319

Biochimica et Biophysica Acta, 447 (1976)319--327 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

BBA 98711

ADDITION BY ATP : RNA ADENYLYLTRANSFERASE FROM E S C H E R I C H I A C O L I OF 3'-LINKED OLIGO(A) TO BACTERIOPHAGE Qfi RNA AND ITS EFFECT ON RNA REPLICATION

RENE DEVOS, JOHN VAN EMMELO, CHRIS SEURINCK-OPSOMER, ELIE GILLIS and WALTER FIERS

Laboratorium voor Fysiologische Scheikunde and Laboratorium voor Moleculaire Biologie, Ledeganckstraat 35, B-9000 Gent (Belgium)

(Received January 21st, 1976) (Revised manuscript received May 18th, 1976)

Summary

An oligo(A) or poly(A) segment was added in a stepwise fashion to the 3'-end of bacteriophage Qfi-RNA with the aid of ATP : RNA adenylyltransferase from E s c h e r i c h i a coli . Nearly all RNA molecules, present in the reaction mix- ture, could be polyadenylated.

For tail lengths not exceeding 200 nucleotide residues, the physical proper- ties of Q~-RNA-poly(A) were found to be only slightly different from those of the original RNA. The polyadenylated RNA was purified by affinity chroma- tography.

The properties of Q~-RNA with oiigo(A) tails of different average lengths were investigated in the in vitro replication reaction. Almost complete abolish- ment of template activity, even by short oligo(A) stretches, was found. Further- more, polyadenylated Q~-RNA inhibited the normal replication reaction of Q~- RNA by removal of host factor HFI, in the same way as does free poly(A).

Int roduct ion

Stepwise addition of a poly(A) segment to the 3'-end of bacteriophage RNAs can be achieved in vitro with an ATP : RNA nucleotidyltransferase from E s c h e r i c h i a co l i [1,2]. The efficiency of the addition reaction is dependent upon the type of primer RNA used. In the case of Qfi-RNA, nearly all mole- cules can be polyadenylated.

The sedimentation properties of Qfi-RNA-poly(A) are very similar to those of normal Qfl-RNA. However, glycerol or sucrose gradient centrifugation can be used for separation of free poly(A) from Q~-RNA and Q~-RNA-poly(A). Poly- adenylated Q~-RNA is selectively retained by oligo(dT)-cellulose in high salt

320

and can be eiuted by lowering the salt concentration [3]. It was of interest to investigate the properties of Qfl-RNA with oligo(A) tails

of different average lengths in the in vitro replication reaction, because the in- fectivity of polyadenylated Qfl-RNA in a sferoplast assay was reported to be almost unaltered [4], whereas the template activity of the same material in the Q~-replicase assay was nearly completely abolished [5].

Since free poly(A) is known to interact specifically with host factor HFI [6], the role of this component of the replication complex was more closely examined.

Materials and Methods

ATP : RNA adenylyltransferase was extracted from E. coli B/r and purified by the method of Sippel [1].

Bacteriophage Q~ was grown on E. coli C3000 and the RNA was extracted from purified phage. Homogeneous 30 S Qfi-RNA was selected after zone sedi- mentat ion through glycerol gradients.

Qfl-RNA-instructed RNA polymerase ("replicase") was extracted from E. coli D l l as described [8].

[3H]ATP and [14C] UTP were purchased from The gadiochemical Centre, Amersham, U.K. Unlabelled nucleoside triphosphates were from P and L Bio- chemicals Inc., Milwaukee, Wisc., U.S.A. or from Sigma, St. Louis, Mo., U.S.A. Oligo(dT)-cellutose was obtained from Collaborative Research, Waltham, Mass., U.S.A. and was used according to Aviv and Leder [3].

(rA)2s and (rA)~-5 oligomers were from Miles, Slough, U.K. The average monomer numbers are derived from intrinsic viscosity measurements and corre- spond to monomer numbers 39 and 78 from sedimentation velocity measure- ments, with asymmetric distributions, the 50% ranges being 25--51 and 62--108, respectively (data from manufacturer).

Poly(A) was from P and L Biochemicals and showed an average sedimenta- tion coefficient (s20.w) of 10.

The adenylyltransferase assay contained 50 mM Tris, pH 7.9, 0.5 mM EDTA, 12.5 mM MgC12, 2 mM MnC12, 1 mM dithiothreitol, 160 mM NaC1 and 0.2 mM [3H] ATP (spec. act. 10--50 Ci/mol). Incubations were at 37°C for the times specified.

The Q~-replicase assay contained 85 mM Tris, pH 7.4, 12.5 mM MgC12, 2.5 mM EDTA, 4 mM phosphoenolpyruvate, 40 pg/ml pyruvate kinase (Boehringer, Mannheim, G.F.R.), 0.5 mM each of ATP, GTP and CTP and 0.2 mM [~4C]- UTP (spec. act. 3 or 15.6 Ci/mol) in addition to Qfl-RNA and host factor HFI as specified. Incubations were at 37°C for 15 min.

Results and Discussion

Physicochemical characterization of the reaction products In a typical experiment four aliquots of Q~-RNA at a level of 50--80 pg/ml,

were incubated with ATP : RNA adenylyltransferase for respectively 2, 4 and 6 min at 37°C in the presence of ATP and for 6 min at 37°C in the absence of ATP.

321

1 0 0 0

5 0 o On L © 0 E

E u

i

.~ a i

- L

- i • ,, ;~,

i i - , , ,, ooo ° ; ,,%°°

1 . . • . .

I

"1 o

1 0 0 0 - I !i'i\

', 5 0 0 k ? ~ ~ ,

0 • . . . . . j • . . . .

- d

T O P ~ 10 15 2'0 25 3'0 0

1.0

0 5

0 £

lO E u

E c

( )5 "~

FFClCt ion n u m b e r

Fig. l . S e d i m e n t a t i o n pat tern of Q~J-RNA, i n c u b a t e d w i t h ATP : R N A a d e n y l t r a n s f e r a s e , a , b , c , 2 , 4 , 6

rain at 3 7 ° C w i t h [ 3 H I A T P ( spec . act . 4 6 . 8 C i / m o l ) ; d , 6 min at 3 7 ° C w i t h o u t A T P , A f t e r i n c u b a t i o n , the R N A was r e c o v e r e d by p h e n o l i z a t i o n , p r e c i p i t a t e d w i t h e t h a n o l and d i s so lved in 0 . 1 M NaC1 , 5 m M Tris .

HCI , PH 7 . 0 , I m M MgCl 2 and 0 .1 m M E D T A . Linear grad ients o f 1 0 - - 3 0 % g l y c e r o l in the s a m e b u f f e r w e r e run for 5 h a t 4 0 0 0 0 r e v . / m i n in a B e c k m a n ro tor S W 4 1 . T h e grad ients were transferred t h r o u g h an ISCO U V - m o n i t o r and f r a c t i o n a t e d , 2 5 - p l a l i q u o t s o f e a c h fract ion w e r e u s e d for rad ioac t iv i ty d e t e r m i n a - t ions . Dashed l ine , actual A 2 5 4 rim; Sol id l ine, [ 3 H ] A M P in ac id -pree ip i tab le mater ia l .

After treatment with phenol /chloroform/isoamylalcohol ( 2 5 : 2 4 : 1 , v/v) and ethanol precipitation, the different samples of RNA were sedimented through 10--30% glycerol gradients (Fig. 1). The bulk of the radioactivity was found near the position of the A260 n~ peak of intact Qfl-RNA. The absorbance and radioactivity peak fractions were pooled and further examined.

Aliquots of the glycerol gradient pools were made 0.5 M in KCI or NaC1 and loaded onto oligo(dT)-cellulose columns, equilibrated with high salt buffer (0.5 M NaC1, 10 mM Tris, pH 7, and 4 mM EDTA) [3] . After washing with the same buffer, the bound material was eluted in low salt, i.e. 10 mM Tris, pH 7 (Fig. 2). About 15--20% of the treated Q~-RNA failed to bind to the column, whereas unmodif ied Qfi-RNA does not bind at all.

Polyadenylated Qfi-RNA with poly(A) tails of different lengths was retained by the column but more than 90% of it could be eluted in low salt. Upon analysis of this RNA by electrophoresis through 2.4% polyacrylamide gels the radioactivity profile coincided with the absorbance profile of the Qfi-RNA present {Fig. 3).

These results indicate a high efficiency of polyadenylation of intact Qfl-RNA and very little synthesis of free poly(A) strands by ATP : RNA adenylyltrans- ferase under the conditions used.

On the other hand polyadenylation of MS2-RNA under the same conditions, yielded a product of which only about 50% was retained by the oligo(dT)-cellu- lose column [2] .

The average length of the poly(A) tails attached to Q/3-RNA could be calcu- lated on the basis of the [3H] AMP incorporation versus A26o ,m units of RNA

322

E c o

08

Q4

1 2 3

i

I I I I I I I I I I I I I I I

I I I

4 5 6 7 8 9 10 11 12 13 14 15 16

FrGc t lot1 n u m b e r

o

q

10 <

o7

E Q u 5 ~

o

F i g . 2. O l i g o ( d T ) - c e l l u l o s e c h r o m a t o g r a p h y o f p o l y a d e n y l a t e d Q f l - R N A . I n c u b a t i o n w a s f o r 6 m i n a t 3 7 ° C in t h e p r e s e n c e o f [ 3 H ] A T P ( s p e c . a c t . 4 6 . 8 C i / m o l ) . A f t e r g l y c e r o l g r a d i e n t c e n t r i f u g a t i o n , as in t h e

l e g e n d t o F i g . 1, t h e p e a k f r a c t i o n s w e r e p o o l e d , p r e c i p i t a t e d w i t h e t h a n o l a n d d i s s o l v e d in 6 0 p l o f 0 . 5 M

NaC1, 10 m M T r i s , p H 7, a n d 4 m M E D T A . T h e R N A w a s c h r o m a t o g r a p h e d o v e r a 0 . 6 X 1.6 c m o l igo - ( d T ) - c e n u l o s e c o l u m n a t r o o m t e m p e r a t u r e a n d f r a c t i o n s o f 3 0 0 p l w e r e c o l l e c t e d . B o u n d m a t e r i a l w a s

e l u t e d in 1 0 m M T r i s , p H 7 ( c h a n g e - o v e r t o l o w sa l t e l u t i o n is i n d i c a t e d b y d a s h e d l ine ) . 2 0 0 pl o f e a c h

f r a c t i o n w a s c o u n t e d in a T r i t o n - t o l u o l l i q u i d s c i n t i l l a t i o n c o u n t i n g m i x t u r e f o r r a d i o a c t i v i t y d e t e r m i n a -

t i o n s . H a t c h e d a r e a , A260 n m , m e a s u r e d d i r e c t l y in a 2 5 0 p l ce l l o f a G i l f o r d s p e c t r o p h o t o m e t e r ; d o t t e d a r e a , [ 3 H ] A M P l abe l . T h e n o n - r e t a i n e d m a t e r i a l r e p r e s e n t e d 2 0 % o f t h e t o t a l a b s o r b a n c e a n d 7 % o f t h e

t o t a l r a d i o a c t i v i t y .

in the oligo(dT)-cellulose fractions and was in reasonable agreement with the values found during the intermediate purification steps (Table I).

Functional characterization o f the reaction products This section contains data on template activity and inhibitory effects of

polyadenylated Q~-RNA in the in vitro RNA replication reaction. Polyadenylated Q/3-RNA, purified by glycerol gradient centrifugation and

oligo(dT)-cellulose chromatography, was used as a template in the Q/3-replicase assay, but none of the preparations showed significant activity (Fig. 4). The template activity of the modified RNA could not be restored by omission or by

T A B L E I

M O L A R R A T I O O F [ 3 H ] A M P I N C O R P O R A T E D I N A C I D - P R E C I P I T A B L E M A T E R I A L T O P R I M E R

Q ~ - R N A D E T E R M I N E D B Y A 2 5 4 n m O R A 2 6 0 n m M E A S U R E M E N T

E a c h i n c u b a t i o n m i x t u r e c o n t a i n e d 6 0 p g / m l o f Q ~ - R N A a n d 0 . 2 m M [ 3 H ] A T P ( s p e c . ac t . 4 6 . 8 o r 5 0 C i / m o l ) . A T P : R N A a d e n y l y l t r a n s f e r a s e w a s u s e d a t a l eve l o f 2 0 b tg /ml ( p h o s p h o c e l l u l o s e f r a c t i o n a c c o r d - i ng to S i p p e l [ 1 ] ). W h e r e e n o u g h i n d e p e n d e n t v a l u e s w e r e a v a i l a b l e , s t a n d a r d d e v i a t i o n s are g i v e n .

I n c u b a t i o n I n c u b a t i o n G r a d i e n t p e a k O l i g o ( d T ) - c e l - t i m e ( m i n ) m i x t u r e f r a c t i o n lu lo se l o w sa l t

e l u t i o n f r a c t i o n

2 4 5 53 4 1 5 8 1 3 4 5 2 4 4 1 5 6 6 2 3 7 + 2 5 1 4 3 ± 41 1 3 5 f 6

323

10-

9

8 a

6

1

i TOP 10

23S 16S

20 30 40 5 ; 6'0 70

Slice number

Fig. 3. Po lyacry la rn ide gel e l ec t rophores i s of p o l y a d e n y l a t e d Qfl-RNA af te r b an d in g in a sucrose g rad ien t and ol igo(dT)-cel lu lose c h r o m a t o g r a p h y . E lec t rophores i s was car r ied o u t in a cyl indr ica l 2.4% po lyac ry l - amide gel in a b u f f e r which c on t a ine d 0.1% s o d i u m d o d e c y l sulfate , 10% glycerol , 40 m M Tr is -ace ta te , 33 mM sod ium ace ta t e and 0.7 mM E D T A , ad jus ted to pH 7.2. Af t e r a p r e ru n , the gels were l oaded and run for 2 h at 5 m A pe r gel. A f t e r scanning in u l t r av io le t wi th a J o y c e - L o e b l C h r o m o s c a n , the gels were cu t in 1 m m slices for coun t ing . Solid l ine, A 2 6 0 n m scan (unca l ib ra t ed ) ; h i s t og ram, [3H] AMP label pe r gel slice, a f t e r e lu t ion in s o d i u m d o d e c y l sulfate b u f f e r and c o u n t i n g in T r i ton - to luo l l iquid scint i l la t ion c o u n t i n g m i x t u r e . A r r o w s ind ica ted the pos i t ion of the r R N A b an d s run in a separa te gel.

10

o

c_ o

c 6

4 ~ J

E Q ~3

0 5 10 #g /m l temp la te RNA

Fig. 4. T e m p l a t e ac t iv i ty of Q~-RNA, i nc uba t e d wi th ATP : R N A adeny ly l t r ans f e r a se and pur i f ied b y g lycerol g rad ien t cen t r i fuga t ion . •, o, c, 2, 4, 6 rain i n c u b a t i o n at 37°C wi th [ 3 H I ATP; m, 6 rain incuba- t ion at 37°C w i t h o u t ATP; e, 6 rain i ncuba t ion at 0°C wi th [ 3 H ] ATP. 15 and 30 pl of the peak f rac t ions f r o m glycerol g rad ien t s as in Fig. I were i n c u b a t e d in the n o r m a l repl icase assay, con ta in ing sa tu ra t ing a m o u n t s of hos t f ac to r H F I . Af t e r 15 rain of i ncuba t ion at 37°C, the [14C] UMP label i n c o r p o r a t e d in ac id-prec ip i tab le ma te r i a l was d e t e r m i n e d . Cor rec t ions for spill-over b e t w e e n 3 H and 14 C coun t ing channe l s

were appl ied .

a

6

0 6

3

mu ~

0 i

5 1 15 20

O6

O6

0 4

0 2

b

L

s ? -o

E

2 0

5 10 15 20

F r a c t i o n n u m b e r

I a

I • I

4 I

I I

3 I I I I

2 I I I

E

0 0

c ~ b &

L

E i •

u i % I 2

2 I

I E

I

I

r - / 7 - / r / - ~ __ , i ~ , B ,

I 5 10 13

FFCIC t I Olq numbe r

Fig . 5. T e m p l a t e a c t i v i t y of Q f i - R N A , i n c u b a t e d w i t h A T P : R N A a d e n y l y l t r a n s f e r a s e a n d p u r i f i e d t h r o u g h s u c r o s e 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 o l i g o ( d T ) - c e n u l o s e c h r o m a t o g r a p h y , a, 5 m i n i n c u b a t i o n a t 3 7 ° C w i t h o u t A T P ; b , 5 r a in i n c u b a t i o n a t 3 7 ° C w i t h | 3 H ] A T P ( s p e c . a c t . 5 0 C i / m o l ) . ( A ) 1 0 - - 3 0 % s u c r o s e g r a d i e n t s , r u n f o r 2 h in a B e c k m a n S W 6 5 r o t o r a t 6 0 0 0 0 r e v . / m i n , e . [ 3 H ] A M P in a c i d - p r e c i p i t a b l e m a t e r i a l f r o m 50 p l o f e a c h f r a c t i o n : | 1 4 C ] U M P i n c o r p o r a t e d in a c i d - p r e c i p i t a b l e m a t e r i a l in a Qfi-

r e p l i c a s e r e a c t i o n m i x t u r e u s i n g 1 0 pl o f e a c h f r a c t i o n as t e m p l a t e . C o n t i n u o u s l ine , A 2 5 4 n m r e g i s t r a - t i o n . (B) O l i g o ( d T ) - c e l l u l o s e c h r o m a t o g r a p h y ( u s i n g 0 . 5 M K C I in t h e h i g h sa l t b u f f e r ) o f t h e g r a d i e n t p e a k f r a c t i o n s . D o t t e d a r e a , [ 3 H } A M P in a c i d - p r e c i p i t a b l e m a t e r i a l f r o m 3 0 p l o f e a c h f r a c t i o n ; h a t c h e d a r e a , [ 14C] U M P i n c o r p o r a t e d in a c i d - p r e c i p i t a h l e m a t e r i a l in a Q f i - r e p l i c a se r e a c t i o n m i x t u r e u s i n g 10 p l

o f e a c h f r a c t i o n as t e m p l a t e . T h e d a s h e d l ine i n d i c a t e s t h e c h a n g e - o w , r f r o m h i g h t o l o w sa l t e l u t i o n .

325

supplementary addition of host factor HFI. As a reference, Q~-RNA incubated for 6 min at 37°C with ATP : RNA adenylyltransferase in the absence of ATP and purified as above (but not retained on the oligo(dT)-cellulose column in high-salt buffer) was found to be fully active as a template (Fig. 5).

When polyadenylated Q~-RNA (up to 10 pg/ml) was added together with unmodified Q~-RNA, the RNA replication reaction proceeded normally pro- vided the host factor HFI concentration was saturating. In these conditions the relative amount of polyadenylated Qfi-RNA had no influence on the result. When factor HFI was present in over-saturating amounts, causing a certain inhibition, the addition of Qfl-RNA-poly(A) even resulted in a slight stimula- tion of the replicase reaction.

However, when only limiting amounts of HFI were present, a severe inhibi- tion by polyadenylated Qfi-RNA was observed. The inhibitory effect could be interpreted as being due to the removal of an equivalent amount of host factor HFI by a given amount of Qfi-RNA-poly(A). Assuming a molecular weight for host factor HFI of 75 000, about 0.15 pmol of Q~-RNA-poly(A) bound approx. 0.28 pmol of host factor HFI. The selective binding of host factor HFI to poly(A) is well known [0].

It was important to know whether the poly(A) tail by itself could account for all or part of this host factor HFI-binding capacity. MS2-RNA-poly(A), pre- pared in a similar way [2] and containing also about 135 AMP residues at the 3'-end, showed an approx. 2-fold lower affinity towards host factor HFI. Non- adenylated MS2-RNA had a very small factor-binding capacity.

Commercial poly(A), oligo (rA)2.~ and oligo (rA)90 were also inhibitory, but it was not possible to formulate a correlation between molar concentration (or 3'-terminal A content) and inhibitory effect, in the first place because these commercial preparations showed a broad size distribution, both upon zone sedimentation and gel electrophoretic analysis. A large proportion of these molecules carried a 3'-phosphate residue. Treatment of poly(A) with bacterial alkaline phosphatase enhanced its ability to act as a primer in the adenylyl- transferase assay by a factor of 30 (unpublished results), but did not change its inhibitory effect in the Q~-replicase assay. On the other hand, reduction of the mean size of a poly(A) preparation markedly enhanced its inhibiting capacity on a weight basis. Yet, considerably higher total molar amounts of the com- mercial oligonucleotides were required in order to produce a specified inhibi- tory effect (Table II).

The effect of enzyme concentration on the inhibition by poly(A)-containing compounds was negligible. Changing the Q~-RNA concentration in the incuba- tion mixture from 1 to 4 pg/ml had no influence on the percent inhibition by a given amount of poly(A)-containing polynucleotide, e.g. (rA)~.

In all cases, a sharp transition from high to low levels of incorporation was found upon addition of inhibitory RNA species. These data indicate a stoichio- metric relationship between host factor HFI and the inhibitory features of oligo(A)-containing compounds, but the exact nature of the interacting groups is not clear.

The portion of adenylated Q~-RNA that was not retained by the oligo(dT)- cellulose column corresponded to molecules with a short poly(A) tail. These were nevertheless poor templates in the replication reaction (Fig. 5). Assuming

326

T A B L E I I

I N H I B I T I O N O F T H E Q ~ - R E P L 1 C A S E R E A C T I O N B Y P O L Y ( A ) - O R O L I G O ( A ) - C O N T A I N I N G R N A

S P E C I E S

T h e s t a n d a r d r e p l i c a s e a s s a y c o n t a i n e d 0 . 2 5 p g o f QI3 -RNA a n d 0 .1 m u n i t o f p u r i f i e d r e p l i c a s e in 0 .1 m l

r e a c t i o n m i x t u r e . I n c u b a t i o n s w e r e at 3 7 ° C f or 15 m i n . S t a n d a r d e r r o r s w e r e c a l c u l a t e d w h e n at l eas t f o u r

i n d e p e n d e n t d e t e r m i n a t i o n s w e r e ava i lab le . T h e p o l y ( A ) tai ls at the 3 ' - e n d o f Ql3- or M S 2 - R N A had an

average l e n g t h o f 1 3 5 A M P r e s i d u e s . 1 u n i t o f Ql3-repl icase i n c o r p o r a t e d 1 p m o l U M P per rain at a t e m - p l a t e l eve l o f 0 . 5 0 p g p e r a s s a y .

H o s t f a c t o r H F I (r ig) R N A s p e c i e s a d d e d ( n g ) [ I 4 C L U M P i n c o r p o r a t e d (% ± ~)

R e f e r e n c e s e t 32 101 +- 3 26 - 1 0 0 ( r e f . v a l u e )

9 .6 - - 67 + 1 4 3 . 2 29 + 13

T e s t ser ies 1 3 2 Q ~ - R N A - p o l y ( A ) 1 9 0 34

M S 2 - R N A - p o h ¢ ( A ) 2 0 0 6 5

M S 2 - R N A - p o l v ( A ) 4 0 0 23

p o l y ( A ) 1 0 0 19

T e s t ser ies 2 26 Q I 3 - R N A - p o l y ( A ) 1 9 0 29 :~: 4

M S 2 - R N A - p o l y ( A ) 2 0 0 50

p o l y ( A ) 50 29

p o l y ( A ) 1 0 0 16 ( r A ) ~ 20 26

( r A ) ~ 5 0 1 5 ( r A ) ~ 1 0 91 4. 6

( r A ) ~ 2 0 i 8 + 6

that all the oligo(A) present was attached to Qfl-RNA, the average chain length for this fraction was calculated as between 10 and 60 residues, depending upon the particular experiment. Most of the material migrated together with intact Q~-RNA in a 2.4% polyacrylamide gel, indicating negligible degradation. After a second incubation with ATP : RNA adenylyltransferase and ATP, further adenylation was observed.

The template test is very specific and sensitive, since removal of more than the 3'-terminal A residue of Qfi-RNA results in the complete loss of template capacity, both in vivo and in vitro [10 ,11] . Therefore, the fraction of Qfl-RNA after adenylation that is not retained by oligo(dT)-cellulose in high salt, may consist of several types of non-template RNA molecules, with sizes very similar to the original Q~-RNA. Selective enzymatic removal of the poly(A) tails present would help to clarify this question.

Conclusions

(1) When Qfi-gNA is used as a primer in vitro for the ATP : RNA adenylyl- transferase from E. coli, at least 90% of the intact Q~-RNA molecules present is adenylated within the first few minutes at 37°C and the remaining material is still able to be adenylated.

3 2 7

(2) About 80% of the Qfl-RNA can be recovered in an adenylated form from an oligo(dT)-cellulose column.

(3) Adenylated Qfl-RNA is no longer a template for Qfl-replicase, whatever the concentrat ion of host factor HFI.

(4) Adenylated Q~-RNA, like oligo- or polyriboadenylic acid, inhibits the replicase reaction by selective removal of host factor HFI.

Acknowledgements

We are grateful to Dr. P. Kaesberg for the gift of Qfl mutant am 86 and to Dr. A. Sippel for the strain E. coli B/r. This research was supported by grants from the I.W.O.N.L. (Centrum voor Fytovirologie) and from the F.K.F.O. (No. 10.300). R.D. received a fellowship from the I.W.O.N.L.

References

1 S ippe l , A. ( 1 9 7 3 ) Eur . J . B i o e h e m . 37 , 3 1 - - 4 0 2 Devos , R. , Gillis, E. a n d Fiefs , W. ( 1 9 7 6 ) Eur . J . B i o e h e m . 62 , 4 0 1 - - 4 1 0 3 Aviv, H. a n d L e d e r , P. ( 1 9 7 2 ) P r o c . Na t l . A c a d . Sci. U.S. 69 , 1 4 0 8 - - 1 4 1 2 4 Gi lvarg , C., B o l l u m , F . J . a n d W e i s s m a n n , C. ( 1 9 7 5 ) P roc . Nat l . A c a d . Sci. U.S. 72, 4 2 8 - - 4 3 2 5 Gi lvarg , C., J o c k u s e h , H. a n d W e i s s m a n n , C. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 1 4 , 3 4 1 - - 3 4 8 6 C a r m i c h a e l , G. , Webe r , K. , Nive leau , A. a n d W a h b a , A.J . ( 1 9 7 5 ) J . Biol . C h e m . 2 5 0 3 6 0 7 - - 3 6 1 2 7 De Boeve r , J . G . , Gillis, E. a n d Fiefs , W. ( 1 9 7 1 ) Meded . R i jk s f ak . L a n d b o u w w e t . 36 , 5 2 2 8 De Boever , J .G . , v a n E m m e l o , J . , Gillis, E. a n d Fiers , W. ( 1 9 7 3 ) Meded . R i jk s f ak . L a n d b o u w w e t . 38 ,

1 6 2 3 - - 1 6 3 0 9 P e r r y , R.P . , L a T o r r e , J . , Ke l l ey , D .E . a n d G r e e n b e r g , J . R . ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 6 2 ,

2 2 0 - - 2 2 6 10 K a m e n , R. ( 1 9 6 9 ) N a t u r e 2 2 1 , 3 2 1 - - 3 2 5 11 R e n s i n g , U. a n d A u g u s t , J .T . ( 1 9 6 9 ) N a t u r e 2 2 4 , 8 5 3 - - 8 5 6