t h e s t a n d a r d m i x ture described under Methods with calf thymus DNA a s t e m p l a t e u s i n g 1 ~tg of PLNA p o l y m e r a s e A o r A*. I n c u b a t i o n w a s f o r 20 m i n at v a r i o u s t e m p e r a t u r e s a s i n d i c a t e d 0 e f t ) . I n c u b a t i o n at v a r i o u s pH w a s p e r f o r m e d i n t h e a p p r o p r i a t e 0.07 M Tris-HC1 buffer.
similar. This is in k e e p i n g w i t h the fact that the two m i s s i n g subunits are not required for d(A-T)= transcription s i n c e both p o l y m e r a s e s transcribe this template w i t h the s a m e efficiency. Therefore
76
J. H u e t a n d c o l l .
i n t h i s c a s e th.e i n a c t i v a t i o n of t h e t w o d i s s o c i a b l e p o l y p e p t i d e s w a s n o t e x p e c t e d to i n f l u e n c e t h e overall thermal inactivation curves. -
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1.6. - - M a p p i n g o[ R N A p o l y m e r a s e s A s u b u n i l s . T o k n u w m o r e a b o u t t h e p r o p e r t i e s of t h e t w o d i s s o c i a b l e p o l y p e p t i . d e s of 48,00,0 a n d 37,000 M W w h i c h a r e a b s e n t i n p o l y m e r a s e A*, t h e c o m p l e t e enzyme was dissociated in 6 M urea and the
BIOCHIMIE,
1 9 7 6 , 58, n ° 1 - 2 .
Fro. 9. - - Two d i m e n s i o n a l mapping of RNA polymerase A subunits. RNA p o l y m e r a s e A (2'0 ~,g) was dissociated wi~h 6M u r e a and subjected to electro focussing in 5 p e r cent p o l y a c r y l a m i d e gel c o n t a i n i n g am.~holines. The pH g r a d i e n t a f t e - e,lectrophoresis ranged f r o m pH 4 to 9.5. The gel strip c o n t a i n i n g the focussed s u b u n i t s was subjected to a second d i m e n s i o n electrophoresis w i t h sodium dodecyl sulfate to i d e n t i f y the s u b u n i t s according to t h e i r m o l e c u l a r weight. The details of the m e t h o d s will be described el s'ewhere [14]. The s u b u n i t s are indicated by t h e i r molecular weight [ × 10-3).
cyl su,lfate [14]. T h e r e s u l t s a r e i l l u s t r a t e d i n f i g u r e 9. A f t e r t h e tv¢o d i m e n s i o n a l f r a c t i o n a t i o n t h e s u b u n i t s a p p e a r as s h a r p s p o t s . O n e r e c o g n i s e s s u b u n i t s of M W 40,000, 37,00L0, 29,000, 24,000 a n d 20,0.00. U n d e r t h e s e c o n d i t i o n s s u b u n i t s o f l o w e r m o l e c u l a r w e i g h t r u n o u t o f t h e gel. T h e i s o e l e c -
Yeast R N A polymerases. t r i c p o i n t of the p r o t e i n s can be d e t e r m i n e d by this t e c h n i q u e and will he r e p o r t e d elsewhere. The 37,0.0'0 daltons subunit has an i s o e l e e t r i c p o i n t of 7.2. C o n c e r n i n g the p o l y p e p t i d e chain of 48,0,00 MW it h a p p e n s that this subunit p r e c i p i tates in u r e a t o g e t h e r w i t h the t`wo largest subunits and does not e n t e r the gel. P r e c i p i t a t i o n of the 37,000 MW com,ponent also o c c u r s f r e q u e n t l y . II. - TRAN:SIERIPTION OF SV40 SUPERHELICAL DNA BY YEAST RNA laOL~/MERASE B. It `was p r e v i o u s l y o b s e r v e d that yeast I~NA polym e r a s e B does not bin,d to a D,NA-cellulose c o l u m n p r e p a r e d "with native cal~f t h y m u s DNA. In contrast the enzyme strongly b i n d s to a d e n a t u r e d DNA-cellulose c o l u m n and can be p u r i f i e d by this t e c h n i q u e [17]. P r e v i o u s e x p e r i m e n t s have also s h o w n that yeast R.N,A p o l y m e r a s e s A and B h a v e a lo`w a c t i v i t y `with intact double s t r a n d e d template due to a d e f i c i e n c y in c h a i n i n i t i a t i o n F5]. It w a s p r o p o s e d a m o d e l of t r a n s c r i p t i o n of eukar y o t i c DcNA in w h i c h the u n p a i r e d D~NA structure r e q u i r e d for c h a i n initiation could be i n d u ced by p r o t e i n factors p r e s e n t in c h r o m a t i n , by a lo.cal h a i r p i n s t r u c t u r e in the DNA or by supercoiling [11]. The folio`wing e x p e r i m e n t s w e r e per~ [
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SV$O DNA form I.
Binding reaction was carried out as described under M,ateria'l and M,etl~ods. ~'A), 0,16 ~g of [3H] SV40 DNA (0.0.5 pmole of DNA) was mixed with varying amounts of enzyme as ind,icated in the figure. (B), 1.2 pmole of RNA polymerase B was ]nixed with varying amounts of [3H] SV40 DNA. P~e'sults are expressed as percent of input D~A retained (O), or as pmoles (× 10~) of DNA molecules retained (Q--Q).
77
II.1. - - Bindit~g of RNA polymerase B to SV40
DNA -FI. In the light of the r e c e n t f i n d i n g that all superhelical DNA p r e s e n t some u n p a i r e d or loosely b a s e - p a i r e d regions it was i n t e r e s t i n g to s t u d y the b i n d i n g of RNA p o l y m e r a s e B to this p a r t i c u l a r template. The c o n v e n t i o n a l m e t h o d of m e m b r a n e filtration was used [18] w h i c h takes a d v a n t a g e of the p r o p e r t y of n i t r o c e l l u l o s e m e m b r a n e s to retain free or DNA b o u n d RNA p o l y m e r a s e whereas the DNA alone is not r e t a i n e d . F i g u r e 10A shows that the anmunt of [3HIlabelled SV40 DNA r e t a i n e d on the m e m b r a n e is p r o p o r t i o n a l to the a m o u n t of enzyme a d d e d until 60 p e r cent of the D,NA is r e t a i n e d . The absence of c o o p e r a t i v i t y i n d i c a t e s that a single molecule of e n z y m e can r e t a i n on the filter one m o l e c u l e of DNA (for discussion see Ref. [18]). The non l i n e a r i t y of the c u r v e after 60 p e r cent DNA retention reflects the fact that one m o l e c u l e of DNA can p r o b a b l y bin, d m o r e than one m o l e c u l e of enzyme. F o l l o w i n g H i n k l e and C h a m b e r l i n calculations, 63 p e r cent o.f the DN,A shoul,d be r e t a i n e d at a ratio of one e n z y m e p e r DNA [18]. This h o w ever, in our case, is obtained at a ratio of e n z y m e to DNA equal to 5. F i g u r e 10 B s h o w s that the a m o u n t of DNA r e t a i n e d by a given a m o u n t of e n z y m e is the same w i t h i n a c e r t a i n r a n g e of DNA c o n c e n t r a t i o n w h i c h at least i n d i c a t e s that all possible p o l y m e r a s e molecules w e r e q u a n t i t a t i v e l y b o u n d to the DNA. T h e r e f o r e the above results coul~d be due either to a l o w effi,ciency of the filter in r e t e n t i o n of the b i n a r y complex, o r to the p r e f e r e n t i a l b i n d i n g of the e n z y m e to p r e v i o u s l y f o r m e d enzyme-DNA c o m p l e x , or to the b i n d i n g of the e n z y m e as a d i m e r or oligomer. Actually it is k n o w n that the efficiency of the m e m b r a n e filtration t e c h n i q u e is not absolute and that it is affected by several factors such as t e m p e r a t u r e , i o n i c strength and extent of washing. On the o t h e r h a n d , the m o l e c u l a r w e i g h t of the b o u n d e n z y m e is not kn(~wn since at l o w salt c o n c e n t r a t i o n s the s e d i m e n t a t i o n constant of the m o l e c u l e is h i g h e r than that e x p e c t e d for the m o n o m e r f o r m [4]. Finally, as it is likely that the b i n d i n g o c c u r s on the u n p a i r e d r e g i o n of the s u p e r h e l i c a l DNA, it is not e x c l u d e d that the b i n d i n g of one m o l e c u l e favors the i n s e r t i o n of a d d i t i o n a l m o l e c u l e s of enzyme. ]I.2 - - Stability of the enzyme-DNA complex.
f o r m e d to investigate the ability of yeast RNA p o l y m e r a s e B to bin, d to and initiate t r a n s c r i p t i o n on a c i r c u l a r s u p e r h e l i c a I D,NA, SV40 DNA f o r m I.
BIOCHIMIE, 1976, 58, n ° 1-2.
The stability of the b i n a r y c o m p l e x was investigated in the p r e s e n c e of an excess of a competitor DNA. The e n z y m e w a s first p r e i n c u b a t e d w i t h [3H] SV40 DNA-FI, then i n c u b a t e d for in-
78
J. H u e t a n d coll.
c r e a s i n g p e r i o d of time w i t h d e n a t u r e d calf thymus DNA and finally f i l t e r e d on m e m b r a n e . As s h o w n in figure 11, the b i n a r y c o m p l e x v e r y
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Dissociation of RNA polgmerase e-SV$O DNA-FI complcxcs. RNA polymerase B (1.5 pmole) and [3H~] SV40 DNA (0.8 !lxg) were incubated for 10 min at 37 ° (~) or at 19° (©) in the binding mixture ~0.25 ml) described under M'ethods. Denaturated calf thymus DNA (15 p,g) was then added and incubation was continued at the same temoerature for increasing period of time. A~iquots (0.05 mt)l were withdrawn at d,ifferent times and processed as described under Methods, The results are given as percentage of the DNA retained, on the membrane in absence of coinpetitor (corresponding to 60 per cent of input DNA). Fro. 1 1 . -
RNA p o l y m e r a s e B. I n d e e d the specific a c t i v i t y of the enzyme w i t h this t e m p l a t e was found to be s i m i l a r to that obtained w i t h p u r i f i e d calf t h y m u s DNA ( w h i c h still contains small u n p a i r e d regions [5, 11]. H o w e v e r optimal con, ditions for RNA synthesis are quite d i f f e r e n t f r o m that p r e v i o u s l y det e r m i n e d veith the latter template. F i g u r e 12 s h o w s t h e RNA synthesis at i n c r e a s i n g enzyme c o n c e n t r a t i o n s . T h e a m o u n t of RNA m a d e r e a c h e d up to 80 p e r cent of the amount of SV40 ,DNA template used in the assay. But this w a s o b l a i n e d at an e x t r e m e l y h i g h e n z y m e to D,NA r a t i o of about 600 e n z y m e molecules p e r m o l e c u l e of DNA. C o m p a r e d to calf t h y m u s RNA p o ] y m e r a s e B [7, 8] the yeast e n z y m e t h e r e f o r e a p p e a r s to be less efficient in t r a n s c r i b i n g this type of template. Nevertheless this study i n d i c a t e s that yeast RNA p o l y m e r a s e B can bin:d to and trans.crib.e to some extent a s u p e r c o i l e d c i r c u l a r DNA, w h e r e a s it is unable to t r a n s c r i b e d l i n e a r double s t r a n d e d p h a g e T 7 or T~ DNA. These results s u p p o r t the h y p o t h e s i s that the e n z y m e needs some loosen or u n p a i r e d D~NA s t r u c t u r e to initiate t r a n s c r i p t i o n .
L,01
. . . . .
T h e p r e s e n c e of an a p p a r e n t l y u n p a i r e d region in the DNA was e x p e c t e d to allow t r a n s c r i p t i o n of this double s l r a n d e d c i r c u l a r template by yeast
BIOCHIMIE, 1976, 58, n ° 1-2.
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r a p i d l y d e c a y e d at 37 ° as well as 19 ° . W i t h i n a f e w seconds m o r e than 80 p e r cent of the complexes w e r e dissociated. It thus a p p e a r s that, in contrast ~vith E. colt RNA p o l y m e r a s e w h i c h forms h i g h l y stable c o m p l e x e s w i t h p r o m o t e r sites at 37 °, the b i n d i n g of RNA p o l y m e r a s e B to SV40 DNA is v e r y unstable. On the other hand, Hossenlop et al. [12], using s o n i c a t e d calf t h y m u s DNA as a c o m p e t i t o r , concl,uded that the c o m p l e x e s b e t w e e n calf t h y m u s enzymes A or B and SV40 DNA-FI w e r e r a t h e r stable. This d i s c r e p a n c y could reflect a difference in the DNA b i n d i n g prop e r t i e s of the yeast and calf t h y m u s enzymes or else could be due to the p o o r e r efficiency of sonicated DNA to b i n d RNA p o l y m e r a s e as c o m p a r e d w i t h a d e n a t u r e d template. In this r e s p e c t it should be noted that the o b s e r v e d i n s t a b i l i t y of the enzyme-DNA c o m p l e x e s could b r i n g about a loss of some DNA molecules d u r i n g w a s h i n g of the m e m b r a n e .
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Fro. 12. - - Transcription of SV$O DNA-FI by yeast RNA polymerase B. P~NA synthesis was carried out as described under Methods with 0.16 ~g of SV40 DNA-FI as template and increasing amounts of enzyme. DIS,CUSS'IO,N. E a r l i e r stu~dies h a v e s h o w n that yeast RNA polymerases A and B are v e r y c o m p l e x m u l t i m e r i c p r o t e i n s [4, 5, 17]. As it is unl'ikely that all the p o l y p e p t i d e c h a i n s associated to the e n z y m e are d i r e c t l y i n v o l v e d in the e n z y m a t i c f o r m a t i o n of the p h o s p h o d i e s t e r bond, it is likely that some of the p u t a t i v e subunits are in fact s p e c i f i c i t y det e r m i n a n t s or r e g u l a t o r y factors. It is t h e r e f o r e
Yeast RNA poigmerases. not s u r p r i s i n g that some of these p r o t e i n s are more loosely associated to the enzyme. This appears to he the case for two p o l y p e p t i d e chains of RNA p o l y m e r a s e A of 48,00~0 a n d 37,000 daltons. The evidence that these po'lypeptides are norreally associated to the enzyme have been presented elsewhere [7]. I n this paper, w e r e investigated in more details the p r o p e r t i e s of the two forms of RNA polymerases, A (or complete enzyme) a n d A*. F r o m the results p r e s e n t e d here, the difference in template specificity of the two enzymes is f i r m l y established. The enzymes can also be differentiated on the basis of their ionic strength a n d divalent cation r e q u i r e m e n t s . The p a r t i c u l a r l y low activity of RNA polymerase A" at high salt a n d low Mg 2÷ c o n c e n t r a t i o n s suggest a r a t h e r weak enzyme-DNA i n t e r a c t i o n . The limiring step in t r a n s c r i p t i o n of calf t h y m u s DNA could be the DNA b i n d i n g or i n i t i a t i o n steps. This hypothesis is s u p p o r t e d by the o c c u r r e n c e of a lag phase in t r a n s c r i p t i o n of d(I-(~) n o r (d T) n (although no such lag phase was seen w i t h native DNA). A somewhat lower efficiency of RNA polymerase A* seen at low t e m p e r a t u r e and at low GTP c o n c e n t r a t i o n [193, could also suggest a deficiency in the process of c h a i n initiation. C o n c e r n i n g the p r o p e r t i e s of the two dissociable p o l y p e p t i d e s it is likely that the 48,0.0.0 MW p o l y p e p t i d e is a very basic p r o t e i n for the following reasons : First after removal of the two polypeptides RNA polymerase A* has a higher electrop h o r e t i c m i g r a t i o n rate t h a n enzyme A, w h i c h c a n n o t be e x p l a i n e d simply by the 15 per cent r e d u c t i o n in molecular weight of the enzyme. Secondly, RNA p o l y m e r a s e A* is eluted from phosphocellulose at lower salt c o n c e n t r a t i o n s than enzyme A. T h i r d l y , the two subunits, of 48,0,0.0 a n d 37,000 MW r e m a i n tightly b o u n d to the phosphecellulose a n d are eluted t a n d e m l y at higher salt c o n c e n t r a t i o n t h a n RNA p o l y m e r a s e A (0.4 M a m m o n i u m sulfate). F i n a l l y , s u b u n i t of 48,000 MW was p a r t i a l l y recovered i n the flow t h r o u g h from DEAE cellulose i n the presence of 8 M urea at pH 8.4. S u b u n i t of 37,000 daltons w i t h an isoelectric p o i n t at pH 7.2 is also one of the more basic s u b u n i t of the enzyme. These observations suggest that the two dissociable s u b u n i t s could he i n v o l v e d in template b i n d i n g or even that these p r o t e i n s are normal,ly associated w i t h the c h r o m a t i n . It is not excluded that the two p o l y p e p t i d e s are i n fact associated to each other w h i c h woul, d e x p l a i n w h y they are eluted tandemly from phosphocellulose. The two forms of RNA p o l y m e r a s e A a n d A" are fonctionaHy distinct. How is p o l y m e r a s e A* related to the multiple forms of I~NA polymerases
BIOCHIMIE,
1 9 7 6 , 58, n ° 1-2.
79
isolated from all eu,karyotic ceils ? F r o m what is k n o w n from the molecular structure of n u c l e a r a n i m a l RNA polymerases it is clear that subclasses of the A or B enzyme m a i n l y arise from a change i n m o l e c u l a r weight of the largest subunit. This is the case for example for enzyme Bo, B~ and BII from c a g t h y m u s [3] a n d for enzyme IIo, II~ a n d IIB - - to follow each author's n o m e n clature - - from m u r i n e p l a s m a c y t o m a cells [20]. The case of enzyme BI, a a n d Buh is somewhat different since it appears to i n v o l v e a difference in charge rather t h a n m o l e c u l a r weight i n one single s u b u n i t [3]. The c o n v e r s i o n of RNA polymerase A to A" b y dissociation of two s u b u n i t s c o r r e s p o n d to a different type of enzyme alteration. I n fact, an observation closely related to ours was made by Schwartz a n d Roeder [21] who separated by p o l y a c r y l a m i d e gel electrophoresis two fractions of RNA polymerase A (or I) from p l a s m a c y t o m a cells. One b a n d of p r o t e i n was l a c k i n g one su,bunit of 6.1,0.00 MW and was appar e n t l y inactive. However it was not clearly reported w h i c h template was used i n the assay. It is not excluded that this enzyme might also have a different template r e q u i r e m e n t t h a n the complete enzyme. The b i p h a s i c curve f o u n d as a f u n c t i o n of the i o n i c strength w i t h yeast RNA p o l y m e r a s e A is r e m i n i s c e n t of those o b t a i n e d w i t h RNA polymerase III [2]. Moreover it is s t r i k i n g that yeast RNA polymerase A shows a low hut significant sensitivity to a - a m a n i t i n [7] as do class C (or III) RNA polymerases [1, 2]. RNA p o l y m e r a s e B i n yeast is about 100 fold less sensitive to a - a m a n i t i n t h a n the c o r r e s p o n d i n g a n i m a l enzymes [22] therefore it w o u l d not he s u r p r i s i n g that class C RNA polymerases from yeast require high c o n c e n t r a tions of the toxic peptide for i n h i b i t i o n . W i t h o u t e n t e r i n g the n o m e n c l a t u r e l a b y r i n t h it was interesting to note these similarities b e t w e e n enzyme A" a n d class C polymerases. C o n c e r n i n g the template r e q u i r e m e n t s of RNA p o l y m e r a s e B, the results r e p o r t e d here u s i n g s u p e r h e l i c a l SV4,0 DNA are i n the line of the p r e v i o u s observations [ l l j w h i c h d e m o n s t r a t e d the r e q u i r e m e n t of this enzyme for an u n p a i r e d DNA structure to perfom RN.A c h a i n i n i t i a t i o n . Circular SV40 DNA-EI is t r a n s c r i b e d by yeast RNA p o l y m e r a s e B and, in this respect, this DNA is a better template t h a n a l i n e a r double s t r a n d e d DNA su'ch as T7 DNA. However if one compares the yeast p o l y m e r a s e activity to that of the calf t h y m u s enzyme B the latter appears to he m u c h more efficient w i t h the supercoiled template [10].
J. Huet and coll.
80
Also t h e b i n d i n g of t h e y e a s t e n z y m e to SV40 D N A - F i is v e r y u n s t a b l e as s h o w n b y t h e c o m p e t i tion experiments. This could mean that although s u p e r c o i l i n g i n DNA d o e s i n d e e d h e l p t h e p o l y m e r a s e b i n d and initiate, additional factors are r e q u i r e d to o b t a i n h i g h t r a n s c r i p t i o n e f f i c i e n c y . T h i s c o n c l u s i o n a g a i n l e a d s u s to f a c e t h e c h r o m a t i n p r o b l e m a n d l o o k f o r DNA b i n d i n g p r o t e i n s w h i c h m i g h t i n d u c e t h e p r o p e r DNA config u r a t i o n r e c o g n i z e d b y e u ~ a r y o t i c RNA p o l y merases. R~SUM~. Deux f o r m e s de RNA p o l y m e r a s e A de levure sont s~par~es pa.r c h r o m a t o g r a p h i e sur phosphoceHu],ose. L'une &entre elles, appelde RNA poIym~rase A*, a perdu d~eux sous~un~it~s de 48 000 et 37 000 daltons. L¢s propri~t6s des deux esp~ces enzyinatiques son¢ comp a r s e s dans le prdsent travail. La RNA polymdrase A*, qui tran:serit le d(A-T),, avee la m~me efficacit~ que l'enzyn~e compl+te, est par contre Men moins active avec du D'NA n~tif comme raatrice. La RNA polym~rase A* est particuli+remen,t peu active h h a u t e force ionique et h faible concentration en Mg2+. Les couches d'inactivation t h e r m i q u e des deux en,zymes sont trbs differences suivant que l'activit6 r~sidueHe est mesur~e avec du DNA n a t i f ou du d(A-T), comme matrice. Ces r~sultats, sugg~rent que les deux chaines polypeptidiques dissoci6es de la RNA polym6rase A jouen¢ un r61e i m p o r t a n t dans la transcription. L'~tude de la sp~cificit6 de la RNA polym~rase B a 6t~ poursuivie en utilisan{ du DNA de SV 40, forine I. La R.NA polym.6rase B est capable de r e t e n i r l.e DNA [3Hi de SV 40 s ur filtre de nitrocell,ulose. Cepend a n t le complexe enzyme-DNA est tr+s instable. Le fair que la RNA polym~rase B puisse t r a n s c r i r e du DNA superenroul~ renforee l'hypoth~se que l'enzyme r e q u i e r t des zor~es d~natur~es dans le DNA pour pouvoir se fixer h la matrice et in,itier la transcription.
BIOCHIMIE, 1976, 58, n ° 1-2.
REFERENCES. l. Seif~art, K. H., Benecke, B. J. a Jaxhasz, P. P. (1972) Arch. Biochem. Biophys., 15], 519-5~)2. 2. Schwartz, L. B., S.klar, V. E. F., Jaehning, J. A., W.einmann, R. ~, R~oeder, K. G. (19'74') J. Biol. Chem., 249, 5889-5897. 3. Chamb'on, P. (1,974) in <> (P. D. Boyer ed.), Vo]. X, pp. 2~1-331, Acad. Pres~ (N. Y). 4. Buhler, J. M., S~entenac, A. ~ Fromageot, P. (1974) J. Biol. Chem., 249, 596,3-59q0. 5. Dezdl~e, S. & Sentenac, A. (1972) Eur. J. Biochem., 34, 41-52. 6. B~hl,er, J. M., Dez~16e, S., H.uet, J., Iborra, F. SenCenac, A. (1975) i*~ <>, FEBS proceedings, Vol. 33 (E. J. H,ihv~gi, J. Siimegi ~, P. Ven'etianer, ed's). N o r t h Holland Pub. Co., A m s t e r d a m and Akad6miai Kiado, Budapesi. 7. Ha*et, J., Buhler, J. M., S~ntenac, A. & From.ageot, P. (1975) Proc. Nat. Acad. Sci. U.S.A., 72, 30343038. 3. D ez~16e, S., S entenae, A. ~ FroInageot, P. (197~4) J. Biol. Chem., 249, 5978-5983. 9. Hirt, B. (1'967) J. Mol. Biol., 26, 3,65-369. 10. M,and.el,, J. L. ~ Chambon., P. (19.74) Eur. J. Biochem., 41, 367'-378. 11. Dcz~l~e, S., Sentenae, A. a Fromageot, P. (1974) J. Biol. Chem., 249, 597,1-5977. 12. Hossenlop, P., Oudet, P. ~ Chambon, P. (19,74) Eur. J. Biochem., 41, 397-411. 13. Laemmli, U. K. (1970) Nature, 227, 680-685. 14. Iborra, F. ~ Bulher, J. M. (1976) Anal. B i o c h e m (in press). 15. Cxissinger, F., Kectinger, C. & Chambon, P. (1974) Biochimie, 56, 319-3:83. 16. Mangel, W. F. ~ Chamberlin, M. J. ~1974) J. Biol. Chem., 249, 3,002-3006. 17. Dez61~e, S., Sentenac, A. a Fromageot, P. (1972) FEBS Lett., 21, 1-6. 18. Hink]e, D. C. a Chamber]in~ M. J. (1¢97~) J. Mol. Biol., 70, 15%185. 19. Huet, J. Unpublished result. 20. Sklar, V. E., Schwartz, L. B. a Roeder, R. G. (1975) Proc. Nat. Acad. Sci. U.S.A., 72, 348-352. 21. Schwartz, L. B. & Roeder, R. G. (1974) J. Biol. Chem., 249, 5898~5906. 22. D'ez~l,~e, S., Sentenac, A. a Fromageot, P. (1970) FEBS Letters, 7, 220-2~22.
Further characterization of yeast RNA polymerases. Efl ct of subunits removale). J a n i n e HUET, S y b i l l e DEZ~L~E, F r a n q o i s IBORRA, J e a n - M a r i e BUHLER, A n d r 6 SENTEN&C a n d P i e r r e FIROMAGEOT <~.
Service de Biocbimie, D~partement de Biologic, Centre d'Etudes Nucldaires de Saclay, B.P. n ° 2, 91190 Gif-sur-Yvette, France. Summary. - - Two f o r m s of yeast RNA polymerase A are resolved by phosphoeellulose c h r o m a t o g r a p h y . One of these, called RNA p o l y m e r a s e A*, is lacking two polypeptide c h a i n s of 48,0'0.0 a n d 37,000 daltons. The properties of the two enzymes are compared in t h e p r e s e n t paper. R~NA p o l y m e r a s e A* t r a n s c r i b e s dfA-T), w i t h a s i m i l a r efficiency as the complete enzyme, b u t it is c o m p a r a t i v e l y much less active w i t h native DNA. The two enzymes can also be differen,tiated on t h e basis of t h e i r ionic s t r e n g t h and divalent cation r e q u i r e m e n t s . RNA p o l y m e r a s e A* h a s a p a r t i c u ] a r l y low activity at high salt and low Mg2+ concentrations. T h e r m a l i n a c t i v a t i o n cur~'es of the two enzymes are different when residual activity is assayed w i t h native DNA. I n c o n t r a s t w i t h d(A-T), as t e m pl,~te the a p p a r e n t i n a c t i v a t i o n curves of the t w o enzymes are identical. The data sugges,t t h a t the two dissociable polypcptide chains play an i m p o r t a n t role in transcri,ption. The t e m p l a t e specificity of yeast RNA .polymerase B was f u r t h e r investigated using SV40 DNA-FI as template. RNA polym,erase B is able to ~etain [3H]SV40 DNA-FI on nitrocellulose filters b u t the enzyme-DNA complex is very unstable. The o b s e r v a t i o n t h a t RNA p o l y m e r a s e B can t r a n s c r i b e to some extent a supercoiled DNA b u t not a l i n e a r double s t r a n d e d t e m p l a t e supports the h y p o t h e s i s t h a t the enzyme needs some u n p a i r e d DNA s t r u c t u r e to i n i t i a t e t r a n s c r i p t i o n .
INTRODUCTION. T h e c o m p l e x i t y of g e n e t r a n s c r i p t i o n i n h i g h e r cells is r e f l e c t e d b y t h e s t r u c t u r a l f e a t u r e s of t h e m u l t i p l e f o r m s of R N A p o l y m e r a s e s . T h r e e c l a s s e s of e n z y m e s , w h i c h c a n b e c o n v e n i e n t l y d i s t i n g u i s h e d o n t h e b a s i s of t h e i r t t - a m a n i t i n s e n s i t i v i t y , a p p e a r to b e r e s p o n s i b l e f o r all n u c l e a r R N A s y n t h e s i s It, 2]. Nu,clear R N A p o l y m e r a s e s , p u r i fied fro~m v a r i o u s o r g a n i s m s , a r e c o m p l e x m u l t i m e r l e p r o t e i n s w h i c h c o n s i s t of t w o 1,arge s u b u n i t s i n e q u i m o l a r a m o u n t , a s s o c i a t e d w i t h a s e r i e s of s m a l l e r p o l y p e p t i d e c h a i n s [3]. A n u m b e r of q u e s tion,s a r i s e w h e n c o n s i d e r i n g t h e s e c o m p l e x p r o tein structures. (1) A r e aH t h e p o l y p e p t i d e c o m p o n e n t s associated with the enzymes required for the basic, template-directed polynlerisation reaction ? (2) A l t e r n a t i v e l y , d o e s e a c h of t h e s e p o l y m e r a s e s c o n s i s t of a f u n d a m e n t a l e n z y m e s u r r o u n d e d w i t h regulatory components or specificity determinants having a specialized role in transcription ? (3) A r e t h e m u l t i p l e f o r m s of R N A p o l y m e r a s e A, B an,d C c o m p , l e t e l y g e n e t i c a l l y d i s t i n c t (*) Th~s. p a p e r is dedicated to the m e m o r y of Huguette de R.obi.chon-Szulmajster. To w h o m all eorrespond.ence should he addressed.
proteins or do they share common subunits which could carry a basic function in DNA transcription ? (4) A r e t h e s u b c l a s s e s of t h e s e e n z y m e s d u e to t h e loss o r a l t e r a t i o n of o n e o r m o r e p o l y p e p t i d e c o m p o n e n t s d u r i n g p u r i f i c a t i o n o r a r e -they i n d e e d i m p l i c a t e d i n s o m e r e g u l a t o r y p r o c e s s at the polymerase level ? W e h a v e d e s c r i b e d [4, 5] t h e p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n of R N A p o l y m e r a s e A a n d B f r o m y e a s t cells Saccharomyces cerevisiae. T h e s t r u c t u r a l p r o p e r t i e s of t h e t w o e n z y m e s w e r e i n v e s t i g a t e d to b r i n g s o m e l i g h t o n s.ome of t h e a b o v e q u e s t i o n s . It w a s f o u n d t h a t a l t h o u g h R N A p o l y m e r a s e s A a n d B a p p e a r lo b e d i s t i n c t p r o t e i n s o n t h e b a s i s of a n u m b e r of c r i t e r i a i n c l u d i n g s o m e i m m u n o l o g i c a l a n d s t r u c t u r a l p r o p e r t i e s , t h e s e enz y m e s s h a r e t h r e e c o m m o n s u b u n i t s of l o w m o l e c u l a r w e i g h t [6]. I n t h e c o u r s e of t h i s .~tudy y e a s t R N A p o l y l n e r a s e A w a s f o u n d to b e c o n v e r t e d u n d e r c e r t a i n c o n d i t i o n s to a n e w f o r m of e n z y m e c a l l e d R N A p o l y m e r a s e A* w h i c h is l a c k i n g t w o p o l y p e p t i d e c h a i n s [6, 7]. H e n c e t h e o p p o r t u n i t y w a s g i v e n to b e g i n to a n s ~ - e r -the f i r s t t w o q u e s t i o n s c o n c e r n i n g t h e r o l e of t h e d i f f e r e n t p o l y p e p t i d e c o m p o n e n t s of t h i s e n z y m e . I t is t h e p u r p o s e of t h i s c o m m u n i c a t i o n to d e s c r i b e a d d i -
72
J. H u r t a n d coll.
tional properties of RNA polymerase A" c o m p a r e d to that of the complete enzyme. I n a d d i t i o n , some n e w results are r e p o r t e d on the template specificity of yeast RNA polymerase B.
MATERIAL AND METHODS.
fected 3T6 cells g r o w n i n the p r e s e n c e of [3H]t h y m i d i n e by the methcvd of Hirt [9, 10]. Native calf t h y m u s DNA was p u r i f i e d on nitrocellulose c o l u m n [5~. [i¢Cl-nucleoside triphosphates were o b t a i n e d from N.E.N. Corp. [3H]UTP a n d Ea32,p]GTP was obtained from C.E.N. Saclay. Other materials were as p r e v i o u s l y described.
"YEAST CELLS.
RNA POLYMERASE A ASSAY. S a c c h a r o m y c e s cerevisiae (4094 B a Ad 2 Ur 1) was obtained from Huguette de Robichon-Szulmajster. The cells were g r o w n and collected as p r e v i o u s l y described [51.
RNA POLYMEnASE:S A, A* AN,D B. E n z y m e s A a n d B were p u r i f i e d as p r e v i o u s l y des.cribe~d [4, 51 a n d were homogeneous p r o t e i n s as seen by gel electrophoresis (Fig. 1). RN,A polyInerase A* was o b t a i n e d by phos.phocellulose c h r o m a t o g r a p h y of pure RNA po.lylne.rase A [7].
•
,?.
S t a n d a r d i n c u b a t i o n m i x t u r e (0.1 ml) c o n t a i n e d : 0.07 M Tris-HC,1 (pH 8), 5 InM M.gCl~, 1 mM dithiothreitol, 1 InM each of AT!P, GT.P, a n d CT~, 0.5 mM [3H]UTP (2,0 to 30 corn per pmole), 12 ~g of native calf t h y m u s DNA a n d 1 to 2 ~g of RNA polymerase A or A*. W h e n s y n t h e t i c p o l y m e r s were used as temp'late, only the c o m p l e m e n t a r y nucleoside triphosphates were added w i t h one labelled nucleotide at 0.5 InM as i n d i c a t e d . After a 20 m i n i n c u b a t i o n at 30 °, acid insoluble radioactivity was d e t e r m i n e d Ell]. As,s'AY Fort TRANSCRIPTION OF SV40 D[N.A-FI BY RI~A POLYME.RASEB. The i n c u b a t i o n m i x t u r e (.0.1 Inl) c o n t a i n e d : 0.07 M Tris-HCl (pH 8), 4.5 InM MnC12, 5 mM dithiothreitol, 10 mM a m m o n i u m su'lfate, 0.4 InM each ATP, CTP, UTP a n d 0.2 mM [ct32PIGTP (65 epm per pmole), enzyme a n d DNA as i n d i c a ted. Reaction was initiated by a d d i t i o n of the salt a n d substrate m i x t u r e ; after 5 Inin i n c u b a t i o n at 37 ° to p e r f o r m RNA c h a i n i n i t i a t i o n , the ammon i u m sulfate concentr,ation was i n c r e a s e d to 80 m,M and i n c u b a t i o n was co n'tinued for 25 rain at 37 ° . Fort B I N D I N G [ZH1 SV49 DNA-FI.
ASSAY
FIG. 1. --Polyacrylamide gel electrophoresis of yeast RNA polymerases A and B under non denaturing conditions. Analytic electrop,h.oresis was performed as indicated under M,ethods. Left : RNA polymcrasc B (5 ~g ; phosphocellu.lose fraction [5]) ; right : RNA polymerase A (4 ~g ; glycerol gradient fraction [4]). NUCLEIC ACIDS
OF R N A
POLYME,RASE B
TO
B i n d i n g r e a c t i o n was c o n d u c t e d as f o l l o w s : [3H] S V 4 0 - D N A (95 per cent form I D N A ; 7500,0, cpm per u,g) an,d RNA p o l y m e r a s e B were naixed i n 50 td of b i n d i n g bu,ffer c o n t a i n i n g 0.05 M Tris-HC1 (pH 8), 1 InM dithiothreit(fl, 0.1 mM EDT~A, 3 mM MnCl,, a n d 3,0 per cenl glycerol. After 1,0 m i n at 37 ° the m i x t u r e was diluted with 1 ml of b i n d i n g buffer at 37 °, i m m e d i a t e l y filtered on nitrocellulose m e m b r a n e (Mil]ipore HAWP 02.5) and r a d i o a c t i v i t y r e t a i n e d on the filter was determ i n e d [12].
AND N U C ~ E O T I D E S .
Synthetic p o l y m e r s d(A-T)n, 4(I-C)n, (d T) n were o b t a i n e d as p r e v i o u s l y described [81. Unlabelled SV40 D,NA FI was a gift of Dr. M. Yaniv (Institut Pasteur). [3H] SV40 DNA was p u r i f i e d from in-
BIOCHIMIE, 1976, 58, n ° 1-2.
P O L Y A C R Y L A M I D E , GEL .~ECTROPHORE,SIS.
The electrophoresis of ,I~NA polylnerase u n d e r n o n .denaturing c o n d i t i o n s was p e r f o r m e d as desc r i b e d [4]. P o l y a c r y l a m i d e gel electrophoresis in
73
Yeast RNA polymerases. the p r e s e n c e of s o d i u m d o d e c y l sulfate w a s essenflatly p e r f o r m e d a c c o r d i n g to the p r o c e d u r e desc r i b e d b y L a e m m l i [13]. T h e t w o d i m e n s i o n a l m a p p i n g of s u b u n i t s w a s c a r r i e d out b y electrof o c u s s i n g in 6M urea f o l l o w e d b y s o d i u m d o d e c y l sulfate gel e l e c t r o p h o r e s i s in the s e c o n d d i m e n sion. T h e m e t h o d w i l l be d e s c r i b e d e l s e w h e r e
C o m p a r e d a c t i v i t y of RNA p o l y m e r a s e s A a n d A* w i t h various templates.
1.1. - -
A p r e l i m i n a r y s c r e e n i n g of r e m e n t s of the t w o f o r m s of that the r e m o v a l of the t w o p o n e n t s m a r k e d l y altered the
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F o l l o w i n g p h o s p h o c e l i u l o s e c h r o m a t o g r a p h y of p u r e R N A p o l y m e r a s e A, t w o e n z y m a t i c fractions are o b t a i n e d w h i c h h a v e the s a m e s p e c i f i c a c t i v i t y w i t h d(A-T)n as t e m p l a t e [7]. One fraction is l a c k i n g t w o p o l y p e p t i d e c o m p o n e n t s of 48,000 a n d 37,0.0.0 daltons. T h i s e n z y m e , calle.d R N A p o l y m e r a s e A* has a v e r y r e d u c e d a c t i v i t y on native calf t h y m u s D N A [7]. T h e r e c o v e r y
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FIG. 3. - - Kinetics of RNA synthesis with different templates by RNA polymerases A and A*. Four standard incubation mixtures described under Methods were scaled up to 1 ml. Template was either 100 ~xg of native calf t h y m u s DNA, 31 ~g d(I-C)~, 30 Ixg d(A-T)n or 30 ~g (dT),,. Incubation with RNA potymerase A (7 ;~g) or RNA polymerase A* (10 ug) was carried out at 30 °. At different times 0.1 ml aliquots were withdrawn and processed to estimate acid-insoluble radioactivity as usuaI. (G--C)), RNA polymerase A ; (Q--O)~ RNA polymerase A*.
Fx6. 2. - - Polyacrylamide gel eleclrophoresis with sodium dodecyl sulfate of RATA polymerases A and A* after phosphocellulose chromatography. Glycerol gradient RNA polymerase A was chromatograp'hed on phosphoeeilul, ose as described [7]. Protein fractions were analysed by electrophoresis with sodium dodecyl sulfate. From l,eft to right fraction I : RNA polymerase A* ; fraction 2' : RNA polym,erasc A ; 3 : the two dissociated po]ypeptides.
f r o m p h o s p h o c e l l u l o s e of the t w o e n z y m e s fractions A a n d A* as wel,1 as the t w o d i s s o c i a t e d p o l y p e p t i d e s is illustrated in figure 2.
BIOCHIMIE, 1976, 58, n ° 1-2.
of the e n z y m e ~7]. F i g u r e 3 s h o w s the k i n e t i c s of R N A s y n t h e s i s u s i n g four different t e m p l a t e s w i t h R N A p o l y m e r a s e A a n d A*. W i t h the alternated c o p o . l y m e r d(A-T) n, both e n z y m e s are e q u a l l y active. An e x t e n s i v e s y n t h e s i s of r(A-U) n o c c u r e d w i t h o u t lag p h a s e a n d lasted for m o r e than one hour. On the other h a n d , w i t h native c a l f t h y m u s D N A as t e m p l a t e , R N A p o l y m e r a s e A* w a s about 6 f o l d less active than the c o m p l e t e e n z y m e . R N A p o l y m e r a s e A* w a s also c o m p , a r a t i v e l y less active w i t h d(I-C) n as t e m p l a t e a n d a m a r k e d lag p h a s e in R N A s y n t h e s i s s u g g e s t e d that the e n z y m e h a d s o m e d i f f i c u l t y in i n i t i a t i n g the p o l y m e r i s a t i o n reaction. W i t h (dT) n as t e m p l a t e , the c o m plete e n z y m e w a s also m o r e active than e n z y m e A* a n d a p r o n o u n c e d lag p h a s e w a s s e e n w i t h
J. H u e t a n d coll.
74
this template. These results c l e a r l y i n d i c a t e s that the two m i s s i n g p o l y p e p t i d e s are not r e q u i r e d for the basic p r o c e s s o,f t r a n s c r i p t i o n . On the o t h e r han~d, since the v a r i o u s templates, e x c e p t for d(A-T)n, are t r a n s c r i b e d w i t h a different efficiency, it is likely that the t w o polype,p,tides play an i m p o r t a n t role in transcri,ption. 1.2. - - Effect of enzyme concentration. The lag p h a s e in RNA synthesis could be due to the p r e s e n c e of trace a m o u n t of a catalytic comp o n e n t in RNA p o l y m e r a s e A* p r e p a r a t i o n . If this
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T h e effect of a m m o n i u m sulfate c o n c e n t r a t i o n u p o n p o l y m e r a s e s activities is i l l u s t r a t e d in figure 6. W i t h calf t h y m u s DNA and My2+ both enzymes are m o r e active at l o w salt c o n c e n t r a tions and h a v e o p t i m a l activity at 5 mM ammon i m n sul, fate. In the p r e s e n c e of Mg 2+ plus Mn2+ ions I={NA p o l y m e r a s e A shows b i p h a s i c p r o f i l e w i t h m a x i m a at 5 mM an,d 50 mM a m m o n i u m sulfate. The i n t e r e s t i n g o b s e r v a t i o n was that RNA p o l y m e r a s e A* w a s p r a c t i c a l l y i n a c t i v e at h i g h
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i o n i c s t r e n g t h u p o n e n z y m e activity [3]. It was t h e r e f o r e of interest to c o m p a r e the d i v a l e n t cations and i o n i c strength o p t i m a of the two enzymes u n d e r o t h e r w i s e well d e f i n e d a n d s i m i l a r conditions. F i g u r e 5 s h o w s RNA p o l y m e r a s e s A and A* activities w i t h native DNA as template, in the p r e s e n c e of Mg 2+ or Mn 2+ i n d i v i d u a l l y or w i t h a c o m b i n a t i o n of the t w o d i v a l e n t cations. The same o p t i m a l c o n c e n t r a t i o n of Mg 2+, about 5 mM, a c t i v a t e d RNA p o l y m e r a s e s A and A* but, s u r p r i s i n g l y , e n z y m e A* w a s p r a c t i c a l l y i n a c t i v e at l o w c o n c e n t r a t i o n s of MgCI 2 (2 to 3 raM) w h i c h stimulated RNA p o l y m e r a s e A. W i t h Mn 2+ ions alone, the activity of b o t h enzymes w a s m u c h r e d u c e d and to the same extent. The c o m b i n a t i o n of 5 mM MgG] 2 w i t h v a r y i n g c o n c e n t r a t i o n s of MnC12 stimulated only to a l i m i t e d extent transc r i p t i o n by the two polymerases.
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FIe. 4. - - Effect of enzyme concentration. RNA synthesis was carried out as described under Nleth'ods wit'h calf thymus DN.A (O, Q) (12 l~g) or d(A-T),. ~A, A) (3 ivg)~as template. RNA polymerases A ar A* were added at varying concentration as indicated (©, Zk), RNA polymerase A ; (O, A), RNA polymerase A*.
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w e r e the case the enzyme a c t i v i t y w o u l d be e x p e c t e d to v a r y as a f u n c t i o n of e n z y m e c o n c e n tration. F i g u r e 4 s h o w s the extent of RNA synthesis w i t h the t w o f o r m s of enzymes at different e n z y m e c o n c e n t r a t i o n s . W i t h i n a large range, RNA synthesis w a s in both cases l i n e a r l y dependent on ihe amount of enzyme. No a p p a r e n t c o o p e r a t i v i t y was o b s e r v e d w i t h d(A-T)~ or native DNA as template.
Fie. 5. - - Effect of divalent cations on RNA polgmerases A and A* activity. RNA synthesis was carried out in 0.1 ml standard mixtures described under Methods using calf thymus DNA and either RNA polymerase A (1.0 !~g) or RNA polyme.rase A* (1.2 ~g). Diva,lent cations were added at varying concentrations as indicated in the figure. Left : (A, Jk); MnC12 alone (©, O) 5 mM~ lVfgCla plus varying concentrations of MInC12 ; right (O, @) MgC12 alone. Open symbols, RNA polymerase A ; closed symbols, RNA po~ymerase A.*.
Divalent cations and ionic strength optima. The v a r i o u s forms of I~NA p o l y m e r a s e s h a v e often been d i f f e r e n t i a t e d by t h e i r p r e f e r e n t i a l a c t i v a t i o n by Mg 2+ or Mn 2÷ and by the effect of
salt c o n c e n t r a t i o n s (above 50 mM a m m o n i u m sulfate w i t h Mg 2+ or w i t h Mg 2+ plus Mn2+). Thus about a 15 to 30 fold d i f f e r e n c e in s p e c i f i c acti-
1.3.-
BIOCHIMIE, 1976, 58, n ° 1-2.
Yeast RNA polymerases. vity of the two e n z y m e s w a s observed at 70 mM salt. The effect of salt concentration w a s also investigated w i t h s y n t h e t i c p o l y m e r s as templates. (fig. 6). One can see that, as p r e v i o u s l y noted by others [15], the effect of i o n i c strength d e p e n d s
m a i n l y influence the e n z y m e D N A interaction in the case of bacterial e n z y m e [16] this suggests that RNA p o l y m e r a s e A* c o u l d have s o m e difficulty for DNA b i n d i n g or c h a i n initiation.
Optimum pH and temperature.
1.4.NATIVE
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p H o p t i m u m for the two e n z y m e s is around p H 8 to 8.5. Practically the s a m e ratio of activity b e t w e e n the p o l y m e r a s e s A and A* w a s f o u n d from p H 6.5 to p H 9. Maximal activity w i t h calf t h y m u s DNA w a s obtained at 3,0 °. RNA p o l y m e rase A* w a s c o m p a r a t i v e l y less active than e n z y m e A at l o w e r and higher temperatures (fig. 7).
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The p a r t i c i p a t i o n of the t w o subunits of 48,000 and 37,000 daltons in transcription of native DNA w a s e x p e c t e d to appear in the thermal inactivation curves of the p o l y m e r a s e s . We therefore c o m pared the heat i n a c t i v a t i o n of RNA p o l y m e r a s e s A and A* u s i n g calf t h y m u s DNA to assay their residual activity. The results are s h o w n in figure 8. It is interesting to observe that first the activity of e n z y m e A d r o p p e d r a p i d l y at 53 ° then the rate of inaetivation and the total r e m a i n i n g activity f o l l o w e d those of RNA p o l y m e r a s e A*. In contrast, w i t h d(A-T) n as template the apparent inactivation curves of the t w o e n z y m e s w a s s t r i k i n g l y
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Fro. 6. - - Effect of ammonium sulfate concentration. RNA synthesis was carried out in 0.1 ml standard mixtures, described under l~ethods vri~h either RNA polymerase A (1.0 It~g) or RNA polymerase A* (1.2' t~g). Ammonium sulfate was present at varying concentratior~s as indicated. T~he templates were calf thymus DNA (6 'l~g) or d(A-T), (3 ~g) or d(I-C)=. RNA synthesis is given as nmoles of total polymerized nucleotides as cal.culated from base composition of the templates. (O), R.NA potymerase A ; (O), P~NA polymerase A*.
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Fro. 7. on the template used. W i t h d(A-T) n or d(I-C)n, m,aximal activity w a s again obtained at l o w salt concentrations. Although RNA p o l y m e r a s e s A and A* have the same activity w i t h d(A-T)= at l o w i o n i c strength, a difference is again observed at about 50 mM a n m l o n i u m sulfate. The above results s h o w that the difference in s p e c i f i c activity betw e e n the t w o forms of e n z y m e s can be m u c h enh a n c e d u n d e r c o n d i t i o n s of h i g h i o n i c strength or 1.ow Mg 2+ concentration. As the i o n i c c o n d i t i o n s
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R N A s y n t h e s i s w a s c a r r i e d o u t in t h e s t a n d a r d m i x ture described under Methods with calf thymus DNA a s t e m p l a t e u s i n g 1 ~tg of PLNA p o l y m e r a s e A o r A*. I n c u b a t i o n w a s f o r 20 m i n at v a r i o u s t e m p e r a t u r e s a s i n d i c a t e d 0 e f t ) . I n c u b a t i o n at v a r i o u s pH w a s p e r f o r m e d i n t h e a p p r o p r i a t e 0.07 M Tris-HC1 buffer.
similar. This is in k e e p i n g w i t h the fact that the two m i s s i n g subunits are not required for d(A-T)= transcription s i n c e both p o l y m e r a s e s transcribe this template w i t h the s a m e efficiency. Therefore
76
J. H u e t a n d c o l l .
i n t h i s c a s e th.e i n a c t i v a t i o n of t h e t w o d i s s o c i a b l e p o l y p e p t i d e s w a s n o t e x p e c t e d to i n f l u e n c e t h e overall thermal inactivation curves. -
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1.6. - - M a p p i n g o[ R N A p o l y m e r a s e s A s u b u n i l s . T o k n u w m o r e a b o u t t h e p r o p e r t i e s of t h e t w o d i s s o c i a b l e p o l y p e p t i . d e s of 48,00,0 a n d 37,000 M W w h i c h a r e a b s e n t i n p o l y m e r a s e A*, t h e c o m p l e t e enzyme was dissociated in 6 M urea and the
BIOCHIMIE,
1 9 7 6 , 58, n ° 1 - 2 .
Fro. 9. - - Two d i m e n s i o n a l mapping of RNA polymerase A subunits. RNA p o l y m e r a s e A (2'0 ~,g) was dissociated wi~h 6M u r e a and subjected to electro focussing in 5 p e r cent p o l y a c r y l a m i d e gel c o n t a i n i n g am.~holines. The pH g r a d i e n t a f t e - e,lectrophoresis ranged f r o m pH 4 to 9.5. The gel strip c o n t a i n i n g the focussed s u b u n i t s was subjected to a second d i m e n s i o n electrophoresis w i t h sodium dodecyl sulfate to i d e n t i f y the s u b u n i t s according to t h e i r m o l e c u l a r weight. The details of the m e t h o d s will be described el s'ewhere [14]. The s u b u n i t s are indicated by t h e i r molecular weight [ × 10-3).
cyl su,lfate [14]. T h e r e s u l t s a r e i l l u s t r a t e d i n f i g u r e 9. A f t e r t h e tv¢o d i m e n s i o n a l f r a c t i o n a t i o n t h e s u b u n i t s a p p e a r as s h a r p s p o t s . O n e r e c o g n i s e s s u b u n i t s of M W 40,000, 37,00L0, 29,000, 24,000 a n d 20,0.00. U n d e r t h e s e c o n d i t i o n s s u b u n i t s o f l o w e r m o l e c u l a r w e i g h t r u n o u t o f t h e gel. T h e i s o e l e c -
Yeast R N A polymerases. t r i c p o i n t of the p r o t e i n s can be d e t e r m i n e d by this t e c h n i q u e and will he r e p o r t e d elsewhere. The 37,0.0'0 daltons subunit has an i s o e l e e t r i c p o i n t of 7.2. C o n c e r n i n g the p o l y p e p t i d e chain of 48,0,00 MW it h a p p e n s that this subunit p r e c i p i tates in u r e a t o g e t h e r w i t h the t`wo largest subunits and does not e n t e r the gel. P r e c i p i t a t i o n of the 37,000 MW com,ponent also o c c u r s f r e q u e n t l y . II. - TRAN:SIERIPTION OF SV40 SUPERHELICAL DNA BY YEAST RNA laOL~/MERASE B. It `was p r e v i o u s l y o b s e r v e d that yeast I~NA polym e r a s e B does not bin,d to a D,NA-cellulose c o l u m n p r e p a r e d "with native cal~f t h y m u s DNA. In contrast the enzyme strongly b i n d s to a d e n a t u r e d DNA-cellulose c o l u m n and can be p u r i f i e d by this t e c h n i q u e [17]. P r e v i o u s e x p e r i m e n t s have also s h o w n that yeast R.N,A p o l y m e r a s e s A and B h a v e a lo`w a c t i v i t y `with intact double s t r a n d e d template due to a d e f i c i e n c y in c h a i n i n i t i a t i o n F5]. It w a s p r o p o s e d a m o d e l of t r a n s c r i p t i o n of eukar y o t i c DcNA in w h i c h the u n p a i r e d D~NA structure r e q u i r e d for c h a i n initiation could be i n d u ced by p r o t e i n factors p r e s e n t in c h r o m a t i n , by a lo.cal h a i r p i n s t r u c t u r e in the DNA or by supercoiling [11]. The folio`wing e x p e r i m e n t s w e r e per~ [
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SV$O DNA form I.
Binding reaction was carried out as described under M,ateria'l and M,etl~ods. ~'A), 0,16 ~g of [3H] SV40 DNA (0.0.5 pmole of DNA) was mixed with varying amounts of enzyme as ind,icated in the figure. (B), 1.2 pmole of RNA polymerase B was ]nixed with varying amounts of [3H] SV40 DNA. P~e'sults are expressed as percent of input D~A retained (O), or as pmoles (× 10~) of DNA molecules retained (Q--Q).
77
II.1. - - Bindit~g of RNA polymerase B to SV40
DNA -FI. In the light of the r e c e n t f i n d i n g that all superhelical DNA p r e s e n t some u n p a i r e d or loosely b a s e - p a i r e d regions it was i n t e r e s t i n g to s t u d y the b i n d i n g of RNA p o l y m e r a s e B to this p a r t i c u l a r template. The c o n v e n t i o n a l m e t h o d of m e m b r a n e filtration was used [18] w h i c h takes a d v a n t a g e of the p r o p e r t y of n i t r o c e l l u l o s e m e m b r a n e s to retain free or DNA b o u n d RNA p o l y m e r a s e whereas the DNA alone is not r e t a i n e d . F i g u r e 10A shows that the anmunt of [3HIlabelled SV40 DNA r e t a i n e d on the m e m b r a n e is p r o p o r t i o n a l to the a m o u n t of enzyme a d d e d until 60 p e r cent of the D,NA is r e t a i n e d . The absence of c o o p e r a t i v i t y i n d i c a t e s that a single molecule of e n z y m e can r e t a i n on the filter one m o l e c u l e of DNA (for discussion see Ref. [18]). The non l i n e a r i t y of the c u r v e after 60 p e r cent DNA retention reflects the fact that one m o l e c u l e of DNA can p r o b a b l y bin, d m o r e than one m o l e c u l e of enzyme. F o l l o w i n g H i n k l e and C h a m b e r l i n calculations, 63 p e r cent o.f the DN,A shoul,d be r e t a i n e d at a ratio of one e n z y m e p e r DNA [18]. This h o w ever, in our case, is obtained at a ratio of e n z y m e to DNA equal to 5. F i g u r e 10 B s h o w s that the a m o u n t of DNA r e t a i n e d by a given a m o u n t of e n z y m e is the same w i t h i n a c e r t a i n r a n g e of DNA c o n c e n t r a t i o n w h i c h at least i n d i c a t e s that all possible p o l y m e r a s e molecules w e r e q u a n t i t a t i v e l y b o u n d to the DNA. T h e r e f o r e the above results coul~d be due either to a l o w effi,ciency of the filter in r e t e n t i o n of the b i n a r y complex, o r to the p r e f e r e n t i a l b i n d i n g of the e n z y m e to p r e v i o u s l y f o r m e d enzyme-DNA c o m p l e x , or to the b i n d i n g of the e n z y m e as a d i m e r or oligomer. Actually it is k n o w n that the efficiency of the m e m b r a n e filtration t e c h n i q u e is not absolute and that it is affected by several factors such as t e m p e r a t u r e , i o n i c strength and extent of washing. On the o t h e r h a n d , the m o l e c u l a r w e i g h t of the b o u n d e n z y m e is not kn(~wn since at l o w salt c o n c e n t r a t i o n s the s e d i m e n t a t i o n constant of the m o l e c u l e is h i g h e r than that e x p e c t e d for the m o n o m e r f o r m [4]. Finally, as it is likely that the b i n d i n g o c c u r s on the u n p a i r e d r e g i o n of the s u p e r h e l i c a l DNA, it is not e x c l u d e d that the b i n d i n g of one m o l e c u l e favors the i n s e r t i o n of a d d i t i o n a l m o l e c u l e s of enzyme. ]I.2 - - Stability of the enzyme-DNA complex.
f o r m e d to investigate the ability of yeast RNA p o l y m e r a s e B to bin, d to and initiate t r a n s c r i p t i o n on a c i r c u l a r s u p e r h e l i c a I D,NA, SV40 DNA f o r m I.
BIOCHIMIE, 1976, 58, n ° 1-2.
The stability of the b i n a r y c o m p l e x was investigated in the p r e s e n c e of an excess of a competitor DNA. The e n z y m e w a s first p r e i n c u b a t e d w i t h [3H] SV40 DNA-FI, then i n c u b a t e d for in-
78
J. H u e t a n d coll.
c r e a s i n g p e r i o d of time w i t h d e n a t u r e d calf thymus DNA and finally f i l t e r e d on m e m b r a n e . As s h o w n in figure 11, the b i n a r y c o m p l e x v e r y
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Dissociation of RNA polgmerase e-SV$O DNA-FI complcxcs. RNA polymerase B (1.5 pmole) and [3H~] SV40 DNA (0.8 !lxg) were incubated for 10 min at 37 ° (~) or at 19° (©) in the binding mixture ~0.25 ml) described under M'ethods. Denaturated calf thymus DNA (15 p,g) was then added and incubation was continued at the same temoerature for increasing period of time. A~iquots (0.05 mt)l were withdrawn at d,ifferent times and processed as described under Methods, The results are given as percentage of the DNA retained, on the membrane in absence of coinpetitor (corresponding to 60 per cent of input DNA). Fro. 1 1 . -
RNA p o l y m e r a s e B. I n d e e d the specific a c t i v i t y of the enzyme w i t h this t e m p l a t e was found to be s i m i l a r to that obtained w i t h p u r i f i e d calf t h y m u s DNA ( w h i c h still contains small u n p a i r e d regions [5, 11]. H o w e v e r optimal con, ditions for RNA synthesis are quite d i f f e r e n t f r o m that p r e v i o u s l y det e r m i n e d veith the latter template. F i g u r e 12 s h o w s t h e RNA synthesis at i n c r e a s i n g enzyme c o n c e n t r a t i o n s . T h e a m o u n t of RNA m a d e r e a c h e d up to 80 p e r cent of the amount of SV40 ,DNA template used in the assay. But this w a s o b l a i n e d at an e x t r e m e l y h i g h e n z y m e to D,NA r a t i o of about 600 e n z y m e molecules p e r m o l e c u l e of DNA. C o m p a r e d to calf t h y m u s RNA p o ] y m e r a s e B [7, 8] the yeast e n z y m e t h e r e f o r e a p p e a r s to be less efficient in t r a n s c r i b i n g this type of template. Nevertheless this study i n d i c a t e s that yeast RNA p o l y m e r a s e B can bin:d to and trans.crib.e to some extent a s u p e r c o i l e d c i r c u l a r DNA, w h e r e a s it is unable to t r a n s c r i b e d l i n e a r double s t r a n d e d p h a g e T 7 or T~ DNA. These results s u p p o r t the h y p o t h e s i s that the e n z y m e needs some loosen or u n p a i r e d D~NA s t r u c t u r e to initiate t r a n s c r i p t i o n .
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T h e p r e s e n c e of an a p p a r e n t l y u n p a i r e d region in the DNA was e x p e c t e d to allow t r a n s c r i p t i o n of this double s l r a n d e d c i r c u l a r template by yeast
BIOCHIMIE, 1976, 58, n ° 1-2.
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r a p i d l y d e c a y e d at 37 ° as well as 19 ° . W i t h i n a f e w seconds m o r e than 80 p e r cent of the complexes w e r e dissociated. It thus a p p e a r s that, in contrast ~vith E. colt RNA p o l y m e r a s e w h i c h forms h i g h l y stable c o m p l e x e s w i t h p r o m o t e r sites at 37 °, the b i n d i n g of RNA p o l y m e r a s e B to SV40 DNA is v e r y unstable. On the other hand, Hossenlop et al. [12], using s o n i c a t e d calf t h y m u s DNA as a c o m p e t i t o r , concl,uded that the c o m p l e x e s b e t w e e n calf t h y m u s enzymes A or B and SV40 DNA-FI w e r e r a t h e r stable. This d i s c r e p a n c y could reflect a difference in the DNA b i n d i n g prop e r t i e s of the yeast and calf t h y m u s enzymes or else could be due to the p o o r e r efficiency of sonicated DNA to b i n d RNA p o l y m e r a s e as c o m p a r e d w i t h a d e n a t u r e d template. In this r e s p e c t it should be noted that the o b s e r v e d i n s t a b i l i t y of the enzyme-DNA c o m p l e x e s could b r i n g about a loss of some DNA molecules d u r i n g w a s h i n g of the m e m b r a n e .
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Fro. 12. - - Transcription of SV$O DNA-FI by yeast RNA polymerase B. P~NA synthesis was carried out as described under Methods with 0.16 ~g of SV40 DNA-FI as template and increasing amounts of enzyme. DIS,CUSS'IO,N. E a r l i e r stu~dies h a v e s h o w n that yeast RNA polymerases A and B are v e r y c o m p l e x m u l t i m e r i c p r o t e i n s [4, 5, 17]. As it is unl'ikely that all the p o l y p e p t i d e c h a i n s associated to the e n z y m e are d i r e c t l y i n v o l v e d in the e n z y m a t i c f o r m a t i o n of the p h o s p h o d i e s t e r bond, it is likely that some of the p u t a t i v e subunits are in fact s p e c i f i c i t y det e r m i n a n t s or r e g u l a t o r y factors. It is t h e r e f o r e
Yeast RNA poigmerases. not s u r p r i s i n g that some of these p r o t e i n s are more loosely associated to the enzyme. This appears to he the case for two p o l y p e p t i d e chains of RNA p o l y m e r a s e A of 48,00~0 a n d 37,000 daltons. The evidence that these po'lypeptides are norreally associated to the enzyme have been presented elsewhere [7]. I n this paper, w e r e investigated in more details the p r o p e r t i e s of the two forms of RNA polymerases, A (or complete enzyme) a n d A*. F r o m the results p r e s e n t e d here, the difference in template specificity of the two enzymes is f i r m l y established. The enzymes can also be differentiated on the basis of their ionic strength a n d divalent cation r e q u i r e m e n t s . The p a r t i c u l a r l y low activity of RNA polymerase A" at high salt a n d low Mg 2÷ c o n c e n t r a t i o n s suggest a r a t h e r weak enzyme-DNA i n t e r a c t i o n . The limiring step in t r a n s c r i p t i o n of calf t h y m u s DNA could be the DNA b i n d i n g or i n i t i a t i o n steps. This hypothesis is s u p p o r t e d by the o c c u r r e n c e of a lag phase in t r a n s c r i p t i o n of d(I-(~) n o r (d T) n (although no such lag phase was seen w i t h native DNA). A somewhat lower efficiency of RNA polymerase A* seen at low t e m p e r a t u r e and at low GTP c o n c e n t r a t i o n [193, could also suggest a deficiency in the process of c h a i n initiation. C o n c e r n i n g the p r o p e r t i e s of the two dissociable p o l y p e p t i d e s it is likely that the 48,0.0.0 MW p o l y p e p t i d e is a very basic p r o t e i n for the following reasons : First after removal of the two polypeptides RNA polymerase A* has a higher electrop h o r e t i c m i g r a t i o n rate t h a n enzyme A, w h i c h c a n n o t be e x p l a i n e d simply by the 15 per cent r e d u c t i o n in molecular weight of the enzyme. Secondly, RNA p o l y m e r a s e A* is eluted from phosphocellulose at lower salt c o n c e n t r a t i o n s than enzyme A. T h i r d l y , the two subunits, of 48,0,0.0 a n d 37,000 MW r e m a i n tightly b o u n d to the phosphecellulose a n d are eluted t a n d e m l y at higher salt c o n c e n t r a t i o n t h a n RNA p o l y m e r a s e A (0.4 M a m m o n i u m sulfate). F i n a l l y , s u b u n i t of 48,000 MW was p a r t i a l l y recovered i n the flow t h r o u g h from DEAE cellulose i n the presence of 8 M urea at pH 8.4. S u b u n i t of 37,000 daltons w i t h an isoelectric p o i n t at pH 7.2 is also one of the more basic s u b u n i t of the enzyme. These observations suggest that the two dissociable s u b u n i t s could he i n v o l v e d in template b i n d i n g or even that these p r o t e i n s are normal,ly associated w i t h the c h r o m a t i n . It is not excluded that the two p o l y p e p t i d e s are i n fact associated to each other w h i c h woul, d e x p l a i n w h y they are eluted tandemly from phosphocellulose. The two forms of RNA p o l y m e r a s e A a n d A" are fonctionaHy distinct. How is p o l y m e r a s e A* related to the multiple forms of I~NA polymerases
BIOCHIMIE,
1 9 7 6 , 58, n ° 1-2.
79
isolated from all eu,karyotic ceils ? F r o m what is k n o w n from the molecular structure of n u c l e a r a n i m a l RNA polymerases it is clear that subclasses of the A or B enzyme m a i n l y arise from a change i n m o l e c u l a r weight of the largest subunit. This is the case for example for enzyme Bo, B~ and BII from c a g t h y m u s [3] a n d for enzyme IIo, II~ a n d IIB - - to follow each author's n o m e n clature - - from m u r i n e p l a s m a c y t o m a cells [20]. The case of enzyme BI, a a n d Buh is somewhat different since it appears to i n v o l v e a difference in charge rather t h a n m o l e c u l a r weight i n one single s u b u n i t [3]. The c o n v e r s i o n of RNA polymerase A to A" b y dissociation of two s u b u n i t s c o r r e s p o n d to a different type of enzyme alteration. I n fact, an observation closely related to ours was made by Schwartz a n d Roeder [21] who separated by p o l y a c r y l a m i d e gel electrophoresis two fractions of RNA polymerase A (or I) from p l a s m a c y t o m a cells. One b a n d of p r o t e i n was l a c k i n g one su,bunit of 6.1,0.00 MW and was appar e n t l y inactive. However it was not clearly reported w h i c h template was used i n the assay. It is not excluded that this enzyme might also have a different template r e q u i r e m e n t t h a n the complete enzyme. The b i p h a s i c curve f o u n d as a f u n c t i o n of the i o n i c strength w i t h yeast RNA p o l y m e r a s e A is r e m i n i s c e n t of those o b t a i n e d w i t h RNA polymerase III [2]. Moreover it is s t r i k i n g that yeast RNA polymerase A shows a low hut significant sensitivity to a - a m a n i t i n [7] as do class C (or III) RNA polymerases [1, 2]. RNA p o l y m e r a s e B i n yeast is about 100 fold less sensitive to a - a m a n i t i n t h a n the c o r r e s p o n d i n g a n i m a l enzymes [22] therefore it w o u l d not he s u r p r i s i n g that class C RNA polymerases from yeast require high c o n c e n t r a tions of the toxic peptide for i n h i b i t i o n . W i t h o u t e n t e r i n g the n o m e n c l a t u r e l a b y r i n t h it was interesting to note these similarities b e t w e e n enzyme A" a n d class C polymerases. C o n c e r n i n g the template r e q u i r e m e n t s of RNA p o l y m e r a s e B, the results r e p o r t e d here u s i n g s u p e r h e l i c a l SV4,0 DNA are i n the line of the p r e v i o u s observations [ l l j w h i c h d e m o n s t r a t e d the r e q u i r e m e n t of this enzyme for an u n p a i r e d DNA structure to perfom RN.A c h a i n i n i t i a t i o n . Circular SV40 DNA-EI is t r a n s c r i b e d by yeast RNA p o l y m e r a s e B and, in this respect, this DNA is a better template t h a n a l i n e a r double s t r a n d e d DNA su'ch as T7 DNA. However if one compares the yeast p o l y m e r a s e activity to that of the calf t h y m u s enzyme B the latter appears to he m u c h more efficient w i t h the supercoiled template [10].
J. Huet and coll.
80
Also t h e b i n d i n g of t h e y e a s t e n z y m e to SV40 D N A - F i is v e r y u n s t a b l e as s h o w n b y t h e c o m p e t i tion experiments. This could mean that although s u p e r c o i l i n g i n DNA d o e s i n d e e d h e l p t h e p o l y m e r a s e b i n d and initiate, additional factors are r e q u i r e d to o b t a i n h i g h t r a n s c r i p t i o n e f f i c i e n c y . T h i s c o n c l u s i o n a g a i n l e a d s u s to f a c e t h e c h r o m a t i n p r o b l e m a n d l o o k f o r DNA b i n d i n g p r o t e i n s w h i c h m i g h t i n d u c e t h e p r o p e r DNA config u r a t i o n r e c o g n i z e d b y e u ~ a r y o t i c RNA p o l y merases. R~SUM~. Deux f o r m e s de RNA p o l y m e r a s e A de levure sont s~par~es pa.r c h r o m a t o g r a p h i e sur phosphoceHu],ose. L'une &entre elles, appelde RNA poIym~rase A*, a perdu d~eux sous~un~it~s de 48 000 et 37 000 daltons. L¢s propri~t6s des deux esp~ces enzyinatiques son¢ comp a r s e s dans le prdsent travail. La RNA polymdrase A*, qui tran:serit le d(A-T),, avee la m~me efficacit~ que l'enzyn~e compl+te, est par contre Men moins active avec du D'NA n~tif comme raatrice. La RNA polym~rase A* est particuli+remen,t peu active h h a u t e force ionique et h faible concentration en Mg2+. Les couches d'inactivation t h e r m i q u e des deux en,zymes sont trbs differences suivant que l'activit6 r~sidueHe est mesur~e avec du DNA n a t i f ou du d(A-T), comme matrice. Ces r~sultats, sugg~rent que les deux chaines polypeptidiques dissoci6es de la RNA polym6rase A jouen¢ un r61e i m p o r t a n t dans la transcription. L'~tude de la sp~cificit6 de la RNA polym~rase B a 6t~ poursuivie en utilisan{ du DNA de SV 40, forine I. La R.NA polym.6rase B est capable de r e t e n i r l.e DNA [3Hi de SV 40 s ur filtre de nitrocell,ulose. Cepend a n t le complexe enzyme-DNA est tr+s instable. Le fair que la RNA polym~rase B puisse t r a n s c r i r e du DNA superenroul~ renforee l'hypoth~se que l'enzyme r e q u i e r t des zor~es d~natur~es dans le DNA pour pouvoir se fixer h la matrice et in,itier la transcription.
BIOCHIMIE, 1976, 58, n ° 1-2.
REFERENCES. l. Seif~art, K. H., Benecke, B. J. a Jaxhasz, P. P. (1972) Arch. Biochem. Biophys., 15], 519-5~)2. 2. Schwartz, L. B., S.klar, V. E. F., Jaehning, J. A., W.einmann, R. ~, R~oeder, K. G. (19'74') J. Biol. Chem., 249, 5889-5897. 3. Chamb'on, P. (1,974) in <