175
Biochimica et Biophysica Acta, 418 (1976) 175--183 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98495
THE BINDING SITE F O R COAT PROTEIN ON BACTERIOPHAGE Q~ R N A
HANS WEBER
Institut fiir Molekularbiologie I, Universith't Ziirich, 8049 Ziirich (Switzerland)
(Received June 23rd, 1975)
Summary The site of interaction of phage Q~ coat protein with Q~ RNA was determined by ribonuclease TI degradation of complexes of coat protein and [ 32p]. R N A obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and b o u n d [ 32p] R N A fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Q~, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.
Introduction Virus-specific protein synthesis in RNA phage-infected cells is subject to a variety of control mechanisms [1,2]. A particular feature of the phage systems appears to be the use of translational repressors, namely proteins which bind to the ribosomal attachment site at the beginning of a cistron and thereby prevent the ribosomes from initiating the synthesis of the corresponding protein. Such a function has been assigned to Q~ replicase, which was shown to bind to Q~ R N A at the coat cistron initiation site [3] and to prevent the initiation of coat protein synthesis [4,5]. For group I phages (R17, MS2, f2), work on coat protein mutants as well as in vitro studies on protein synthesis suggested a role for coat protein as a repressor of the synthesis of the replicase protein late in infection [6,7]. This role was confirmed by the finding that R17 coat protein indeed binds to R17 RNA at the beginning of the replicase cistron [8]. Furthermore, RNA-fragment binding studies [9] as well as measurements of the relaxation kinetics of the melting o f fragment-protein complexes indicated that the R17 RNA-coat pro-
176 tein interaction occurs precisely on the small hairpin helix structure containing the initiator codon [ 10]. In the Qj3 system, results obtained with coat protein mutants suggested a similar regulatory role for coat protein [11]. We demonstrate here that the Qfl coat protein, too, is capable of a specific interaction at the initiation site for the replicase cistron of Q~3 RNA. Using techniques similar to those employed previously, we have degraded coat protein-RNA complexes with ribonuclease T1 and isolated three protein-bound RNA fragments. Sequence analysis showed all of these to be derived from the replicase cistron initiation site. As in the case of R17, the smallest fragment characterized (27 nucleotides) appears to consist of a small hairpin helix containing the initiator codon. Results and Discussion Since Qfi coat protein has a very low solubility in aqueous buffers, complex formation was carried o u t by codialysis from concentrated urea solution into buffer solutions. The reaction was followed by determining the fraction of [ 32p] Qfi RNA retained on Millipore filters due to its binding to coat protein. Preliminary experiments showed that in presence of a 35- to 45-fold molar excess of coat protein, up to about 40% of the RNA acquired the ability to bind to Millipore within 1--3 h of dialysis at room temperature. Further prolongation of dialysis did n o t increase the extent of reaction, while reduction of the excess of coat protein decreased it very considerably. Ribonuclease T1 degradation of the complexes was done in presence of a 4- to 7-fold excess of unlabeled Qfl RNA in order to reduce the a m o u n t of weakly {and non-specifically) bound [ 32p] RNA material by competition. The e x t e n t of degradation was arbitrarily selected so as to yield a b o u t 1% of the total initial RNA. The degraded complexes were collected by binding to Millipore filters, from which the [ 3 2 P ] R N A fragments were extracted using phenol/buffer mixtures. Fig. 1 shows an autoradiographic pattern obtained by electrophoretic separation of such fragment mixtures on 20% polyacrylamide gels. The three most p r o m i n e n t fragments, C-l, C-2 and C-3 (together comprising about 0.15% of the total initial radioactivity) were extracted from the gels, one part of each was digested with ribonuclease T1 and another with ribonuclease A. The products of these digestions were fractionated by twodimensional ionophoresis [ 12]. The autoradiograms obtained showed very conspicuous similarities to those reported for a fragment derived from the region around the replicase cistron ribosomal binding site by Porter et al. [13]. In particular, the RNAase T1 fingerprint of fragment C-1 (Fig. 2A) is identical to the one reported for fragment 2 in reference 13, with the exception of one additional spot, designated A. Accordingly, the RNAase A fingerprint obtained from C-1 (Fig. 2B) is identical to the one reported for fragment 2 except for the disappearance of spot No. 7 and the appearance of a new spot, designated B (along with t w o additional mononucleotides, Gp and Up). The analytical data of the various digestion products of the fragments C-I, C-2 and C-3 are compiled in Tables I and II. They leave no d o u b t that C-1 is a fragment of 88 nucleotides comprising the whole sequence of fragment 2 of
177
Cm
m~ ,
C2
C ~3
~
b|-~ Fig. 1. Gel e l e c t r o p h o r e s i s o f [ 3 2 p ] Q ~ R N A f r a g m e n t s r e t a i n e d on Millipore b y c o a t p r o t e i n b i n d i n g . [ 3 2 p ] Q#3 R N A ( 1 5 0 #g, 0.1 n m o l , specific r a d i o a c t i v i t y 3 • 106 e p m / ~ g ) a n d Q~ c o a t p r o t e i n (50/~g, 3.6 n m o l ) , in 50 m M T r i s / a c e t a t e ( p H 7 . 8 ) / 1 0 m M m e r c a p t o e t h a n o l / 9 . 4 M u r e a (0.5 m l ) w e r e d i a l y z e d against 5 0 0 m l of a b u f f e r c o n t a i n i n g 1 0 0 m M Tris • HC1 ( p H 7 . 5 ) , 1 5 0 m M NaCl, 10 m M MgCI2, 10 m M m e r c a p t o e t h a n o l a n d 1 m M E D T A at 25 °. A f t e r 1 h a 2/~1 a l i q u o t was d i l u t e d to 0.5 m l w i t h dialysis b u f f e r , filtered t h r o u g h Mfllipore ( H A W P , p o r e size 0 . 4 5 /~m) a n d w a s h e d w i t h t h r e e 1-ml p o r t i o n s of dialysis b u f f e r : a b o u t 37% of the R N A w a s b o u n d to t h e filter. T h e m a i n s a m p l e w a s t r a n s f e r r e d f r o m t h e dialysis b a g i n t o a t u b e at 2 5 ° C , a n d 1 m g o f u n l a b e l e d Q#3 R N A a n d 1 0 0 0 u n i t s [ 2 0 ] o f R N A a s e T I w e r e a d d e d . A f t e r 5 rain at 25QC, t h e first h a l f o f the s a m p l e ( A ) w a s filtered t h r o u g h Millipore (3 X 1 m l washings). T h e r a d i o a c t i v i t y r e t a i n e d as d e t e r m i n e d b y C h e r e n k 0 v c o u n t i n g w a s a b o u t 1.0% of t h e t o t a l R N A a p p l i e d to t h e filter. T h e filter w a s i m m e d i a t e l y e x t r a c t e d b y v i g o r o u s s h a k i n g in a m i x t u r e c o n t a i n ing 1 m l o f e x t r a c t i o n b u f f e r (20 m M Tris • HC1 ( p H 7 . 5 ) , 2 m M E D T A , 0.1% s o d i u m d o d e c y l sulfate, 50 ~tg o f y e a s t c a r r i e r R N A ) a n d 1 m l o f redistilled p h e n o l e q u i l i b r a t e d w i t h e x t r a c t i o n b u f f e r . T h e s e c o n d h a l f o f t h e s a m p l e (B) w a s f i l t e r e d a f t e r 10 rain o f i n c u b a t i o n a n d a b o u t 0.8% of t h e original r a d i o a c t i v i t y w a s r e t a i n e d . F i l t e r B w a s e x t r a c t e d in t h e s a m e w a y as filter A, t h e e x t r a c t i o n m i x t u r e s w e r e c o o l e d to 0°C a n d c e n t r i f u g e d in a clinical c e n t r i f u g e . T h e R N A w a s p r e c i p i t a t e d f r o m the a q u e o u s l a y e r s b y t h e a d d i t i o n of 0.1 vol o f 0.5 M Tris • HCI ( p H 7 . 5 ) , 1.0 M NaC1, 0 . 0 5 M E D T A , a n d 2 v o l u m e s o f cold e t h a n o l . A f t e r s t o r a g e o v e r n i g h t at - - 2 5 ° C , t h e R N A w a s c o l l e c t e d b y c e n t r i f u g a t i o n , d r i e d in v a c u o a n d dissolved in a s o l u t i o n o f 12% s u c r o s e a n d 0 . 0 4 % b r o m o p h e n o l b l u e ( 5 0 /~1). T h e s a m p l e s (A, 3.2 • 106 c p m ; B, 1.6 • 106 c p m ) w e r e i n s e r t e d i n t o t h e slots o f a 20% p o l y a c r y l a m i d e gel p r e p a r e d a n d r u n as d e s c r i b e d in Materials a n d M e t h o d s . T h e s y m b o l bl i n d i c a t e s t h e p o s i t i o n o f t h e b r o m o p h e n o l blue marker.
178
Porter et al. [13] plus an additional pentanucleotide A-A-U-U-Gp at its 3' end {Fig. 3). Fragment C-2 has a length of 71 nucleotides and corresponds to C-1 lacking the twelve 5'-terminal and the five 3'-terminal nucleotides. Fragment C-3, finally, consists of the 27 nucleotides at the 5' end of fragment C-2. The finding that all three fragments contain the initiator c o d o n of the replicase cistron demonstrates the affinity of coat protein for this site. We presume that in the Q~ system, as in the case of R17, the coat protein blocks the synthesis of replicase protein late in infection by direct binding to the ribosomal initiation site for this cistron. The possibility that contaminating amounts of the minor capsid proteins, A1 and A2 [ 1 4 ] , could be involved in
PH35_5.
qlP
! :DEAE
i ea
08
.p
bl
• !
130 !O
8 e
14~
D
015
t
17 Fig. 2 A .
179 the binding of this site is considered unlikely because: (a) most of the minor proteins had been removed during the purification of the coat protein; and (b) control experiments using proteins A1 or A2 instead of coat protein under otherwise identical conditions for the formation and degradation of complexes with [ 3 2p] Q~ RNA did not result in any specific fragment patterns (data not shown).
lO t
D
g
g
~
J
Fig. 2B.
Fig. 2. A u t o r a d i o g r a m s o f t w o - d i m e n s i o n a l f i n g e r p r i n t s o f f r a g m e n t C-1. (A) P r o d u c t s o f t h e d i g e s t i o n o f f r a g m e n t C-1 b y R N A a s e T 1 . F i r s t d i m e n s i o n : E l e c t r o p h o r e s i s o n c e l l u l o s e a c e t a t e in 7 M u r e a a t p H 3 . 5 . S e c o n d d i m e n s i o n : E l e c t r o p h o r e s i s o n D E A E - p a p e r in 7% f o r m i c a c i d . (B) P r o d u c t s o f t h e d i g e s t i o n o f f r a g m e n t C-1 b y R N A a s e A. F i r s t d i m e n s i o n : E l e c t r o p h o r e s i s o n c e l l u l o s e a c e t a t e in 7 M u r e a a t p H 3 . 5 . S e c o n d d i m e n s i o n : E l e c t r o p h o r e s i s o n D E A E - p a p e r a t p H 1 . 9 . T h e n u m b e r e d s p o t s are i d e n t i f i e d in T a b l e s I a n d II. T h e s y m b o l bl i n d i c a t e s t h e p o s i t i o n o f t h e b l u e d y e m a r k e r ( x y l e n e c y a n o l F F ) .
1.4 1.2 0.8 1.0 1.8 2.3 5.1 2.4 0.9
A--U--Gp C--A--U--Gp A--A---C--A--C--A--A--Gp
a b c d e
[1] [1] [1] [1] [1] [2] [4] [1] [1]
-1.3 0.6 1.0 1.8 2.4 5.3 1.0 -[1] [1] [1] [1] [2] [4] [0]
1.0 [I] 0.9 [I]
-1.0 [ 1 ]
0.9 [ 1 ] 1.1 [1]
C-2
-1.2 -1.0 --0.2 0.5 -[0] [0]
[1]
[1]
1.0 [I] 1.0 [I]
---
-0.9 [1]
C-3
A--A---Gp, 1.0 A - - A - C p , 2.0 Up A - - A - - U p , 0.9 A - - C p , 1.0 G p Cp, 0.9 U p A--A---Gp, 1.2 Cp, 2.0 U p A - - A - - A - - U p , 0.9 G p A - - U p , 1.0 G p A - - U p , 1.1 Gp, 0.9 Cp A--A---Gp, 1.0 A - - A - - C p , 0.9 A - - C p A - - G p , 0.9 A---Cp
1.0 A - - A - - U p , 1.3 Gp) 0.8 U p
1.0 1.1 1.1 1.0 1.0 1.0 1.0 0.9 1.1
1.0 A - - A - - C p , 1.0 A--.Gp, 2.8 Cp, 4.3 Up 1.0 A - - U p , 1.0 G p , 3.0 Cp, 2.1 U p
P r o d u c t s of d i g e s t i o n b y R N A a s e A c
U - - U - - G p , 1.7 A p
(C,U 2 ) A p , G p , A p U - - G p , 2.9 A p U--Gp, Ap U--Gp, C--Ap G p , 2.1 C - - A p , 2.8 A p G p , C--AD, A p
( C 4 , U 3 ) A P , U - - A p , G p , 1.6 A p (C 2 ,U 3 )Gp, ( C , U 2 ) G p e, U---Cp e, C - - A p ( C , U ) A p , U - - A p , G p , 1.9 A p U - - U - - G p , 2.1 C - - A p , Ap
Products of digestion by R N A a s e U2 d
T h e s p o t n u m b e r s a n d t h e s e q u e n c e s of t h e o l i g o n u c l e o t i d e s are t h o s e given b y P o r t e r et al. [ 1 3 ] , e x c e p t for s p o t A. E x p e c t e d values are given in p a r e n t h e s e s . Identification by electrophotetic mobility only. I d e n t i f i c a t i o n b y m o b i l i t y a n d b y m o n o n u c l e o t i d e analysis using r i b o n u c l e a s e T 2 . T h e s e f r a g m e n t s are o b t a i n e d f r o m o l i g o n u c l e o t i d e No. 5 b y o v e r d i g e s t i o n w i t h r i b o n u c l e a s e U2, w h i c h u n d e r t h e s e c o n d i t i o n s i n t r o d u c e s a f u r t h e r split at t h e C p U b o n d of t h e n o r m a l digestion p r o d u c t U-C-U-U-C-Gp.
A--Gp c C--C--Gp c C--Gp c Gp c A--A--U--U--Gp
A--C--A--Gp
1.0 [1] 1.0 [1]
U--C--U--A--A--Gp A--A--A--U--Gp
7
8 10 11 12 13 14 15 16 17 A
0.7 [1] 1.0 [ 1 ]
U--A--A--C--U--A--A---Gp C--A--C--A--A--U--U--Gp
6a 6b
0.9 [1] 1.3 [1]
C-1
M o l a r yield in f r a g m e n t b
U--A--A--C--U--C--U--C--U--C--A--Gp C--A--U--C--U--U---C--Gp
Sequence a
1 5
S p o t No. a
C A T A L O G OF O L I G O N U C L E O T I D E S O B T A I N E D BY R N A a s e T 1 D I G E S T I O N OF F R A G M E N T S C - l , C-2 A N D C-3
TABLE I
181
C-3
(27
N.)
C-2
(71N.)
C-1
(88
N.)
r
~o 2O 3O 40 U.A-A- C.U .A-A- G-G-A-U-G-A-A-A-U-G-C-A-U-G-U- C-U-A-A- G-A- C-A- G-C-A-U-C-U-U-C-G-C-G-U-A-A-C-fMet
-
Set
-
Lys
-
Thr
-
Ala
-
Ser
-
Ser
-
Arg
-
Ash
-
C-2 C-1 50 60 70 8O . _U.C.U.Co0- C-A-G-C. G-C - ~ C-A-A-U-U-G-C-G.CoC- G-AoG-C-C.G. CoG-A-AoC-A-C-A°AoG-AoAoU-U-Gp
---
Ser
-
Leu
-
Ser
-
Ala
-
Gin
-
Leu
-
Arg
-
Arg
-
Ala
-
Ala
-
Asn
-
Th¢
~
Arg
-
lie
-
Fig. 3. R N A s e q u e n c e a r o u n d the b e g i n n i n g o f t h e replicase c i s t r o n c o v e r e d b y f r a g m e n t s C-1, C-2 a n d
C-3.
T A B L E II C A T A L O G O F O L I G O N U C L E O T I D E S O B T A I N E D BY R N A a s e A D I G E S T I O N O F F R A G M E N T C - l , C-2 a n d C-3 Spot No.
Sequence a
M o l a r yield in f r a g m e n t b C-2
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 B C D
A--A--G--G--A--Up Cr-A--A--A--Up G--A--G--Cp A--A--Cr--A--Cp G--Up c G--A--A--Cp A--A--Gpc A--A--Up c A--G--Cp A--Upc G--Cp c A--A--Cp c A---Cp c Up c Cp c A--A--G--A--A--Up A--A--A--Upc Gp c
1.0 0.9 1.0 0.8 2.7 0.9 -1.0 1.8 2.3 5.9 2.0 2.6 10.2 8.4 1.0 -1.2
[1] [1] [1] [1] [2] [1] [I] [2] [2] [6] [2] [2] [10] [S] [1] [1]
C-2
C-3
--1.1 0.9 2.4 1.0 0.7 1.0 2.0 2.5 6.2 1.0 2.6 5.7 7.0 -1.0
---1.0 1.1 ---1.0 2.1 1.1 --2.7 2.6 -1.1 0.9
--
[1] [1] [2] [1] [1] [1] [2] [2] [6] [1] [2] [7] [8] [1]
P r o d u c t s of d i g e s t i o n b y R N A a s e T ! c
[1] [1]
1.0 1.0 1.2 1.0
A - - A - - G p , 1.1 A - - U p , 0.8 G p A - - A - - A - - U p , 0.9 G p A - - G p , 0.9 G p , 0.7 Cp A - - A - - G p , 1.0 A - - C p
1.0 A--A---Cp, 1.0 G p
[1] [2] [1]
1.0 A - - G p , 1.0 Cp
[3] [3] 0.9 A - - A - - G p , 1.1 A - - A - - U p [I] [I]
a T h e s p o t n u m b e r s a n d o l i g o n u c l e o t i d e s e q u e n c e s are t h o s e g i v e n b y P o r t e r et al. [ 1 3 ] , e x c e p t for s p o t s B, C a n d D. b E x p e c t e d v a l u e s are given in p a r e n t h e s e s . c Identification by electrophoretic mobility only.,
182
A
oI ~$
C'gi o lA" G 7 C-3 Initiation
G.C
A,U-A G.C ,o.# A.U, i ~5"-3'~U-A-A-C-U-A-A-G-G-A-U-GA-A" C J tC-A-C-A-A-G-A-A-U-U-Gp "UtA o", CG
G:¢ c.9
A-A-U-GoC
c' c b-c-u-c.d ,C.G,
¢., ,G.C C.G A.U, C" U ~A_At Fig. 4. Possible s e c o n d a r y s t r u c t u r e of R N A n e a r the c o a t p r o t e i n b i n d i n g site. On the basis of the AG v a l u e s g i v e n b y T i n o c o et al. [ 2 1 ] the free energies of helix f o r m a t i o n f o r the small a n d the large l o o p are a b o u t - - 6 . 6 a n d - - 2 3 . 6 kcal ( - - 2 7 . 6 a n d - - 9 8 . 7 J) r e s p e c t i v e l y .
Examination of the RNA sequence around the start of the replicase cistron reveals the potential for considerable secondary structure (Fig. 4). Of particular interest is the probable presence of a helical hairpin structure comprising the initiation codon. This structure forms the major portion of C-3, the smallest of the fragments isolated. Convincing physical evidence has been given [10] that on R17 RNA a similar helical hairpin element is the site of direct interaction with coat protein. By analogy it seems likely that in the case of Qfl, the element recognized by coat protein is the helical structure contained in fragment C-3. Materials and Methods Uniformly labeled [32p]Qfl RNA was prepared essentially as described earlier [15]. The specific radioactivity was up to 12 • l 0 s cpm/pg. The material showed considerable degradation within several days of storage at --25 ° C. The preparation of purified Q~ coat protein was described by Hofstetter et al. [14]. The preparation used contained no detectable (i.e. less than 1%) A2 protein and only trace amounts (less than 4%) of A1 protein. In order to obtain the protein in a solution free of sodium dodecyl sulfate, the preparation was precipitated by addition of 3 volumes of ethanol. After storage for several hours at --25 ° C, the precipitate was recovered by centrifugation, washed with 80% ethanol and centrifuged again. The protein pellet was dried in vacuo and dissolved in a solution containing 0.05 M Tris/acetate (pH 7.8), 0.01 M mercap-
183
toethanol and 9.4 M urea. Removal of remaining traces of sodium dodecyl sulfate by filtration through Dowex-1 [ 16 ] did not have any effect. Ribonuclease T1 and ribonuclease U2 were obtained from Calbiochem, ribonuclease A from Worthington. Partially purified ribonuclease T2 was prepared from Taka Diastase (Sankyo) as described by Hiramaru et al. [ 17]. Gel electrophoresis w.as carried out on 200 × 400 × 4 mm slabs using concentrations of 20% acrylamide and 1% bisacrylamide in the buffer system described by Peacock and Dingman [ 1 8 ] . The gels were run overnight at 700 V. Autoradiographs were prepared using Fuji RX Medical X-ray film. Slices corresponding to the radioactive bands were cut from the gel and the RNA was recovered by extraction of the crushed gel pieces with 0.4 M NaC1, followed by precipitation with 2.5 volumes of ethanol in presence of 10--20 pg/ml carrier yeast RNA. Ribonuclease digestions of [ 3 :p] RNA fragments, two-dimensional ionophoretic separations of [ 32 p] oligonucleotides and further techniques were used for RNA sequence determination as described by Brownlee [ 19]. Acknowledgements I am indebted to Dr. Charles Weissmann for his support thank Ms. Sylvia Schmidlin for excellent technical assistance Monstein for the preparation of Q~ coat protein. This project was the Schweizerische Nationalfonds (No. 3.132.73) and the Jane Fund (No. 243).
and advice. I and Mr. H.J. supported by Coffin Childs
References 1 Weissmann, C., Biileter, M.A., Goodman, H.M., Hindley, J. and Weber, H. (1973) Annu. Rev. Binchem. 42, 303--328 2 Sugiyama, T., Korant, B.D. and Lonberg-Holm, K.K. (1972) Annu. Rev. Microbiol. 26, 467--502 3 Weber, H., BiHeter, M., Kahane, S., Hindley, J., Porter, A. and Weissmann, C. (1972) Nat. New Biol. 237, 166--170 4 Kolakofsky, D. and Weissmann, C. (1971) Nat. New Biol. 231, 42--46 5 Kolakofsky, D. and Weissmann, C. (1971) Biochim. Biophys. Acta 2 4 6 , 5 9 6 - - 5 9 9 6 Lodish, H.F. and Zinder, N.D. (1966) J. Mol. Biol. 19, 333--348 7 Sugiyama, T. and Nakada, D. (1967) Proc. Natl. Aead. Sci. U.S. 57, 1 7 4 4 - - 1 7 5 0 8 Bernardi, A. and Spahr, P.F. (1972) Proe. Natl. Acad. Sci. U.S. 69, 3 0 3 3 - - 3 0 3 7 9 Steitz, J.A. (1974) Natttre 248, 223--225 10 Gralla, J., Steitz, J.A. and Crothers, D.M. (1974) NattLre 2 4 8 , 2 0 4 - - 2 0 8 11 Horiuchi, K. and Matsuhashi, S. (1970) Virology 42, 49--60 12 Sanger, F., Brownlee, G.G. and Barrell, B.G. (1985) J. Mol. Biol. 1 3 , 3 7 3 - - 3 9 8 13 Porter, A.G., Hindley, J. and Billeter, M.A. (1974) Eur. J. Biochem. 4 1 , 4 1 3 - - 4 2 0 14 Hofstetter, H., Monstein, H.-J. and Weissmann, C. (1974) Biochim. Biophys. Acta 374, 238--251 15 Weissmann, C., Colthart, L. and Libonati, M. (1968) Biochemistry 7 , 8 6 5 - - 8 7 4 16 Weber, K. and Kuter, D.J. (1971) J. Biol. Chem. 246, 4 5 0 4 - - 4 5 0 9 17 Hiramaru, M., Uchida, T. and Egami. F. (1966) Anal. Biochem. 17, 135--142 18 Peacock, A.C. and Dingman, C.W. (1968) Biochemistry 7 , 6 6 8 - - 6 7 4 19 Brownlee, G.G. (1972) Determination of sequences in R N A , North Holland, Amsterdam 20 Uchida, T. and Egami, F. (1966) in Procedures in Nucleic Acid Rese~Lrch (Cantoni, G.L. and Davies, D.R., eds), pp. 3--13, Harper & R o w , New York 21 Tinoco, Jr, I., Borer, P.N., Dengler, B., Levine, M.D., Uhlenbeck, O.C., Crothers, D.M. and Gralla, J. (1973) Nat. New Biol. 246, 40--41
Biochimica et Biophysica Acta, 418 (1976) 175--183 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98495
THE BINDING SITE F O R COAT PROTEIN ON BACTERIOPHAGE Q~ R N A
HANS WEBER
Institut fiir Molekularbiologie I, Universith't Ziirich, 8049 Ziirich (Switzerland)
(Received June 23rd, 1975)
Summary The site of interaction of phage Q~ coat protein with Q~ RNA was determined by ribonuclease TI degradation of complexes of coat protein and [ 32p]. R N A obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and b o u n d [ 32p] R N A fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Q~, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.
Introduction Virus-specific protein synthesis in RNA phage-infected cells is subject to a variety of control mechanisms [1,2]. A particular feature of the phage systems appears to be the use of translational repressors, namely proteins which bind to the ribosomal attachment site at the beginning of a cistron and thereby prevent the ribosomes from initiating the synthesis of the corresponding protein. Such a function has been assigned to Q~ replicase, which was shown to bind to Q~ R N A at the coat cistron initiation site [3] and to prevent the initiation of coat protein synthesis [4,5]. For group I phages (R17, MS2, f2), work on coat protein mutants as well as in vitro studies on protein synthesis suggested a role for coat protein as a repressor of the synthesis of the replicase protein late in infection [6,7]. This role was confirmed by the finding that R17 coat protein indeed binds to R17 RNA at the beginning of the replicase cistron [8]. Furthermore, RNA-fragment binding studies [9] as well as measurements of the relaxation kinetics of the melting o f fragment-protein complexes indicated that the R17 RNA-coat pro-
176 tein interaction occurs precisely on the small hairpin helix structure containing the initiator codon [ 10]. In the Qj3 system, results obtained with coat protein mutants suggested a similar regulatory role for coat protein [11]. We demonstrate here that the Qfl coat protein, too, is capable of a specific interaction at the initiation site for the replicase cistron of Q~3 RNA. Using techniques similar to those employed previously, we have degraded coat protein-RNA complexes with ribonuclease T1 and isolated three protein-bound RNA fragments. Sequence analysis showed all of these to be derived from the replicase cistron initiation site. As in the case of R17, the smallest fragment characterized (27 nucleotides) appears to consist of a small hairpin helix containing the initiator codon. Results and Discussion Since Qfi coat protein has a very low solubility in aqueous buffers, complex formation was carried o u t by codialysis from concentrated urea solution into buffer solutions. The reaction was followed by determining the fraction of [ 32p] Qfi RNA retained on Millipore filters due to its binding to coat protein. Preliminary experiments showed that in presence of a 35- to 45-fold molar excess of coat protein, up to about 40% of the RNA acquired the ability to bind to Millipore within 1--3 h of dialysis at room temperature. Further prolongation of dialysis did n o t increase the extent of reaction, while reduction of the excess of coat protein decreased it very considerably. Ribonuclease T1 degradation of the complexes was done in presence of a 4- to 7-fold excess of unlabeled Qfl RNA in order to reduce the a m o u n t of weakly {and non-specifically) bound [ 32p] RNA material by competition. The e x t e n t of degradation was arbitrarily selected so as to yield a b o u t 1% of the total initial RNA. The degraded complexes were collected by binding to Millipore filters, from which the [ 3 2 P ] R N A fragments were extracted using phenol/buffer mixtures. Fig. 1 shows an autoradiographic pattern obtained by electrophoretic separation of such fragment mixtures on 20% polyacrylamide gels. The three most p r o m i n e n t fragments, C-l, C-2 and C-3 (together comprising about 0.15% of the total initial radioactivity) were extracted from the gels, one part of each was digested with ribonuclease T1 and another with ribonuclease A. The products of these digestions were fractionated by twodimensional ionophoresis [ 12]. The autoradiograms obtained showed very conspicuous similarities to those reported for a fragment derived from the region around the replicase cistron ribosomal binding site by Porter et al. [13]. In particular, the RNAase T1 fingerprint of fragment C-1 (Fig. 2A) is identical to the one reported for fragment 2 in reference 13, with the exception of one additional spot, designated A. Accordingly, the RNAase A fingerprint obtained from C-1 (Fig. 2B) is identical to the one reported for fragment 2 except for the disappearance of spot No. 7 and the appearance of a new spot, designated B (along with t w o additional mononucleotides, Gp and Up). The analytical data of the various digestion products of the fragments C-I, C-2 and C-3 are compiled in Tables I and II. They leave no d o u b t that C-1 is a fragment of 88 nucleotides comprising the whole sequence of fragment 2 of
177
Cm
m~ ,
C2
C ~3
~
b|-~ Fig. 1. Gel e l e c t r o p h o r e s i s o f [ 3 2 p ] Q ~ R N A f r a g m e n t s r e t a i n e d on Millipore b y c o a t p r o t e i n b i n d i n g . [ 3 2 p ] Q#3 R N A ( 1 5 0 #g, 0.1 n m o l , specific r a d i o a c t i v i t y 3 • 106 e p m / ~ g ) a n d Q~ c o a t p r o t e i n (50/~g, 3.6 n m o l ) , in 50 m M T r i s / a c e t a t e ( p H 7 . 8 ) / 1 0 m M m e r c a p t o e t h a n o l / 9 . 4 M u r e a (0.5 m l ) w e r e d i a l y z e d against 5 0 0 m l of a b u f f e r c o n t a i n i n g 1 0 0 m M Tris • HC1 ( p H 7 . 5 ) , 1 5 0 m M NaCl, 10 m M MgCI2, 10 m M m e r c a p t o e t h a n o l a n d 1 m M E D T A at 25 °. A f t e r 1 h a 2/~1 a l i q u o t was d i l u t e d to 0.5 m l w i t h dialysis b u f f e r , filtered t h r o u g h Mfllipore ( H A W P , p o r e size 0 . 4 5 /~m) a n d w a s h e d w i t h t h r e e 1-ml p o r t i o n s of dialysis b u f f e r : a b o u t 37% of the R N A w a s b o u n d to t h e filter. T h e m a i n s a m p l e w a s t r a n s f e r r e d f r o m t h e dialysis b a g i n t o a t u b e at 2 5 ° C , a n d 1 m g o f u n l a b e l e d Q#3 R N A a n d 1 0 0 0 u n i t s [ 2 0 ] o f R N A a s e T I w e r e a d d e d . A f t e r 5 rain at 25QC, t h e first h a l f o f the s a m p l e ( A ) w a s filtered t h r o u g h Millipore (3 X 1 m l washings). T h e r a d i o a c t i v i t y r e t a i n e d as d e t e r m i n e d b y C h e r e n k 0 v c o u n t i n g w a s a b o u t 1.0% of t h e t o t a l R N A a p p l i e d to t h e filter. T h e filter w a s i m m e d i a t e l y e x t r a c t e d b y v i g o r o u s s h a k i n g in a m i x t u r e c o n t a i n ing 1 m l o f e x t r a c t i o n b u f f e r (20 m M Tris • HC1 ( p H 7 . 5 ) , 2 m M E D T A , 0.1% s o d i u m d o d e c y l sulfate, 50 ~tg o f y e a s t c a r r i e r R N A ) a n d 1 m l o f redistilled p h e n o l e q u i l i b r a t e d w i t h e x t r a c t i o n b u f f e r . T h e s e c o n d h a l f o f t h e s a m p l e (B) w a s f i l t e r e d a f t e r 10 rain o f i n c u b a t i o n a n d a b o u t 0.8% of t h e original r a d i o a c t i v i t y w a s r e t a i n e d . F i l t e r B w a s e x t r a c t e d in t h e s a m e w a y as filter A, t h e e x t r a c t i o n m i x t u r e s w e r e c o o l e d to 0°C a n d c e n t r i f u g e d in a clinical c e n t r i f u g e . T h e R N A w a s p r e c i p i t a t e d f r o m the a q u e o u s l a y e r s b y t h e a d d i t i o n of 0.1 vol o f 0.5 M Tris • HCI ( p H 7 . 5 ) , 1.0 M NaC1, 0 . 0 5 M E D T A , a n d 2 v o l u m e s o f cold e t h a n o l . A f t e r s t o r a g e o v e r n i g h t at - - 2 5 ° C , t h e R N A w a s c o l l e c t e d b y c e n t r i f u g a t i o n , d r i e d in v a c u o a n d dissolved in a s o l u t i o n o f 12% s u c r o s e a n d 0 . 0 4 % b r o m o p h e n o l b l u e ( 5 0 /~1). T h e s a m p l e s (A, 3.2 • 106 c p m ; B, 1.6 • 106 c p m ) w e r e i n s e r t e d i n t o t h e slots o f a 20% p o l y a c r y l a m i d e gel p r e p a r e d a n d r u n as d e s c r i b e d in Materials a n d M e t h o d s . T h e s y m b o l bl i n d i c a t e s t h e p o s i t i o n o f t h e b r o m o p h e n o l blue marker.
178
Porter et al. [13] plus an additional pentanucleotide A-A-U-U-Gp at its 3' end {Fig. 3). Fragment C-2 has a length of 71 nucleotides and corresponds to C-1 lacking the twelve 5'-terminal and the five 3'-terminal nucleotides. Fragment C-3, finally, consists of the 27 nucleotides at the 5' end of fragment C-2. The finding that all three fragments contain the initiator c o d o n of the replicase cistron demonstrates the affinity of coat protein for this site. We presume that in the Q~ system, as in the case of R17, the coat protein blocks the synthesis of replicase protein late in infection by direct binding to the ribosomal initiation site for this cistron. The possibility that contaminating amounts of the minor capsid proteins, A1 and A2 [ 1 4 ] , could be involved in
PH35_5.
qlP
! :DEAE
i ea
08
.p
bl
• !
130 !O
8 e
14~
D
015
t
17 Fig. 2 A .
179 the binding of this site is considered unlikely because: (a) most of the minor proteins had been removed during the purification of the coat protein; and (b) control experiments using proteins A1 or A2 instead of coat protein under otherwise identical conditions for the formation and degradation of complexes with [ 3 2p] Q~ RNA did not result in any specific fragment patterns (data not shown).
lO t
D
g
g
~
J
Fig. 2B.
Fig. 2. A u t o r a d i o g r a m s o f t w o - d i m e n s i o n a l f i n g e r p r i n t s o f f r a g m e n t C-1. (A) P r o d u c t s o f t h e d i g e s t i o n o f f r a g m e n t C-1 b y R N A a s e T 1 . F i r s t d i m e n s i o n : E l e c t r o p h o r e s i s o n c e l l u l o s e a c e t a t e in 7 M u r e a a t p H 3 . 5 . S e c o n d d i m e n s i o n : E l e c t r o p h o r e s i s o n D E A E - p a p e r in 7% f o r m i c a c i d . (B) P r o d u c t s o f t h e d i g e s t i o n o f f r a g m e n t C-1 b y R N A a s e A. F i r s t d i m e n s i o n : E l e c t r o p h o r e s i s o n c e l l u l o s e a c e t a t e in 7 M u r e a a t p H 3 . 5 . S e c o n d d i m e n s i o n : E l e c t r o p h o r e s i s o n D E A E - p a p e r a t p H 1 . 9 . T h e n u m b e r e d s p o t s are i d e n t i f i e d in T a b l e s I a n d II. T h e s y m b o l bl i n d i c a t e s t h e p o s i t i o n o f t h e b l u e d y e m a r k e r ( x y l e n e c y a n o l F F ) .
1.4 1.2 0.8 1.0 1.8 2.3 5.1 2.4 0.9
A--U--Gp C--A--U--Gp A--A---C--A--C--A--A--Gp
a b c d e
[1] [1] [1] [1] [1] [2] [4] [1] [1]
-1.3 0.6 1.0 1.8 2.4 5.3 1.0 -[1] [1] [1] [1] [2] [4] [0]
1.0 [I] 0.9 [I]
-1.0 [ 1 ]
0.9 [ 1 ] 1.1 [1]
C-2
-1.2 -1.0 --0.2 0.5 -[0] [0]
[1]
[1]
1.0 [I] 1.0 [I]
---
-0.9 [1]
C-3
A--A---Gp, 1.0 A - - A - C p , 2.0 Up A - - A - - U p , 0.9 A - - C p , 1.0 G p Cp, 0.9 U p A--A---Gp, 1.2 Cp, 2.0 U p A - - A - - A - - U p , 0.9 G p A - - U p , 1.0 G p A - - U p , 1.1 Gp, 0.9 Cp A--A---Gp, 1.0 A - - A - - C p , 0.9 A - - C p A - - G p , 0.9 A---Cp
1.0 A - - A - - U p , 1.3 Gp) 0.8 U p
1.0 1.1 1.1 1.0 1.0 1.0 1.0 0.9 1.1
1.0 A - - A - - C p , 1.0 A--.Gp, 2.8 Cp, 4.3 Up 1.0 A - - U p , 1.0 G p , 3.0 Cp, 2.1 U p
P r o d u c t s of d i g e s t i o n b y R N A a s e A c
U - - U - - G p , 1.7 A p
(C,U 2 ) A p , G p , A p U - - G p , 2.9 A p U--Gp, Ap U--Gp, C--Ap G p , 2.1 C - - A p , 2.8 A p G p , C--AD, A p
( C 4 , U 3 ) A P , U - - A p , G p , 1.6 A p (C 2 ,U 3 )Gp, ( C , U 2 ) G p e, U---Cp e, C - - A p ( C , U ) A p , U - - A p , G p , 1.9 A p U - - U - - G p , 2.1 C - - A p , Ap
Products of digestion by R N A a s e U2 d
T h e s p o t n u m b e r s a n d t h e s e q u e n c e s of t h e o l i g o n u c l e o t i d e s are t h o s e given b y P o r t e r et al. [ 1 3 ] , e x c e p t for s p o t A. E x p e c t e d values are given in p a r e n t h e s e s . Identification by electrophotetic mobility only. I d e n t i f i c a t i o n b y m o b i l i t y a n d b y m o n o n u c l e o t i d e analysis using r i b o n u c l e a s e T 2 . T h e s e f r a g m e n t s are o b t a i n e d f r o m o l i g o n u c l e o t i d e No. 5 b y o v e r d i g e s t i o n w i t h r i b o n u c l e a s e U2, w h i c h u n d e r t h e s e c o n d i t i o n s i n t r o d u c e s a f u r t h e r split at t h e C p U b o n d of t h e n o r m a l digestion p r o d u c t U-C-U-U-C-Gp.
A--Gp c C--C--Gp c C--Gp c Gp c A--A--U--U--Gp
A--C--A--Gp
1.0 [1] 1.0 [1]
U--C--U--A--A--Gp A--A--A--U--Gp
7
8 10 11 12 13 14 15 16 17 A
0.7 [1] 1.0 [ 1 ]
U--A--A--C--U--A--A---Gp C--A--C--A--A--U--U--Gp
6a 6b
0.9 [1] 1.3 [1]
C-1
M o l a r yield in f r a g m e n t b
U--A--A--C--U--C--U--C--U--C--A--Gp C--A--U--C--U--U---C--Gp
Sequence a
1 5
S p o t No. a
C A T A L O G OF O L I G O N U C L E O T I D E S O B T A I N E D BY R N A a s e T 1 D I G E S T I O N OF F R A G M E N T S C - l , C-2 A N D C-3
TABLE I
181
C-3
(27
N.)
C-2
(71N.)
C-1
(88
N.)
r
~o 2O 3O 40 U.A-A- C.U .A-A- G-G-A-U-G-A-A-A-U-G-C-A-U-G-U- C-U-A-A- G-A- C-A- G-C-A-U-C-U-U-C-G-C-G-U-A-A-C-fMet
-
Set
-
Lys
-
Thr
-
Ala
-
Ser
-
Ser
-
Arg
-
Ash
-
C-2 C-1 50 60 70 8O . _U.C.U.Co0- C-A-G-C. G-C - ~ C-A-A-U-U-G-C-G.CoC- G-AoG-C-C.G. CoG-A-AoC-A-C-A°AoG-AoAoU-U-Gp
---
Ser
-
Leu
-
Ser
-
Ala
-
Gin
-
Leu
-
Arg
-
Arg
-
Ala
-
Ala
-
Asn
-
Th¢
~
Arg
-
lie
-
Fig. 3. R N A s e q u e n c e a r o u n d the b e g i n n i n g o f t h e replicase c i s t r o n c o v e r e d b y f r a g m e n t s C-1, C-2 a n d
C-3.
T A B L E II C A T A L O G O F O L I G O N U C L E O T I D E S O B T A I N E D BY R N A a s e A D I G E S T I O N O F F R A G M E N T C - l , C-2 a n d C-3 Spot No.
Sequence a
M o l a r yield in f r a g m e n t b C-2
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 B C D
A--A--G--G--A--Up Cr-A--A--A--Up G--A--G--Cp A--A--Cr--A--Cp G--Up c G--A--A--Cp A--A--Gpc A--A--Up c A--G--Cp A--Upc G--Cp c A--A--Cp c A---Cp c Up c Cp c A--A--G--A--A--Up A--A--A--Upc Gp c
1.0 0.9 1.0 0.8 2.7 0.9 -1.0 1.8 2.3 5.9 2.0 2.6 10.2 8.4 1.0 -1.2
[1] [1] [1] [1] [2] [1] [I] [2] [2] [6] [2] [2] [10] [S] [1] [1]
C-2
C-3
--1.1 0.9 2.4 1.0 0.7 1.0 2.0 2.5 6.2 1.0 2.6 5.7 7.0 -1.0
---1.0 1.1 ---1.0 2.1 1.1 --2.7 2.6 -1.1 0.9
--
[1] [1] [2] [1] [1] [1] [2] [2] [6] [1] [2] [7] [8] [1]
P r o d u c t s of d i g e s t i o n b y R N A a s e T ! c
[1] [1]
1.0 1.0 1.2 1.0
A - - A - - G p , 1.1 A - - U p , 0.8 G p A - - A - - A - - U p , 0.9 G p A - - G p , 0.9 G p , 0.7 Cp A - - A - - G p , 1.0 A - - C p
1.0 A--A---Cp, 1.0 G p
[1] [2] [1]
1.0 A - - G p , 1.0 Cp
[3] [3] 0.9 A - - A - - G p , 1.1 A - - A - - U p [I] [I]
a T h e s p o t n u m b e r s a n d o l i g o n u c l e o t i d e s e q u e n c e s are t h o s e g i v e n b y P o r t e r et al. [ 1 3 ] , e x c e p t for s p o t s B, C a n d D. b E x p e c t e d v a l u e s are given in p a r e n t h e s e s . c Identification by electrophoretic mobility only.,
182
A
oI ~$
C'gi o lA" G 7 C-3 Initiation
G.C
A,U-A G.C ,o.# A.U, i ~5"-3'~U-A-A-C-U-A-A-G-G-A-U-GA-A" C J tC-A-C-A-A-G-A-A-U-U-Gp "UtA o", CG
G:¢ c.9
A-A-U-GoC
c' c b-c-u-c.d ,C.G,
¢., ,G.C C.G A.U, C" U ~A_At Fig. 4. Possible s e c o n d a r y s t r u c t u r e of R N A n e a r the c o a t p r o t e i n b i n d i n g site. On the basis of the AG v a l u e s g i v e n b y T i n o c o et al. [ 2 1 ] the free energies of helix f o r m a t i o n f o r the small a n d the large l o o p are a b o u t - - 6 . 6 a n d - - 2 3 . 6 kcal ( - - 2 7 . 6 a n d - - 9 8 . 7 J) r e s p e c t i v e l y .
Examination of the RNA sequence around the start of the replicase cistron reveals the potential for considerable secondary structure (Fig. 4). Of particular interest is the probable presence of a helical hairpin structure comprising the initiation codon. This structure forms the major portion of C-3, the smallest of the fragments isolated. Convincing physical evidence has been given [10] that on R17 RNA a similar helical hairpin element is the site of direct interaction with coat protein. By analogy it seems likely that in the case of Qfl, the element recognized by coat protein is the helical structure contained in fragment C-3. Materials and Methods Uniformly labeled [32p]Qfl RNA was prepared essentially as described earlier [15]. The specific radioactivity was up to 12 • l 0 s cpm/pg. The material showed considerable degradation within several days of storage at --25 ° C. The preparation of purified Q~ coat protein was described by Hofstetter et al. [14]. The preparation used contained no detectable (i.e. less than 1%) A2 protein and only trace amounts (less than 4%) of A1 protein. In order to obtain the protein in a solution free of sodium dodecyl sulfate, the preparation was precipitated by addition of 3 volumes of ethanol. After storage for several hours at --25 ° C, the precipitate was recovered by centrifugation, washed with 80% ethanol and centrifuged again. The protein pellet was dried in vacuo and dissolved in a solution containing 0.05 M Tris/acetate (pH 7.8), 0.01 M mercap-
183
toethanol and 9.4 M urea. Removal of remaining traces of sodium dodecyl sulfate by filtration through Dowex-1 [ 16 ] did not have any effect. Ribonuclease T1 and ribonuclease U2 were obtained from Calbiochem, ribonuclease A from Worthington. Partially purified ribonuclease T2 was prepared from Taka Diastase (Sankyo) as described by Hiramaru et al. [ 17]. Gel electrophoresis w.as carried out on 200 × 400 × 4 mm slabs using concentrations of 20% acrylamide and 1% bisacrylamide in the buffer system described by Peacock and Dingman [ 1 8 ] . The gels were run overnight at 700 V. Autoradiographs were prepared using Fuji RX Medical X-ray film. Slices corresponding to the radioactive bands were cut from the gel and the RNA was recovered by extraction of the crushed gel pieces with 0.4 M NaC1, followed by precipitation with 2.5 volumes of ethanol in presence of 10--20 pg/ml carrier yeast RNA. Ribonuclease digestions of [ 3 :p] RNA fragments, two-dimensional ionophoretic separations of [ 32 p] oligonucleotides and further techniques were used for RNA sequence determination as described by Brownlee [ 19]. Acknowledgements I am indebted to Dr. Charles Weissmann for his support thank Ms. Sylvia Schmidlin for excellent technical assistance Monstein for the preparation of Q~ coat protein. This project was the Schweizerische Nationalfonds (No. 3.132.73) and the Jane Fund (No. 243).
and advice. I and Mr. H.J. supported by Coffin Childs
References 1 Weissmann, C., Biileter, M.A., Goodman, H.M., Hindley, J. and Weber, H. (1973) Annu. Rev. Binchem. 42, 303--328 2 Sugiyama, T., Korant, B.D. and Lonberg-Holm, K.K. (1972) Annu. Rev. Microbiol. 26, 467--502 3 Weber, H., BiHeter, M., Kahane, S., Hindley, J., Porter, A. and Weissmann, C. (1972) Nat. New Biol. 237, 166--170 4 Kolakofsky, D. and Weissmann, C. (1971) Nat. New Biol. 231, 42--46 5 Kolakofsky, D. and Weissmann, C. (1971) Biochim. Biophys. Acta 2 4 6 , 5 9 6 - - 5 9 9 6 Lodish, H.F. and Zinder, N.D. (1966) J. Mol. Biol. 19, 333--348 7 Sugiyama, T. and Nakada, D. (1967) Proc. Natl. Aead. Sci. U.S. 57, 1 7 4 4 - - 1 7 5 0 8 Bernardi, A. and Spahr, P.F. (1972) Proe. Natl. Acad. Sci. U.S. 69, 3 0 3 3 - - 3 0 3 7 9 Steitz, J.A. (1974) Natttre 248, 223--225 10 Gralla, J., Steitz, J.A. and Crothers, D.M. (1974) NattLre 2 4 8 , 2 0 4 - - 2 0 8 11 Horiuchi, K. and Matsuhashi, S. (1970) Virology 42, 49--60 12 Sanger, F., Brownlee, G.G. and Barrell, B.G. (1985) J. Mol. Biol. 1 3 , 3 7 3 - - 3 9 8 13 Porter, A.G., Hindley, J. and Billeter, M.A. (1974) Eur. J. Biochem. 4 1 , 4 1 3 - - 4 2 0 14 Hofstetter, H., Monstein, H.-J. and Weissmann, C. (1974) Biochim. Biophys. Acta 374, 238--251 15 Weissmann, C., Colthart, L. and Libonati, M. (1968) Biochemistry 7 , 8 6 5 - - 8 7 4 16 Weber, K. and Kuter, D.J. (1971) J. Biol. Chem. 246, 4 5 0 4 - - 4 5 0 9 17 Hiramaru, M., Uchida, T. and Egami. F. (1966) Anal. Biochem. 17, 135--142 18 Peacock, A.C. and Dingman, C.W. (1968) Biochemistry 7 , 6 6 8 - - 6 7 4 19 Brownlee, G.G. (1972) Determination of sequences in R N A , North Holland, Amsterdam 20 Uchida, T. and Egami, F. (1966) in Procedures in Nucleic Acid Rese~Lrch (Cantoni, G.L. and Davies, D.R., eds), pp. 3--13, Harper & R o w , New York 21 Tinoco, Jr, I., Borer, P.N., Dengler, B., Levine, M.D., Uhlenbeck, O.C., Crothers, D.M. and Gralla, J. (1973) Nat. New Biol. 246, 40--41
