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On thermal sensitivities of KB cell DNA-dependent RNA polymerases
BIOCHIMIE, 1974, 56, 1293-1296.
On thermal sensitivities of KB cell DNA-dependent RNA polymerases. A. SERGEANT (*) a n d V. KRSMANOVIC (" "). (*) Laboraioire de Chimie Biologique, Universitd des Sciences et Techniques de Lille I, 59650 Villeneuve d'Ascq, France, (**) and Unitd de Recherche de Virologic, U 102 de I'INSERM, 2, Place de Verdun, 59045 Lille Cedex, France.
(18-9-197/D.
INTRODUCTION. E u k a r y o t i c cell nuclei c o n t a i n m u l t i p l e DNA-depend e n t RNA p o l y m e r a s e s [1, 2] o r i g i n a l l y d e s i g n a t e d by Roeder a n d R u t t e r as e n z y m e s 2, II a n d III [1] c o r r e s p o n d i n g to f o r m s AI / A I I , BI/BII a n d AIII, respectively [31. The f o r m I is p r e s e n t in nucleoli, w h e r e a s the f o r m s II a n d III are in n u c l e o p l a s m [4] ; these e n z y m e s s y n t h e s i z e r i b o s o m a l RNA [5, 6, 7], n u c l e a r h e t e r o g e n o u s RNA [7] a n d 4 S/5 S species [8], respectively. RNA p o l y m e r a s e II is s e n s i t i v e to (~-amanitine, while RNA p o l y m e r a s e s I a n d III are i n s e n s i tive to t h i s d r u g at low c o n c e n t r a t i o n s , a l t h o u g h the h i g h c o n c e n t r a t i o n s of the t o x i n i n h i b i t RNA p o l y m e r a s e III [81. Each f o r m is different f r o m t h e o t h e r s w i t h respect to o p t i m a l c o n c e n t r a t i o n of a m m o n i u m sulfate, Mg2+ a n d Mn~+ [1, 0]. R e c e n t l y Shields a n d T a t a [10] h a v e s h o w n t h a t RNA p o l y m e r a s e I f r o m r a t liver nuclei is m o r e affected by t h e r m a l shock t h a n is RNA p o l y m e r a s e II (and III [11]). The differential h e a t s e n s i t i v i t y of RNA p o l y m e r a s e s could be r e l a t e d to t h e i r i n t r i n s i c properties, since the p r e h e a t i n g of t h e r a t liver nuclei also i n h i b i t s m u c h m o r e RNA p o l y m e r a s e I [10]. T h e m e a n i n g of t h e r m a l i n a c t i v a t i o n in the case of complex e n z y m e s s u c h as eulearyotic p o l y m e r a s e s , is not easily u n d e r s t a n d a b l e . Nevertheless, t h e i r differ e n t i a l t h e r m a l s e n s i t i v i t i e s offer a n a d d i t i o n a l possibility to d i s c r i m i n a t e each f o r m f r o m t h e others. D u r i n g our s t u d y on t h e r m a l s e n s i t i v i t i e s of KB cell RNA p o l y m e r a s e s we h a v e observed t h a t e n z y m e II is less i n a c t i v a t e d by t h e h e a t t h a n t h e e n z y m e s I a n d III are, w h i c h is c o m p a t i b l e w i t h t h e r e s u l t s for r a t liver e n z y m e s [10, 11]. F u r t h e r m o r e , we s h o w t h a t relative h e a t s e n s i t i v i t y of e n z y m e I is different w h e t h e r n a t i v e or d e n a t u r e d KB DNA is u s e d for a s s e s s m e n t of RNA p o l y m e r a s e a c t i v i t y after h e a t i n g , s u g g e s t i n g existence of a d d i t i o n a l p r o t e i n / s involved in d o u b l e - s t r a n d e d DNA t r a n s c r i p t i o n . In a d d i t i o n , we h a v e observed t h a t t h e relative h e a t s e n s i t i v i t i e s of RNA p o l y m e r a s e s u n d e r g o a d r a m a t i c c h a n g e dep e n d i n g on storage t e m p e r a t u r e . As will be s h o w n in t h i s p a p e r the RNA p o l y m e r a s e s II a n d lII e x h i b i t a h i g h e r h e a t s e n s i t i v i t y t h a n RNA p o l y m e r a s e I if stored at - - 7 0 ° C or - - 2 0 ° C i n s t e a d of --196°C, xvhieh e x p l a i n o u r p r e v i o u s r e s u l t s o b t a i n e d ~,ith e n z y m e s stored at - - 7 0 ° C for several m o n t h s [12]. MATERIALS AND METHODS. RNA p o l y m e r a s e s were e x t r a c t e d f r o m KB cells nuclei, e s s e n t i a l l y by the m e t h o d of Roeder a n d R u t t e r A'bbreviations : TGMED : Tris, glycerol, MgCI~. EDTA, d i t h i o t h r e i t o l . (**) To w h o m all c o r r e s p o n d a n e e s h o u l d be addressed.
[4], as p r e v i o u s l y described [12], t h e n t h e e n z y m i c e x t r a c t ~¢¢as applied to a DEAE-Sephadex A-25 c o l u m n a n d eluted ~vith a l i n e a r g r a d i e n t of 0.04-0.5 M (NH4)~ SO4 in TGMED (*) buffer (0.05 M Tris-HCi pH 8,25 p. cent v / v glycerol, 5 mM MgCl2 0.1 mM EDTA, 0,5 mM d i t h i o t h r e i t o l ) , as s h o w n in figure 1. U n l e s s specified, isolated e n z y m e s are stored in l i q u i d n i t r o g e n (--196°C) i n e l u t i n g TGMED buffer c o n t a i n i n g 1 m g / m l s e r u m a l b u m i n . P r e - h e a t i n g of RNA p o l y m e r a s e s w a s p e r f o r m e d for 10 m i n at i n d i c a t e d t e m p e r a t u r e s . E n z y m e activity ,~'as a s s a y e d , at o p t i m a l s a l t concent r a t i o n s , as described i n the legends, a - a m a n i t i n w a s u s e d at 0.8 !~g/ml. After i n c u b a t i o n for 30 m i n at 37°C t h e r e a c t i o n s w e r e p r e c i p i t a t e d as described elsewere [12]. DNA ~vas s o l u b i l i s e d f r o m KB cells by S a r k o s y l P r o n a s e t r e a t m e n L isolated by CsC1 e q u i l i b r i u m g r a d i e n t c e n t r i f u g a t i o n a n d stored at 4°C in 0,1 × SSC (0.015 NaCl, 0.0015 M s o d i u m citrate) b y m e t h o d s s i m i l a r to t h o s e p r e v i o u s l y described [13].
RESULTS AND DISCUSSION. KB cell RNA p o l y m e r a s e s I, II a n d III isolated by DEAE-Sephadex c o l u m n c h r o m a t o g r a p h y (fig. 1) e x h i b i t differences in t h e degree of s e n s i t i v i t y to heat, as s h o w n on figure 2. The e n z y m e II is less affected by t h e r m a l shock t h a n are t h e e n z y m e s I a n d III w h i c h is c o n s i s t e n t w i t h p r e v i o u s r e s u l t s for r a t liver e n z y m e s I a n d II [10]. The h i g h e r overall h e a t stability, in o u r conditions, is p r e s u m a b l y due to the protecting effect of s e r u m a l b u m i n (added to p r e v e n t e n z y m e i n a c t i v a t i o n d u r i n g f r a e t i o n a t i o n a n d storage) since t h e p r e - h e a t e d r a t liver nuclei s h o w a s i m i l a r r e s u l t [10]. F u r t h e r m o r e , t h e h e a t i n a c t i v a t i o n curve of KB RNA p o l y m e r a s e I a c t i v i t y i n t h e presence of n a t i v e KB cell DNA a p p e a r s to be biphasic. The early h e a t inactiv a t i o n b e t w e e n 37°C a n d 43°C for e n z y m e I could be related to h e a t effect on specific p r o t e i n / s r e q u i r e d for or acting m o r e e x t e n s i v e l y in d o u b l e - s t r a n d e d DNA transcription, while enzymic moiety transcribing d e n a t u r e d DNA b e e o m e s affected at h i g h e r t e m p e r a t u r e s (see fig. 3). F u r t h e r s u p p o r t for t h i s n o t i o n is the f a c t t h a t t h e e n z y m i c activities ratio in t h e presence of n a t i v e a n d d e n a t u r e d KB cell DNA decreases simultaneously for heated and unheated enzyme I p r e v i o u s l y t r e a t e d b y P r o n a s e , as will be described elsewhere. The presence of a c o n t a m i n a t i n g e n d o n u c l e a s e acting p r e f e r e n t i a l l y on n a t i v e DNA (and i n a c t i v a t e d b e t w e e n 37°C a n d 43°C) could also be considered as possible r e a s o n for t h i s b e h a v i o r of e n z y m e I. To t e s t t h i s a s s u m p t i o n , RNA p o l y m e r a s e I was d i l u t e d 2 a n d
A. Sergeant and V. Krsmanovic.
] 294
5 t i m e s . T h e r e s i d u a l a c t i v i t y ( w i t h n a t i v e KB DNA) a f t e r h e a t i n g a t 42°C ( e x p r e s s e d as p e r c e n t of t h e c o n t r o l v a l u e s a t 37°C) a s ~vell f o r d i l u t e d a s f o r u n d i l u t e d e n z y m e I, v a r i e d f r o m 73-77 p. cent. T h e s a m e
Q.
experiments were done with different DEAE-Sephadex f r a c t i o n s c o n s t i t u t i n g t h e p e a k of R N A p o l y m e r a s e I. The residual activity of aliquots (whatever was their e l u t i n g p o s i t i o n ) p r e h e a t e d a t 42°C v a r i e d a s l i t t l e a s
X
L) I.U nO O. nO 0
_z
Fin. 1. - - D E A E - S e p h a d e x A-25 c o l u m n c h r o m a t o g r a p h y (1 X 15 cm) o f K B cell R N A p o l y m e r a s e s . T h e e n z y m e s w e r e e l u t e d w i t h a 160 m l l i n e a r g r a d i e n t of 0.04-0.5 M (NH~)2SO~ i n T G M E D b u f f e r [12] a n d collect e d i n s e r u m a l b u m i n (1 m g / m l final conc). C o l u m n f r a c t i o n s (1.3 m l ) were a s s a y e d (30 m i n at 37°C) in a final v o l u m e of 150 i~1 c o n t a i n i n g 50 ul of e a c h f r a c t i o n ; 0.4 m M A T P , GTP, CTP; 0.015 m M (3H) U T P (1 C i / m M ) ; 10 ,txl/ml of KB DNA ; 6 m M MgCl.~ ; 3 m M MnCl., ; 50 m M T r i s - H C l p H 8.0 ; 4 m M d i t h i o t h r e i t o l ; (NH4)2SO~ c o n c e n t r a t i o n w a s o n e - t h i r d of e l u t i n g c o n c e n t r a t i o n s , a l l o w i n g o p t i m a l a c t i v i t y of e a c h of t h r e e e n z y m e s [12]. T h e a c t i v i t y is e~(pressed i n c p m ( c o u n t s p e r rain) o f (3H) U M P i n c o r p o r a t e d b y a l i q u o t s (1 p m o l e of U M P c o r r e s p o n d s to a p p r o x i m a t i v e l l y 600 c p m ) . R N A p o l y m e r a s e a c t i v i t y w a s a s s a y e d i n t h e above conditions with ( ©--© ), n a t i v e KB DNA (a) ; I e--e ) n a t i v e KB DNA a n d 0.8 !~g/ml a - a m a n i t i n (b). F o r t h e r m a l s e n s i t i v i e s e x p e r i m e n t s , t h e f r a c t i o n s c o r r e s p o n d i n g to e n z y m e I, II a n d III 'were p o o l e d as indicated.
Q.
=E I
FRACTION
NUMBER
TABLE I.
Thermal effect on KB cell RNA polymerases stored at --196°C, --70°C and --20°C. Storage tempera tare
-
-
KB cell DNA template
Residual activities in p. cent Enzyme I
Enzyme II
Enzyme
III
196oC
Native Denatured
50 72
95 93
700 C
Native Denatured
51 (55*) 77
88 (37*) 90
69 62 48 (16") 43
200 C
Native Denatured
43 38
38 34
18 2O
(*) E n z y m e s s t o r e d s e v e r a l m o n t h s at - - 7 0 ° C . KB cell R N A p o l y m e r a s e s 'were p r e p a r e d a n d s t o r e d a t - - 1 9 6 ° C as d e s c r i b e d i n t h e t e x t a n d figure 1. E n z y m e s w e r e e x p o s e d to - - 7 0 ° C d u r i n g 5 'weeks, a n d to - - 2 0 ° C d u r i n g 12 d a y s f o r R N A p o l y m e r a s e s I , a n d II, 3 d a y s f o r R N A p o l y m e r a s e III. P r e - h e a t i n g of e n z y m e s ~vas f o r 10 m i n elt 37°C a n d 46°C, t h e n p o l y m e r a s e a c t i v i t i e s w e r e a s s a y e d at 37°C f o r 30 m i n as i n d i c a t e d in fig. 1 a n d 2 ; t h e KB DNA c o n c e n t r a t i o n f o r e n z y m e I a n d II w a s i n c r e a s e d to 50 lxg/ml. R e s i d u a l a c t i v i t i e s at 46°C a r e e x p r e s s e d a s p e r c e n t of t h e c o n t r o l v a l u e s a t 37°C.
BIOCHIMIE, 1974, 56, n ° 9.
KB cell DNA-dependent RNA polymerases. 76-78 p. cent. These data s h o w t h a t first step i n a c t i v a tion b e t w e e n 37°C a n d 43°C for e n z y m e I activity in the presence of n a t i v e DNA (as s h o w n on fig. 3) could not be due to i n a c t i v a t i o n of a c o n t a m i n a t i n g e n d o n u clease, except a n activity closely associated or belonging to RNA p o l y m e r a s e I. W h e n t h e RNA p o l y m e r a s e activities II a n d III are followed as a f u n c t i o n of the pre-heating temperatures, rather similar inactivation curves are obtained e i t h e r n a t i v e or d e n a t u r e d KB DNA is u s e d to a s s e s s e n z y m i c activity, i n d i c a t i n g t h a t h e a t effect does not act p r e f e r e n t i a l l y on additional p r o t e i n / s r e s p o n s i b l e for native DNA t r a n s c r i p tion (fig. 2).
)b-
100'
1295
tions. However, the e n z y m e s exposed to - - 7 0 ° C d u r i n g 5 w e e k s s h o w a net relative decrease of t h e r m a l s t a bility at 46°C for RNA p o l y m e r a s e III, b u t storage a t - - 7 0 ° C d u r i n g longer periods (several m o n t h s ) e x e r t s on e n z y m e s II a n d I I I a c o n s i d e r a b l e decrease of t h e r m a l s t a b i l i t y (see table I), w i t h lowest r e s i d u a l activ i t y for RNA p o l y m e r a s e III, in a g r e e m e n t w i t h d a t a s h o w n p r e v i o u s l y I12]. E n z y m i c p r e p a r a t i o n s exposed to - - 2 0 ° C for s h o r t e r t i m e (12 d a y s for RNA p o l y m e r a s e s I a n d II a n d 3 d a y s for RNA p o l y m e r a s e III) a g a i n s h o w a n i m p o r t a n t decrease of t h e r m a l s t a b i lity, w i t h lovcest r e s i d u a l a c t i v i t y for RNA p o l y m e rases III. (Obviously, we could store t h e f r o z e n
1
b.J
5O
"It
50
TEMPERATURE Fro. 2.
°C FIG. 3.
FIG. 2. T h e r m a l s e n s i t i v i t y curves of KB RNA p o l y m e r a s e s I, II a n d III. E n z y m e s were isolated by DEAE-Sephadex c o l u m n c h r o m a t o g r a p h y as described i n M a t e r i a l s a n d Methods a n d fig. 1. 100 I~1 of e n z y m e f r a c t i o n , c o n t a i n i n g 1 m g / m l s e r u m a l b u m i n , w e r e i n c u b a t e d at indicated t e m p e r a t u r e s for 10 rain, cooled on ice [10], t h e n r e m a i n i n g e n z y m e activity ~vas a s s a y e d as i n d i c a t e d in fig. 1, except t h e ionic s t r e n g t h for e n z y m e III 'which w a s a d j u s t e d to 200 mM (NH~)2 SO4 [12]. R e s i d u a l activity is e x p r e s s e d as percent of the control v a l u e s at 37°C activity r e m a i n i n g following p r e - h e a t i n g to 58°C w a s considered as blank). C o r r e s p o n d i n g average control v a l u e s ~vere : 18,720 epm 'with n a t i v e KB DNA, for e n z y m e I ; 9,565 e p m w i t h n a t i v e and 95,300 c p m w i t h d e n a t u r e d KB DNA, f o r e n z y m e II :2.575 e p m w i t h n a t i v e a n d 1,555 c p m w i t h d e n a t u r e d KB DNA, for e n z y m e III. The e n z y m e activities I an(i III were a s s a y e d in the presence of 0.8 ~ g / m l ¢t-amanthin, w h e r e a s e n z y m e II activity w a s corrected by s u b t r a c t i n g the a - a m a n i t i n r e s i s t a n t a c t i v i t y p r e s e n t as c o n t a m i n a n t . RNA p o l y m e r a s e I a n d II ~vere a s s a y e d vcith 20 ixg/ml KB DNA : e n z y m e I w i t h n a t i v e ( O - - O I DNA, e n z y m e II 'with n a t i v e ( A - - A ) a n d d e n a t u r e d ( A - - A ) DNA. E n z y m e III w a s a s s a y e d w i t h 10 u g / m l native ( n - - [ 3 ) and denatured ( s - - I ) KB DNA. FIG. 3. T h e r m a l s e n s i t i v i t y curves of KB RNA p o l y m e r a s e I. E n z y m e 'was a s s a y e d , as described in fig. 2, w i t h 20 ~4.g/ml n a t i v e ( Q ~ O ) a n d d e n a t u r e d ( o - - o ) KB DNA in the presence of 0,8 u g / m l u - a m a n i t i n . Average control v a l u e s a t 37°C (defined as in fig. 2) w e r e 18,720 cpm w i t h n a t i v e a n d 12,970 c p m 'with d e n a t u r e d KB DNA. Hence t h e t~NA p o l y m e r a s e I a p p e a r s to be m o r e s e n s i t i v e to h e a t t h a n are t h e RNA p o l y m e r a s e s II a n d III. However, in a p r e v i o u s s t u d y we h a v e observed t h e opposite r e s u l t w i t h KB cell RNA p o l y m e r a s e s stored at --70°C, as well as c h a n g e of h e a t s e n s i t i vities d e p e n d i n g on storage t e m p e r a t u r e [12]. In order to a s c e r t a i n t h i s f e a t u r e the KB cell RNA p o l y m e r a s e s were stored in liquid n i t r o g e n (--196°C) a n d t h e i r h e a t s e n s i t i v i t i e s c o m p a r e d to those of the e n z y m e s exposed to - - 7 0 ° C a n d - - 2 0 ° C f o r s h o r t periods of time. As s h o w n in table I, t h e RNA p o l y m e r a s e II stored at - - 1 9 6 ° C (see Materials a n d Methods), t h e n p r e - h e a t e d at 46°C f o r 10 rain, is h i g h l y r e s i s t a n t , w h e r e a s RNA p o l y m e r a s e s I a n d III r e t a i n respectively a b o u t 50 p. cent a n d 69 p. cent of t h e i r i n i t i a l activities in t h e presence of n a t i v e DNA (72 p. cent a n d 62 p. cent in the presence of d e n a t u r e d DNA) u n d e r t h e s a m e condi-
BIOCHIMIE, 1974,
56, n ° 9.
e n z y m e s at - - 2 0 ° C only for a l i m i t e d t i m e since u n d e r these c o n d i t i o n s each of the t h r e e e n z y m e s u n d e r g o a r a p i d i n a c t i v a t i o n , especially p r o n o u n c e d for enzym e IIl). Modification of t h e r m a l s e n s i t i v i t i e s p r e s e n t e d here m i g h t be e x p l a i n e d by the fact t h a t d e n a t u r a t i o n p h e n o m e n a car occur d u r i n g frozen storage of proteins r e l a t e d p r e s u m a b l y to the presence of "water. For instance, q u a l i t a t i v e m e a s u r e m e n t s at low t e m p e r a t u r e s s h o w t h a t l i q u i d - w a t e r n u c l e a r m a g n e t i c resonance s p e c t r u m exists in frozen t i s s u e s n e a r to - - 7 0 ° C [14]. The storage at - - 1 9 6 ° C does n o t c h a n g e relative h e a t s e n s i t i v i t y of RNA p o l y m e r a s e s p r o b a b l y because of total absence of liquid w a t e r at t h i s t e m p e r a t u r e . In conclusion, the d a t a described in t h i s paper s h o w a f u r t h e r evidence t h a t t h e r m a l s e n s i t i v i t y test allows a n accurate detection of s o m e f u n c t i o n a l f e a t u r e s of
A. Sergeant and V. Krsmanovic.
1296
KB cell RNA polymerases. The first evidence for two steps heat inactivation of RNA p o l y m e r a s e I transcribing native KB DNA could be ascribed to the higher heat sensitivity for the s u b u n i t / s required for or acting more extensively on double stranded-DNA t r a n s c r i p tion. It is of i n t e r e s t t h a t the e x p e r i m e n t s in progress w i t h b o t h Ia (AI) and Ib (AII) enzymes, purified on phosphocellulose column f r o m DEAE-Sephadex RNA polymerase I, sho'w again a higher h e a t sensitivity for proteins involved p r e f e r e n t i a l l y in double s t r a n d e d DNA transcription, although t h e i r inactivation curves are s o m h o w modified. Finally, the results concerning the storage t e m p e r a t u r e s , w h i c h w e r e not been previously reported show clearly an altered h e a t sensitivity for RNA polymerases exposed to --20°C as well as to --70°C instead of --196°C, w h i c h explain our previous data obtained w i t h enzymes stored at --70°C for long time period [12].
Avkno~vledgments. We w o u l d like to t h a n k Drs. P. Chambon and J. Tata for helpful discussions and Dr. J. Samaille for providing research facilities. We are grateful to Dr. Th. Wieland for the gift of a - a m a n i t i n . This vcork was supported by the C.N.R.S. (ERA 225), I.N.S.E.R.M. (Unit6 U 102 and A.T. n ° 18) and Universit6 du Droit et de la Sant6 (U.E.R. III) de Lille.
BIOCHIM1E, 1974, 56, n ° 9.
REFERENCES. 1. Roeder, R. G. ,~ Rutter, W. J. (1969) Nature, 224, 234-449. 2. Kedinger, C., Gniazdowski, M., Mandel, Jr, J. L., Gissinger, F. & Chambon, P. (1970) Biochem. Biophys. Res. Commun., 38, 165-171. 3. Kedinger, C., Nuret, P. & Chambnn, P. (1972) FEBS Letters, 15, 169-174. 4. Rocder, R. G. ~ Rutter, ~V. J. (1970) Proc. Natl. Acad. Sci. U.S., 65, 675-682. 5. Widnel], C. C. & Tara, J. R. (1964) Biochim. Biophys. Acta, 87, 531-533. 6. Widnell, C. G..& Tara, J. R. (1966) Biochim. Biophys. Acta, 123, 478-492. 7. Zylber, E. A. z~ P e n m a n , S. (1971) Proc. Natl. Aead. Sci. U.S., ~;:8, 2861-2865. 8. W e i n m a n n , R. & Roeder, R. G. (1974) Proc. Natl. Acad. Sci. U.S., 71, 1790-1794. 9. See n u m e r o u s articles i n : Cold Spring Harbor Syrup. Quant. Biol., (1970), 35, 641-742. 10. Shields, D. z, Tata, J. R. (1973) F E B S Letters, 31, 209-213. 11. Tara, J. R., personal communication. 12. Sergeant, A. ,~ Krsmanovic, V. (1973) F E B S Letters, 35, 331-335. 13. Young, E. T. ~ Sinsheimer, R. L. (1967) J. Mol. Biol., 30, 147-164. 14. Sussman, M. V., Chin, L. e K a r a k u r u m , O. (1967) Chemical Engineering in Medicine and Biology (Hershey, D., ed.), pp. 495-504, P l e n u m Pres, New Yor~.
On thermal sensitivities of KB cell DNA-dependent RNA polymerases. A. SERGEANT (*) a n d V. KRSMANOVIC (" "). (*) Laboraioire de Chimie Biologique, Universitd des Sciences et Techniques de Lille I, 59650 Villeneuve d'Ascq, France, (**) and Unitd de Recherche de Virologic, U 102 de I'INSERM, 2, Place de Verdun, 59045 Lille Cedex, France.
(18-9-197/D.
INTRODUCTION. E u k a r y o t i c cell nuclei c o n t a i n m u l t i p l e DNA-depend e n t RNA p o l y m e r a s e s [1, 2] o r i g i n a l l y d e s i g n a t e d by Roeder a n d R u t t e r as e n z y m e s 2, II a n d III [1] c o r r e s p o n d i n g to f o r m s AI / A I I , BI/BII a n d AIII, respectively [31. The f o r m I is p r e s e n t in nucleoli, w h e r e a s the f o r m s II a n d III are in n u c l e o p l a s m [4] ; these e n z y m e s s y n t h e s i z e r i b o s o m a l RNA [5, 6, 7], n u c l e a r h e t e r o g e n o u s RNA [7] a n d 4 S/5 S species [8], respectively. RNA p o l y m e r a s e II is s e n s i t i v e to (~-amanitine, while RNA p o l y m e r a s e s I a n d III are i n s e n s i tive to t h i s d r u g at low c o n c e n t r a t i o n s , a l t h o u g h the h i g h c o n c e n t r a t i o n s of the t o x i n i n h i b i t RNA p o l y m e r a s e III [81. Each f o r m is different f r o m t h e o t h e r s w i t h respect to o p t i m a l c o n c e n t r a t i o n of a m m o n i u m sulfate, Mg2+ a n d Mn~+ [1, 0]. R e c e n t l y Shields a n d T a t a [10] h a v e s h o w n t h a t RNA p o l y m e r a s e I f r o m r a t liver nuclei is m o r e affected by t h e r m a l shock t h a n is RNA p o l y m e r a s e II (and III [11]). The differential h e a t s e n s i t i v i t y of RNA p o l y m e r a s e s could be r e l a t e d to t h e i r i n t r i n s i c properties, since the p r e h e a t i n g of t h e r a t liver nuclei also i n h i b i t s m u c h m o r e RNA p o l y m e r a s e I [10]. T h e m e a n i n g of t h e r m a l i n a c t i v a t i o n in the case of complex e n z y m e s s u c h as eulearyotic p o l y m e r a s e s , is not easily u n d e r s t a n d a b l e . Nevertheless, t h e i r differ e n t i a l t h e r m a l s e n s i t i v i t i e s offer a n a d d i t i o n a l possibility to d i s c r i m i n a t e each f o r m f r o m t h e others. D u r i n g our s t u d y on t h e r m a l s e n s i t i v i t i e s of KB cell RNA p o l y m e r a s e s we h a v e observed t h a t e n z y m e II is less i n a c t i v a t e d by t h e h e a t t h a n t h e e n z y m e s I a n d III are, w h i c h is c o m p a t i b l e w i t h t h e r e s u l t s for r a t liver e n z y m e s [10, 11]. F u r t h e r m o r e , we s h o w t h a t relative h e a t s e n s i t i v i t y of e n z y m e I is different w h e t h e r n a t i v e or d e n a t u r e d KB DNA is u s e d for a s s e s s m e n t of RNA p o l y m e r a s e a c t i v i t y after h e a t i n g , s u g g e s t i n g existence of a d d i t i o n a l p r o t e i n / s involved in d o u b l e - s t r a n d e d DNA t r a n s c r i p t i o n . In a d d i t i o n , we h a v e observed t h a t t h e relative h e a t s e n s i t i v i t i e s of RNA p o l y m e r a s e s u n d e r g o a d r a m a t i c c h a n g e dep e n d i n g on storage t e m p e r a t u r e . As will be s h o w n in t h i s p a p e r the RNA p o l y m e r a s e s II a n d lII e x h i b i t a h i g h e r h e a t s e n s i t i v i t y t h a n RNA p o l y m e r a s e I if stored at - - 7 0 ° C or - - 2 0 ° C i n s t e a d of --196°C, xvhieh e x p l a i n o u r p r e v i o u s r e s u l t s o b t a i n e d ~,ith e n z y m e s stored at - - 7 0 ° C for several m o n t h s [12]. MATERIALS AND METHODS. RNA p o l y m e r a s e s were e x t r a c t e d f r o m KB cells nuclei, e s s e n t i a l l y by the m e t h o d of Roeder a n d R u t t e r A'bbreviations : TGMED : Tris, glycerol, MgCI~. EDTA, d i t h i o t h r e i t o l . (**) To w h o m all c o r r e s p o n d a n e e s h o u l d be addressed.
[4], as p r e v i o u s l y described [12], t h e n t h e e n z y m i c e x t r a c t ~¢¢as applied to a DEAE-Sephadex A-25 c o l u m n a n d eluted ~vith a l i n e a r g r a d i e n t of 0.04-0.5 M (NH4)~ SO4 in TGMED (*) buffer (0.05 M Tris-HCi pH 8,25 p. cent v / v glycerol, 5 mM MgCl2 0.1 mM EDTA, 0,5 mM d i t h i o t h r e i t o l ) , as s h o w n in figure 1. U n l e s s specified, isolated e n z y m e s are stored in l i q u i d n i t r o g e n (--196°C) i n e l u t i n g TGMED buffer c o n t a i n i n g 1 m g / m l s e r u m a l b u m i n . P r e - h e a t i n g of RNA p o l y m e r a s e s w a s p e r f o r m e d for 10 m i n at i n d i c a t e d t e m p e r a t u r e s . E n z y m e activity ,~'as a s s a y e d , at o p t i m a l s a l t concent r a t i o n s , as described i n the legends, a - a m a n i t i n w a s u s e d at 0.8 !~g/ml. After i n c u b a t i o n for 30 m i n at 37°C t h e r e a c t i o n s w e r e p r e c i p i t a t e d as described elsewere [12]. DNA ~vas s o l u b i l i s e d f r o m KB cells by S a r k o s y l P r o n a s e t r e a t m e n L isolated by CsC1 e q u i l i b r i u m g r a d i e n t c e n t r i f u g a t i o n a n d stored at 4°C in 0,1 × SSC (0.015 NaCl, 0.0015 M s o d i u m citrate) b y m e t h o d s s i m i l a r to t h o s e p r e v i o u s l y described [13].
RESULTS AND DISCUSSION. KB cell RNA p o l y m e r a s e s I, II a n d III isolated by DEAE-Sephadex c o l u m n c h r o m a t o g r a p h y (fig. 1) e x h i b i t differences in t h e degree of s e n s i t i v i t y to heat, as s h o w n on figure 2. The e n z y m e II is less affected by t h e r m a l shock t h a n are t h e e n z y m e s I a n d III w h i c h is c o n s i s t e n t w i t h p r e v i o u s r e s u l t s for r a t liver e n z y m e s I a n d II [10]. The h i g h e r overall h e a t stability, in o u r conditions, is p r e s u m a b l y due to the protecting effect of s e r u m a l b u m i n (added to p r e v e n t e n z y m e i n a c t i v a t i o n d u r i n g f r a e t i o n a t i o n a n d storage) since t h e p r e - h e a t e d r a t liver nuclei s h o w a s i m i l a r r e s u l t [10]. F u r t h e r m o r e , t h e h e a t i n a c t i v a t i o n curve of KB RNA p o l y m e r a s e I a c t i v i t y i n t h e presence of n a t i v e KB cell DNA a p p e a r s to be biphasic. The early h e a t inactiv a t i o n b e t w e e n 37°C a n d 43°C for e n z y m e I could be related to h e a t effect on specific p r o t e i n / s r e q u i r e d for or acting m o r e e x t e n s i v e l y in d o u b l e - s t r a n d e d DNA transcription, while enzymic moiety transcribing d e n a t u r e d DNA b e e o m e s affected at h i g h e r t e m p e r a t u r e s (see fig. 3). F u r t h e r s u p p o r t for t h i s n o t i o n is the f a c t t h a t t h e e n z y m i c activities ratio in t h e presence of n a t i v e a n d d e n a t u r e d KB cell DNA decreases simultaneously for heated and unheated enzyme I p r e v i o u s l y t r e a t e d b y P r o n a s e , as will be described elsewhere. The presence of a c o n t a m i n a t i n g e n d o n u c l e a s e acting p r e f e r e n t i a l l y on n a t i v e DNA (and i n a c t i v a t e d b e t w e e n 37°C a n d 43°C) could also be considered as possible r e a s o n for t h i s b e h a v i o r of e n z y m e I. To t e s t t h i s a s s u m p t i o n , RNA p o l y m e r a s e I was d i l u t e d 2 a n d
A. Sergeant and V. Krsmanovic.
] 294
5 t i m e s . T h e r e s i d u a l a c t i v i t y ( w i t h n a t i v e KB DNA) a f t e r h e a t i n g a t 42°C ( e x p r e s s e d as p e r c e n t of t h e c o n t r o l v a l u e s a t 37°C) a s ~vell f o r d i l u t e d a s f o r u n d i l u t e d e n z y m e I, v a r i e d f r o m 73-77 p. cent. T h e s a m e
Q.
experiments were done with different DEAE-Sephadex f r a c t i o n s c o n s t i t u t i n g t h e p e a k of R N A p o l y m e r a s e I. The residual activity of aliquots (whatever was their e l u t i n g p o s i t i o n ) p r e h e a t e d a t 42°C v a r i e d a s l i t t l e a s
X
L) I.U nO O. nO 0
_z
Fin. 1. - - D E A E - S e p h a d e x A-25 c o l u m n c h r o m a t o g r a p h y (1 X 15 cm) o f K B cell R N A p o l y m e r a s e s . T h e e n z y m e s w e r e e l u t e d w i t h a 160 m l l i n e a r g r a d i e n t of 0.04-0.5 M (NH~)2SO~ i n T G M E D b u f f e r [12] a n d collect e d i n s e r u m a l b u m i n (1 m g / m l final conc). C o l u m n f r a c t i o n s (1.3 m l ) were a s s a y e d (30 m i n at 37°C) in a final v o l u m e of 150 i~1 c o n t a i n i n g 50 ul of e a c h f r a c t i o n ; 0.4 m M A T P , GTP, CTP; 0.015 m M (3H) U T P (1 C i / m M ) ; 10 ,txl/ml of KB DNA ; 6 m M MgCl.~ ; 3 m M MnCl., ; 50 m M T r i s - H C l p H 8.0 ; 4 m M d i t h i o t h r e i t o l ; (NH4)2SO~ c o n c e n t r a t i o n w a s o n e - t h i r d of e l u t i n g c o n c e n t r a t i o n s , a l l o w i n g o p t i m a l a c t i v i t y of e a c h of t h r e e e n z y m e s [12]. T h e a c t i v i t y is e~(pressed i n c p m ( c o u n t s p e r rain) o f (3H) U M P i n c o r p o r a t e d b y a l i q u o t s (1 p m o l e of U M P c o r r e s p o n d s to a p p r o x i m a t i v e l l y 600 c p m ) . R N A p o l y m e r a s e a c t i v i t y w a s a s s a y e d i n t h e above conditions with ( ©--© ), n a t i v e KB DNA (a) ; I e--e ) n a t i v e KB DNA a n d 0.8 !~g/ml a - a m a n i t i n (b). F o r t h e r m a l s e n s i t i v i e s e x p e r i m e n t s , t h e f r a c t i o n s c o r r e s p o n d i n g to e n z y m e I, II a n d III 'were p o o l e d as indicated.
Q.
=E I
FRACTION
NUMBER
TABLE I.
Thermal effect on KB cell RNA polymerases stored at --196°C, --70°C and --20°C. Storage tempera tare
-
-
KB cell DNA template
Residual activities in p. cent Enzyme I
Enzyme II
Enzyme
III
196oC
Native Denatured
50 72
95 93
700 C
Native Denatured
51 (55*) 77
88 (37*) 90
69 62 48 (16") 43
200 C
Native Denatured
43 38
38 34
18 2O
(*) E n z y m e s s t o r e d s e v e r a l m o n t h s at - - 7 0 ° C . KB cell R N A p o l y m e r a s e s 'were p r e p a r e d a n d s t o r e d a t - - 1 9 6 ° C as d e s c r i b e d i n t h e t e x t a n d figure 1. E n z y m e s w e r e e x p o s e d to - - 7 0 ° C d u r i n g 5 'weeks, a n d to - - 2 0 ° C d u r i n g 12 d a y s f o r R N A p o l y m e r a s e s I , a n d II, 3 d a y s f o r R N A p o l y m e r a s e III. P r e - h e a t i n g of e n z y m e s ~vas f o r 10 m i n elt 37°C a n d 46°C, t h e n p o l y m e r a s e a c t i v i t i e s w e r e a s s a y e d at 37°C f o r 30 m i n as i n d i c a t e d in fig. 1 a n d 2 ; t h e KB DNA c o n c e n t r a t i o n f o r e n z y m e I a n d II w a s i n c r e a s e d to 50 lxg/ml. R e s i d u a l a c t i v i t i e s at 46°C a r e e x p r e s s e d a s p e r c e n t of t h e c o n t r o l v a l u e s a t 37°C.
BIOCHIMIE, 1974, 56, n ° 9.
KB cell DNA-dependent RNA polymerases. 76-78 p. cent. These data s h o w t h a t first step i n a c t i v a tion b e t w e e n 37°C a n d 43°C for e n z y m e I activity in the presence of n a t i v e DNA (as s h o w n on fig. 3) could not be due to i n a c t i v a t i o n of a c o n t a m i n a t i n g e n d o n u clease, except a n activity closely associated or belonging to RNA p o l y m e r a s e I. W h e n t h e RNA p o l y m e r a s e activities II a n d III are followed as a f u n c t i o n of the pre-heating temperatures, rather similar inactivation curves are obtained e i t h e r n a t i v e or d e n a t u r e d KB DNA is u s e d to a s s e s s e n z y m i c activity, i n d i c a t i n g t h a t h e a t effect does not act p r e f e r e n t i a l l y on additional p r o t e i n / s r e s p o n s i b l e for native DNA t r a n s c r i p tion (fig. 2).
)b-
100'
1295
tions. However, the e n z y m e s exposed to - - 7 0 ° C d u r i n g 5 w e e k s s h o w a net relative decrease of t h e r m a l s t a bility at 46°C for RNA p o l y m e r a s e III, b u t storage a t - - 7 0 ° C d u r i n g longer periods (several m o n t h s ) e x e r t s on e n z y m e s II a n d I I I a c o n s i d e r a b l e decrease of t h e r m a l s t a b i l i t y (see table I), w i t h lowest r e s i d u a l activ i t y for RNA p o l y m e r a s e III, in a g r e e m e n t w i t h d a t a s h o w n p r e v i o u s l y I12]. E n z y m i c p r e p a r a t i o n s exposed to - - 2 0 ° C for s h o r t e r t i m e (12 d a y s for RNA p o l y m e r a s e s I a n d II a n d 3 d a y s for RNA p o l y m e r a s e III) a g a i n s h o w a n i m p o r t a n t decrease of t h e r m a l s t a b i lity, w i t h lovcest r e s i d u a l a c t i v i t y for RNA p o l y m e rases III. (Obviously, we could store t h e f r o z e n
1
b.J
5O
"It
50
TEMPERATURE Fro. 2.
°C FIG. 3.
FIG. 2. T h e r m a l s e n s i t i v i t y curves of KB RNA p o l y m e r a s e s I, II a n d III. E n z y m e s were isolated by DEAE-Sephadex c o l u m n c h r o m a t o g r a p h y as described i n M a t e r i a l s a n d Methods a n d fig. 1. 100 I~1 of e n z y m e f r a c t i o n , c o n t a i n i n g 1 m g / m l s e r u m a l b u m i n , w e r e i n c u b a t e d at indicated t e m p e r a t u r e s for 10 rain, cooled on ice [10], t h e n r e m a i n i n g e n z y m e activity ~vas a s s a y e d as i n d i c a t e d in fig. 1, except t h e ionic s t r e n g t h for e n z y m e III 'which w a s a d j u s t e d to 200 mM (NH~)2 SO4 [12]. R e s i d u a l activity is e x p r e s s e d as percent of the control v a l u e s at 37°C activity r e m a i n i n g following p r e - h e a t i n g to 58°C w a s considered as blank). C o r r e s p o n d i n g average control v a l u e s ~vere : 18,720 epm 'with n a t i v e KB DNA, for e n z y m e I ; 9,565 e p m w i t h n a t i v e and 95,300 c p m w i t h d e n a t u r e d KB DNA, f o r e n z y m e II :2.575 e p m w i t h n a t i v e a n d 1,555 c p m w i t h d e n a t u r e d KB DNA, for e n z y m e III. The e n z y m e activities I an(i III were a s s a y e d in the presence of 0.8 ~ g / m l ¢t-amanthin, w h e r e a s e n z y m e II activity w a s corrected by s u b t r a c t i n g the a - a m a n i t i n r e s i s t a n t a c t i v i t y p r e s e n t as c o n t a m i n a n t . RNA p o l y m e r a s e I a n d II ~vere a s s a y e d vcith 20 ixg/ml KB DNA : e n z y m e I w i t h n a t i v e ( O - - O I DNA, e n z y m e II 'with n a t i v e ( A - - A ) a n d d e n a t u r e d ( A - - A ) DNA. E n z y m e III w a s a s s a y e d w i t h 10 u g / m l native ( n - - [ 3 ) and denatured ( s - - I ) KB DNA. FIG. 3. T h e r m a l s e n s i t i v i t y curves of KB RNA p o l y m e r a s e I. E n z y m e 'was a s s a y e d , as described in fig. 2, w i t h 20 ~4.g/ml n a t i v e ( Q ~ O ) a n d d e n a t u r e d ( o - - o ) KB DNA in the presence of 0,8 u g / m l u - a m a n i t i n . Average control v a l u e s a t 37°C (defined as in fig. 2) w e r e 18,720 cpm w i t h n a t i v e a n d 12,970 c p m 'with d e n a t u r e d KB DNA. Hence t h e t~NA p o l y m e r a s e I a p p e a r s to be m o r e s e n s i t i v e to h e a t t h a n are t h e RNA p o l y m e r a s e s II a n d III. However, in a p r e v i o u s s t u d y we h a v e observed t h e opposite r e s u l t w i t h KB cell RNA p o l y m e r a s e s stored at --70°C, as well as c h a n g e of h e a t s e n s i t i vities d e p e n d i n g on storage t e m p e r a t u r e [12]. In order to a s c e r t a i n t h i s f e a t u r e the KB cell RNA p o l y m e r a s e s were stored in liquid n i t r o g e n (--196°C) a n d t h e i r h e a t s e n s i t i v i t i e s c o m p a r e d to those of the e n z y m e s exposed to - - 7 0 ° C a n d - - 2 0 ° C f o r s h o r t periods of time. As s h o w n in table I, t h e RNA p o l y m e r a s e II stored at - - 1 9 6 ° C (see Materials a n d Methods), t h e n p r e - h e a t e d at 46°C f o r 10 rain, is h i g h l y r e s i s t a n t , w h e r e a s RNA p o l y m e r a s e s I a n d III r e t a i n respectively a b o u t 50 p. cent a n d 69 p. cent of t h e i r i n i t i a l activities in t h e presence of n a t i v e DNA (72 p. cent a n d 62 p. cent in the presence of d e n a t u r e d DNA) u n d e r t h e s a m e condi-
BIOCHIMIE, 1974,
56, n ° 9.
e n z y m e s at - - 2 0 ° C only for a l i m i t e d t i m e since u n d e r these c o n d i t i o n s each of the t h r e e e n z y m e s u n d e r g o a r a p i d i n a c t i v a t i o n , especially p r o n o u n c e d for enzym e IIl). Modification of t h e r m a l s e n s i t i v i t i e s p r e s e n t e d here m i g h t be e x p l a i n e d by the fact t h a t d e n a t u r a t i o n p h e n o m e n a car occur d u r i n g frozen storage of proteins r e l a t e d p r e s u m a b l y to the presence of "water. For instance, q u a l i t a t i v e m e a s u r e m e n t s at low t e m p e r a t u r e s s h o w t h a t l i q u i d - w a t e r n u c l e a r m a g n e t i c resonance s p e c t r u m exists in frozen t i s s u e s n e a r to - - 7 0 ° C [14]. The storage at - - 1 9 6 ° C does n o t c h a n g e relative h e a t s e n s i t i v i t y of RNA p o l y m e r a s e s p r o b a b l y because of total absence of liquid w a t e r at t h i s t e m p e r a t u r e . In conclusion, the d a t a described in t h i s paper s h o w a f u r t h e r evidence t h a t t h e r m a l s e n s i t i v i t y test allows a n accurate detection of s o m e f u n c t i o n a l f e a t u r e s of
A. Sergeant and V. Krsmanovic.
1296
KB cell RNA polymerases. The first evidence for two steps heat inactivation of RNA p o l y m e r a s e I transcribing native KB DNA could be ascribed to the higher heat sensitivity for the s u b u n i t / s required for or acting more extensively on double stranded-DNA t r a n s c r i p tion. It is of i n t e r e s t t h a t the e x p e r i m e n t s in progress w i t h b o t h Ia (AI) and Ib (AII) enzymes, purified on phosphocellulose column f r o m DEAE-Sephadex RNA polymerase I, sho'w again a higher h e a t sensitivity for proteins involved p r e f e r e n t i a l l y in double s t r a n d e d DNA transcription, although t h e i r inactivation curves are s o m h o w modified. Finally, the results concerning the storage t e m p e r a t u r e s , w h i c h w e r e not been previously reported show clearly an altered h e a t sensitivity for RNA polymerases exposed to --20°C as well as to --70°C instead of --196°C, w h i c h explain our previous data obtained w i t h enzymes stored at --70°C for long time period [12].
Avkno~vledgments. We w o u l d like to t h a n k Drs. P. Chambon and J. Tata for helpful discussions and Dr. J. Samaille for providing research facilities. We are grateful to Dr. Th. Wieland for the gift of a - a m a n i t i n . This vcork was supported by the C.N.R.S. (ERA 225), I.N.S.E.R.M. (Unit6 U 102 and A.T. n ° 18) and Universit6 du Droit et de la Sant6 (U.E.R. III) de Lille.
BIOCHIM1E, 1974, 56, n ° 9.
REFERENCES. 1. Roeder, R. G. ,~ Rutter, W. J. (1969) Nature, 224, 234-449. 2. Kedinger, C., Gniazdowski, M., Mandel, Jr, J. L., Gissinger, F. & Chambon, P. (1970) Biochem. Biophys. Res. Commun., 38, 165-171. 3. Kedinger, C., Nuret, P. & Chambnn, P. (1972) FEBS Letters, 15, 169-174. 4. Rocder, R. G. ~ Rutter, ~V. J. (1970) Proc. Natl. Acad. Sci. U.S., 65, 675-682. 5. Widnel], C. C. & Tara, J. R. (1964) Biochim. Biophys. Acta, 87, 531-533. 6. Widnell, C. G..& Tara, J. R. (1966) Biochim. Biophys. Acta, 123, 478-492. 7. Zylber, E. A. z~ P e n m a n , S. (1971) Proc. Natl. Aead. Sci. U.S., ~;:8, 2861-2865. 8. W e i n m a n n , R. & Roeder, R. G. (1974) Proc. Natl. Acad. Sci. U.S., 71, 1790-1794. 9. See n u m e r o u s articles i n : Cold Spring Harbor Syrup. Quant. Biol., (1970), 35, 641-742. 10. Shields, D. z, Tata, J. R. (1973) F E B S Letters, 31, 209-213. 11. Tara, J. R., personal communication. 12. Sergeant, A. ,~ Krsmanovic, V. (1973) F E B S Letters, 35, 331-335. 13. Young, E. T. ~ Sinsheimer, R. L. (1967) J. Mol. Biol., 30, 147-164. 14. Sussman, M. V., Chin, L. e K a r a k u r u m , O. (1967) Chemical Engineering in Medicine and Biology (Hershey, D., ed.), pp. 495-504, P l e n u m Pres, New Yor~.