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SEVERO O C H O A- -
Enzymat ic syn thes i s o f r ibonuc le ic ac id
N obel Lect u re, D ecem ber 11 , 195 9
I a m d e e p l y c o n s c i o u s o f t h e g r e a t d i s t i n c t i o n w i t h w h i c h I h a v e b e e n h o n -
ored and deem i t a specia l pr iv i lege to review the recent s tudies of the bio-
s y n t h e s i s o f r i b o n u c l e i c a c i d o n t h i s o c c a s i o n .
The nucle ic ac ids have considerable b iologica l impor tance because of the i r
role in ce l l growth and in the t ransmiss ion of heredi tary characters . As f i r s t
s u g g e s t e d b y t h e p i o n e e r w o r k o f C a s p e r s s o n a n d B r a c h e t t h e f o r m e r f u n c -
t i o n i s p e r f o r m e d b y r i b o n u c l e ic a ci d ( R N A ) t h r o u g h i t s p a r t i c i p a t i o n i n
t h e b i o s y n t h e s i s o f p r o t e i n s . T h e s e c o n d i s c a r r i e d o u t b y d e o x y r i b o n u c l e i c
a c i d (D N A) , t h e m a i n c o m p o n e n t o f t h e n u c l e a r c h r o m o s o m e s . I t i s o f i n -
teres t , however , tha t in cer ta in v i ruses such as tobacco mosaic , inf luenza , and
p o l i o m y e l i t i s v i r u s , w h i c h c o n s i s t o f R N A a n d p r o t e i n , R N A i s t h e c a r r i e r
o f g e n e t i c i n f o r m a t i o n .
M o s t o f t h e c e l l ’ s R N A i s p r e s e n t i n t h e c y t o p l a s m . T h e r e a r e t w o k i n d s
o f c y t o p l a s m i c R N A . O n e , o f r e l a t i v e l y s m a l l m o l e c u l a r s i z e , i s f o u n d i n
the cytoplasmic f lu id and i s , therefore , refer red to as soluble RNA; the other ,
o f m u c h h i g h e r m o l e c u l a r w e i g h t , i s a c o m p o n e n t o f t h e m i c r o s o m a l r i b o -
n u c l e o - p r o t e i n p a r t i c l e s . B o t h p l a y a n e s s e n t i a l r o l e i n p r o t e i n s y n t h e s i s .
T h e r e i s , i n a d d i t i o n , a s m a l l a m o u n t o f R N A i n t h e c e l l n u c l e u s , m o s t o f
i t l o c a t e d i n t h e n u c l e o l u s . T h e r e a r e i n d i c a t i o n s t h a t m o s t , i f n o t a l l , o f
t h e c y t o p l a s m i c R N A i s s y n t h e s i z e d i n t h e n u c l e u s a n d s u b s e q u e n t l y t r a n s -
p o r t e d t o t h e c y t o p l a s m . I n t r a n s m i t t i n g g e n e t i c i n f o r m a t i o n , n u c l e a r D N Ai s s u p p o s e d t o d e t e r m i n e t h e n a t u r e o f t h e n u c l ea r R N A w h i c h , o n e n -
t e r i n g t h e c y t o p l a s m , d e t e r m i n e s i n t u r n t h e n a t u r e o f t h e p r o t e i n s s y n t h e -
s ized.
A l t h o u g h n o t a b l e a d v a n c e s h a d b e e n m a d e i n o u r k n o w l e d g e o f t h e w a y
i n w h i c h t h e n u c l e o t i d e s , t h e n u c l e i c a c i d b u i l d i n g s t o n e s , a r e s y n t h e s i z e d ,
l i t t l e w a s k n o w n u n t i l r e c e n t l y o f t h e m e c h a n i s m o f s y n t h e s i s o f t h e g i a n t
m o l e c u l e s o f t h e n u c l e i c a c i d s t h e m s e l v e s . W e o w e o u r p r e s e n t i n f o r m a t i o n
to the discovery of enzymes capable of ca ta lyzing the synthes is of RNA and
D N A i n t h e t e s t t u b e f r o m s i m p l e , n a t u r a l l y o c c u r r i n g p r e c u r s o r s . T h e s e
p r e c u r s o r s a r e t h e n u c l e o s i d e d i - a n d t r i p h o s p h a t e s , t h e n u c l e o t i d e m o i e t i e s
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6 4 6 1 9 5 9 L O C H O A
of w hich unde rgo po lym er i za t ion w i th r e l ease o f o r thophospha te in t he f i r s t
c a s e o r o f p y r o p h o s p h a t e i n t h e s e c o n d .
In 1955 we isola ted a bacter ia l enzyme capable of ca ta lyzing the synthes is of
h i g h m o l e cu l a r w e i g h t p o l y r i b on u c le o t i d e s fr o m n u c l e o s id e d i p h o s p h a t e s
w i t h r e l e a s e o f o r t h o p h o s p h a t e1, 2
. T h e r e a ct i o n , w h i c h r e q u i r e s m a g n e s i u m
i o n s a n d i s r e v e r s i b l e , c a n b e f o r m u l a t e d b y t h e e q u a t i o n
w h e r e R s t a n d s f o r r i b o s e , P - P f o r p y r o p h o s p h a t e , P f o r o r t h o p h o s p h a t e ,
a n d X f o r o n e o r m o r e b a s e s i n c lu d i n g , a m o n g o t h e r s , a d e n i n e , h y p o x a n -
thine , guanine , urac i l , or cytos ine . In the reverse di rec t ion the enzyme br ings
about a c leavage of polyr ibonucleot ides by phosphate , i . e . a phosphorolys is ,
to y ie ld r ibonucleos ide diphosphates . The react ion i s s imi lar to the revers ible
s y n t h e s i s a n d c l e a v a g e o f p o l y s a c c h a r i d e s , c a t a l y z e d b y p h o s p h o r y l a s e ; f o r
t h i s r e a s o n t h e n e w e n z y m e w a s n a m e d p o l y n u c l e o t i d e p h o s p h o r y l a s e . B e -
cause of its reversibility, the reaction leads to an incorporation or "exchange"
o f o r t h o p h o s p h a t e i n t o t h e t e r m i n a l p h o s p h a t e g r o u p o f n u c l e o s i d e d i p h o s -
pha te s . I t w as th rough th i s exchange tha t po lynuc leo t ide phosphory la se w as
d i s c o v e r e d b y t h e u s e o f r a d i o a c t iv e p h o s p h a t e . In o u r e a r l y w o r k w i t h
G r u n b e r g - M a n a g o , p o l y n u c l e o t i d e p h o s p h o r y l a s e w a s p a r t i a l l y p u r i f i e d , b y
u s e o f t h e r a d i o p h o s p h a t e e x c h a n g e r e a c t i o n , f r o m t h e m i c r o o r g a n i s m A z o-
tobacter vinelandii. The enzyme has the unique fea ture of ca ta lyzing not only
t h e s y n t h e s i s o f R N A f r o m m i x t u r e s o f t h e f o u r n a t u r a l l y o c c u r r i n g r i b o -
n u c l e o s i d e d i p h o s p h a t e s , b u t a l s o t h a t o f n o n - n a t u r a l l y o c c u r r i n g p o l y r i b o -
nucleot ides conta ining only one , two, or three di f ferent k inds of nucleot ides
i n t h e i r c h a i n s . T h e n a t u r e o f t h e p r o d u c t d e p e n d s o n t h e k i n d a n d v a r i e t y
o f t h e n u c l e o s i d e d i p h o s p h a t e s u b s t r a t e s u t i l i z e d f o r t h e s y n t h e s i s3, 4
. Table 1
l i s t s t h e m a i n t y p e s o f p o l y r i b o n u c l e o t i d e s w h i c h h a v e b e e n p r e p a r e d w i t h
p o l y n u c l e o t i d e p h o s p h o r y l a s e . T h e p r e p a r a t i o n o f p o l y r i b o t h y m i d y l i c a c i d
f r o m s y n t h e t i c r i b o t h y m i d i n e d i p h o s p h a t e h a s r e c e n t l y b e e n r e p o r t e d5
.
Structure of polynucleotides - I n j o i n t e x p e r i m e n t s w i t h L . A . H e p p e 16-8
i t
h a s b e e n e s t a b l i s h e d t h a t t h e s y n t h e t i c p o l y r i b o n u c l e o t i d e s c o n f o r m i n a l l
r e s p e c t s t o t h e s t r u c t u r a l p a t t e r n o f n a t u r a l R N A . T h u s , i t w a s f o u n d b y
d e g r a d a t i o n w i t h a l k a l i , o r s u c h e n z y m e s a s s n a k e v e n o m a n d s p l e e n p h o s -
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E N Z Y M A T I C S Y N T H E S I S O F R I B O N U C L E I C A C I D 6 4 7
phodies terase or pancreat ic r ibonuclease , tha t they consis t of l inear chains in
w h i ch t h e c o m p o n e n t n u c l e os i d e u n i t s a r e l i n k e d t o o n e a n o t h e r t h r o u g h
3 ’ , 5 ’ - p h o s p h o d i e s t e r b r i d g e s . T h e s t r u c t u r a l i d e n t i t y w i t h n a t u r a l R N A c a n
br ief ly be i l lus t ra ted by the ef fec t of pancreat ic r ibonuclease on the synthet ic
p o l y m e r p o l y A U . F r o m w h a t i s k n o w n o f t h e a c t i o n o f r i b o n u c l e a s e o n
R N A , p o l y A U w o u l d b e d e g r a d e d a t t h e p o i n t s i n d i c a t e d b y t h e a r r o w s i n
F i g . 1 . U r i d y l i c a c i d ( u r i d i n e 3 ’ - m o n o p h o s p h a t e ) w o u l d b e r e l e a s e d a s t h e
Fig. 1. Scheme of cleavage of poly AU by p ancreatic ribonu clease. The vertical lines
represent the ribose residues; A and U represent the bases adenine and uracil, respec-
tively. The cleavage points are indicated by arrows3
.
o n l y m o n o n u c l e o t id e , a l o n g w i t h a s e r i e s o f s m a l l o l i g o n u c l e o t i d e s , e a c h
c o n s i s t i n g o f o n e u r i d y l i c a c i d r e s i d u e a n d o n e o r m o r e a d e n y l i c a c i d r e s -
idues . Fig . 2 i s an ul t raviole t pr in t of a chromatogram i l lus t ra t ing the separa-
t i o n o f m o n o - , d i , - t r i - , t e t r a - , a n d p e n t a n u c l e o t i d e s e a c h w i t h d e c r e a s i n g R f
v a l u e s f r o m a r i b o n u c l e a s e d i g e s t o f p o l y A U . T h e i n d i v i d u a l s p o t s w e r e
e l u t e d a n d t h e r e s p e c t i v e o l i g o n u c l e o t i d e s i d e n t i f i e d f o l l o w i n g h y d r o l y s i s
wi th a lkal i . Diges t ion of synthet ic RNA with r ibonuclease yie lds , a long wi th
u r i d y l i c a n d c y t i d y l i c a c i d s , m i x t u r e s o f o l i g o n u c l e o t i d e s w h i c h , a s f a r a s
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1 9 5 9 S . O C H O A
Fig. 2. Products of hydrolysis of poly AU by ribonuclease (L. A. Heppel). Origin is
at top of chromatogram. Spot at bottom center is a marker of uridine 3’-phosphate3
.
they have been ident i f ied , a re ident ica l to those obta ined f rom natura l RNA
u n d e r t h e s a m e c o n d i t i o n s .
The question whether a given nucleotide species is linked to the various
o t h e r n u c l e o t i d e s i n t h e p o l y n u c l e o t i d e c h a i n s , a s i s t h e c a s e w i t h n a t u r a l
R N A , c a n b e e a s i l y a n s w e r e d t h r o u g h d e g r a d a t i o n o f s y n t h e t i c R N A l a b e l e d
w i t h r a d i o a c t i v e p h o s p h a t e9
. F ig . 3 p re sen t s t he s t ruc tu re o f a po lynuc leo t ide
( p o l y A *G U C ) , p r e p a r e d f r o m a m i x t u r e o f a d e n o s i n e d i p h o s p h a t e l a b e l e d
w i t h32
P i n t h e f i r s t p h o s p h a t e g r o u p ( a d e n o s i n e -32
P - P ) a n d n o n - l a b e l e d
guanosine- , ur id ine- , and cyt id ine diphosphates . I f the labeled adenyl ic ac id
i s r a n d o m l y d i s t r i b u t e d a s s h o w n , h y d r o l y s i s o f s u c h a p o l y m e r w i t h s n a k e
v e n o m p h o s p h o d i e s t e r a s e ( F i g . 3 A ) w i l l y i e l d n u c l e o s i d e 5 ’ - m o n o p h o s -
p h a t e s o f w h i c h o n l y a d e n o s i n e 5 ’ - m o n o p h o s p h a t e w i l l b e l a b e l e d . H y d r o l -
y s i s w i t h s p l e e n p h o s p h o d i e s t e r a s e , o n t h e o t h e r h a n d ( F i g . 3 B ) w i l l r e l e a s e
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E N Z Y M A T I C S Y N T H E S I S O F R I B O N U C L E I C A C I D 6 4 9
Fig. 3. Scheme of hydrolysis of 32
P-labeled RNA (poly A*GUC) by snake venom
(A ) or spleen (B) phosphodiesterase. The bonds hydrolyzed are indicated by dashed
lines. The asterisk denotes32
P labeling. Ad, Gu, Ur and Cy represent the bases adenine,
guanine, uracil and cytosine, respectively9
.
n u c l e o s i d e 3 ’ - m o n o p h o s p h a t e s a l l o f w h i c h w i l l b e l a b e l e d . T h a t s u c h i s i n -
deed the case i s shown in Fig . 4 . The separa t ion of the hydrolys is products in
t h i s e x p e r i m e n t w a s e f f e c t e d b y i o n - e x c h a n g e c h r o m a t o g r a p h y .
A l t h o u g h l a r g e v a r i a t i o n s i n t h e r e l a t i v e p r o p o r t i o n s o f t h e d i f f e r e n t n u -
c l eos ide d iphospha te subs t r a t e s has a r a the r m arked in f luence on the nuc leo -
t i d e c o m p o s i t i o n o f t h e r e s u l t i n g p o l y m e r , w h e n s y n t h e t i c R N A i s p r e p a r e d
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6 5 01 9 5 9 S . O C H O A
Fig. 4. Hydrolysis of poly A*GUC with snake venom (top) or spleen (bottom) p h o s -
phodiesterase with separation of mononucleotides by ion-exchange chromatography.
Plot of nucleotide concentration (solid lines -o-o-o-) and radioactivity (dashed lines
-o-o-o-) against eff luent fract ion number12
.
f r o m e q u i m o l a r m i x t u r e s o f a d e n o s i n e , g u a n o s i n e , u r i d i n e , a n d c y t i d i n e
d i p h o s p h a t e , t h e n u c l e o t i d e c o m p o s i t i o n o f t h e p r o d u c t i s v e r y s i m i l a r t o
t h a t o f n a t u r a l A z ot ob ac t er RNA. Th i s i s sh o wn in Tab le 2 wh ich g iv es t h e
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E N Z Y M A T I C S Y N T H E S I S O F R I B O N U C L E I C A C I D 651
base ra t ios o f A z ot obact er RNA and of two d i f fe ren t samples o f the syn the t ic
p r o d u c t . I t i s n o t e w o r t h y t h a t t h e b a s e r a t i o s d i f f e r w i d e l y f r o m u n i t y i n
sp i te o f the fac t tha t equ imola r concen t ra t ions o f the nuc leos ide d iphospha te
p r e c u r s o r s w e r e u s e d .
T h e s y n t h e t i c p o l y r i b o n u c l e o t i d e s a l s o r e s e m b l e n a t u r a l R N A i n s i z e .
T h e i r m o l e c u l a r w e i g h t v a r i e s b e t w e e n a b o u t 3 0 , 0 0 0 a n d o n e t o t w o m i l -
l i o n s . T h e s e d i m e n t a t i o n c o n s t a n t o f s a m p l e s o f s y n t h e t i c R N A w a s s i m i l a r
t o t h a t o f R N A i s o l a t e d f r o m w h o l e A z ot obact er c e l l s . P o l y n u c l e o t i d e s c o n -
t a i n i n g o n l y o n e k i n d o f n u c l e o t i d e u n i t , s u c h a s p o l y a d e n y l i c o r p o l y c y t i -
d y l i c a c i d , a r e o f t e n o f v e r y l a r g e s i z e a n d c o n f e r h i g h v i s c o s i t y t o t h e i r
s o l u t i o n s . I t i s p o s s i b l e t o f o l l o w t h e c o u r s e o f s y n t h e s i s v i s u a l l y b y t h e
marked inc rease in v i scos i ty tha t t akes p lace on incuba t ion of nuc leos ide d i -
p h o s p h a t e s w i t h a f e w m i c r o g r a m s o f e n z y m e . N a t u r a l R N A - h a s a n o n -
s p e c i f i c b i o l o g i c a l activity w h i c h i s a l s o e x h i b i t e d b y s y n t h e t i c R N A . T h e
latter is as effective as the former in stimulating the formation of streptolysin S
( a l e c i t h i n a s e ) b y h e m o l y t i c s t r e p t o c o c c i10
.
Reaction mechanism - To study the mechanism of action of polynucleotide
p h o s p h o r y l a s e i t w a s e s s e n t i a l t o o b t a i n h i g h l y p u r i f i e d p r e p a r a t i o n s o f t h e
e n z y m e . T h e l a s t s t e p ( F i g . 5 ) w a s c h r o m a t o g r a p h y o n a h y d r o x y l a p a t i t e
c o l u m n , a m e t h o d d e v e l o p e d b y T i s e l i u s a n d c o l l a b o r a t o r s11
. O n l y t h r o u g h
c h r o m a t o g r a p h y w a s i t p o s s i b l e t o s e p a r a t e t h e e n z y m e f r o m a c o n t a m -
i n a t i n g y e l l o w p r o t e i n o f u n k n o w n n a t u r e w h i c h , a s s e e n i n t h e f i g u r e , i s
e l u t e d a t h i g h e r b u f f e r c o n c e n t r a t i o n s t h a n i s p h o s p h o r y l a s e . T h e p u r i f i c a -
t i o n o f t h e e n z y m e a f t e r c h r o m a t o g r a p h y i s s o m e s i x h u n d r e d f o l d o v e r t h e
i n i t i a l e x t r a c t o f A z ot ob ac t er c e l l s . T h e h i g h l y p u r i f i e d e n z y m e c o n t a i n s a
f i r m l y b o u n d o l i g o n u c l e o t i d e12
w h i c h c a n n o t b e r e m o v e d b y s u c h m e t h o d s
as t rea tment wi th charcoa l o r r ibonuc lease . S ince i t has so fa r no t been pos-
s i b l e t o r e m o v e t h e o l i g o n u c l e o t i d e w i t h o u t d e s t r o y i n g t h e e n z y m e p r o t e i n
i t r e m a i n s u n d e c i d e d w h e t h e r t h i s c o m p o u n d , w h i c h r e p r e s e n t s a b o u t 3 . 5
p e r c e n t o f t h e e n z y m e , i s a p r o s t h e t i c g r o u p o r a c o n t a m i n a n t . T h e o l i g o -
nuc leo t ide cons i s t s o f about twe lve nuc leo t ide res idues o f adenyl ic , guanyl ic ,
u r idy l ic and cy t idy l ic ac id in roughly the same mola r ra t ios as in A z ot obact er
R N A . I t c a n b e i s o l a t e d a f t e r d e n a t u r a t i o n o f t h e p r o t e i n w i t h p e r c h l o r i c
a c i d o r p h e n o l .
S i n c e t h e A z t obact er e n z y m e c a n s y n t h e s i z e R N A a s w e l l a s p o l y n u c l e o -
t ides wi th on ly one nuc leo t ide spec ies , i t i s impor tan t to dec ide whe ther one
is dea l ing wi th a mix ture o f enzymes , each reac t ing wi th a d i f fe ren t nuc leo-
s ide d iphospha te , o r wi th a s ing le enzyme. Al though th i s ques t ion cannot be
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6 5 2 1 9 5 9 S . O C H O A
answered unequivocal ly , i f the act iv i ty toward each of several nucleoside d i -
phosphates increases to the same ex ten t on pur i f icat ion , i t i s very l ikely thata s ing le enzyme i s invo lved . That th is i s so i s shown in Table 3 in which the
* Results, micromoles of 32
P-exchange per mg of enzyme protein, 15 minutes at 30º.
act iv i ty toward each of f ive nucleoside d iphosphates , as assayed by the rad io-
a c t i v e p h o s p h a t e e x c h a n g e m e t h o d , i s s e e n t o i n c r e a s e a p p r o x i m a t e l y t o t h e
s a m e e x t e n t b e t w e e n t w o a d v a n c e d p u r i f i c a t i o n s t e p s . T h e s a m e w a s t r u e
for ear l ier s tages o f pur i f icat ion .
W i t h p a r t i a l l y p u r i f i e d p r e p a r a t i o n s o f p o l y n u c l e o t i d e p h o s p h o r y l a s e p o -
Fig. 5. Chromatography of A zotobacter poiynucleotide phosp horylase on hyd roxyl
apatite (S. Ochoa and S. Mii, unpublished).
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E N Z Y M A T I C S Y N T H E S I S O F R I B O N U C L E I C A C I D 6 5 3
l y n u c l e o t i d e s y n t h e s i s s t a r t s i m m e d i a t e l y a f t e r a d d i n g t h e e n z y m e t o a n
o t h e r w i s e c o m p l e t e s y s t e m . T h i s i s n o t t h e c a s e , h o w e v e r , w i t h h i g h l y p u r -
i f ied preparat ions . In th is case , there i s most ly a more or less p ronounced lag
per iod a l though even tual ly the react ion s tar t s and gradual ly increases in ra te .
E q u i l i b r i u m i s n o t r e a c h e d e v e n a f t e r m a n y h o u r s o f i n c u b a t i o n . T h e r e a c -
t i o n r a t e i s m a r k e d l y s t i m u l a t e d b y a d d i t i o n o f s m a l l a m o u n t s o f o l i g o - o r
p o l y n u c l e o t i d e s w h i c h , a s i s t h e c a s e w i t h g l y c o g e n i n p o l y s a c c h a r i d e s y n -
t h e s i s f r o m g l u c o s e - r - p h o s p h a t e b y ( p o l ys a c c h a r i d e ) p h o s p h o r y l a s e , s e r v e
a s p r i m e r s o f t h e r e a c t i o n . T h e p r i m i n g e f f e c t o f o l i g o r i b o n u c l e o t i d e s w a s
d i s co v e r ed b y H e p p e l a n d c ol la b o r a to r s
1 3
, t h e p r im i n g b y p ol yn u c le ot id e sw a s d i s c l o s e d i n o u r l a b o r a t o r y
1 4
. Th e m a i n o l i go n u c le o t i d e p r im e r s u s e d
h a v e b e e n d i - , t r i - , o r t e t r a a d e n y l i c a c i d s i s o l a t e d a s r e a c t i o n p r o d u c t s o f
h y d r o l y s i s o f p o l y a d e n y l i c a c i d b y a n u c l e a s e f r o m l i v e r n u c l e i15
;
Th e p r imin g b y o l ig o n u c leo t id es i s n o t sp ec i f i c . Th e o l ig o ad en y l i c ac id s
can pr ime the syn thes is o f po lyadenyl ic and po lyur idy l ic acid as wel l as that
o f R N A o r a n y o f t h e o t h e r p o l y n u c l e o t i d e s . P r i m i n g b y p o l y n u c l e o t i d e s ,
on the o ther hand , shows a cer ta in degree o f speci f ic i ty . Thus , po lyadenyl ic
acid p r imes on ly i t s own syn thes is and the same i s t rue o f po lyur idy l ic acid .
O n t h e o t h e r h a n d R N A , w h e t h e r n a t u r a l o r s y n t h e t i c , p r i m e s t h e s y n t h e s i s
o f R N A a s w e l l a s t h a t o f p o l y a d e n y l i c a c i d o r p o l y u r i d y l i c a c i d . V e r y c u -
r i o u s a n d c o m p l e t e l y u n e x p l a i n e d i s t h e e f f e c t o f p o l y c y t i d y l i c a c i d w h i c h
primes the synthesis of al l the polynucleotides os far tested (Table 4) . Fig. 6
s h o w s t h e p r i m i n g o f R N A s y n t h e s i s w i t h h i g h l y p u r i f i e d p o l y n u c l e o t i d e
p h o s p h o r y l a s e b y t r i a d e n y l i c a c i d ( T A A ) , l i v e r R N A , a n d p o l y c y t i d y l i c
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6 5 4 1 9 5 9 S . O C H O A
Fig. 6. Priming of RNA synthesis (S. Ochoa and Mii, unpublished).
D i p h o s p h a t e s ( A + G + U + C ) , 5 moles each; Mg++
, 2.0 moles ; enzyme, g;
TRIS, pH 8.1, moles; volume, 1.0 ml.
acid (poly C). The effect of different p o l y n u c l e o t i d e s o n t h e s y n t h e t i c r e a c -
t i o n i s i l l u s t r a t e d i n T a b l e 4 . I t i s t o b e n o t e d t h a t p o l y a d e n y l i c a c i d i s n o t
only without effect but actual ly inhibi ts the slow synthesis of polyuridylic acid
w h i c h o c c u r s i n t h e a b s e n c e o f a d d e d p r i m e r ; t h e c o n v e r s e i s a l s o t r u e . I t
s h o u l d f u r t h e r b e n o t e d t h a t t h e s y n t h e s i s o f p o l y c y t i d y l i c a c i d i s p r i m e d
o n ly b y p o ly cy t id y l i c ac id i t s e l f .
T h e m e c h a n i s m o f p r i m i n g b y p o l y n u c l e o t i d e s a n d t h e c a u s e a n d s i g n i f -
icance of the specif ici ty just described are as yet unexplained. The possibi l i ty
t h a t p o l y r i b o n u c l e o t i d e s m i g h t f u n c t i o n a s t e m p l a t e s f o r t h e i r o w n r e p l i c a -t ion has no t been substan t ia ted exper imental ly . On the o ther hand , the mode
of act ion of the o l igonucleo t ide p r imers has been e lucidated in e legan t exper-
i m e n t s b y H e p p e l a n d h i s c o l l a b o r a t o r s1 3 , 1 6
. T h e y p r o v e d t h a t t h e o l i g o n u -
cleotides serve as nuclei for growth of the polynucleotide chains by successive
a d d i t i o n o f m o n o n u c l e o t i d e u n i t s . I t m a y b e r e c a l l e d t h a t p o l y s a c c h a r i d e
phosphory lase acts in a s imi lar way by cata lyzing the success ive add i t ion of
g l u c o s y l r e s i d u e s t o t h e t e r m i n a l u n i t s o f a p o l y s a c c h a r i d e p r i m e r . W h e n
t h e s y n t h e s i s o f p o l y u r i d y l i c a c i d i s p r i m e d b y d i - o r t r i a d e n y l i c a c i d , t h e
n e w p o l y n u c l e o t i d e c h a i n s s h o u l d c o n s i s t o f a n u m b e r o f u r i d y l i c a c i d r e s -
idues p receded by two or th ree adenyl ic acid res idues . This i s shown schemat-
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E N Z Y M A T I C S Y N T H E S I S O F R I B O N U C L E I C A C I D 655
- -
Fig. 7. Mechanism of priming by oligonucleotides (From data of M. F. Singer, L. A.
Heppel, and R. J. Hilmoe13
).
ica l ly in the upper and middle por t ions of Fig . 7 . That such i s in fac t the case
was proved fol lowing r ibonuclease diges t ion of polyur idyl ic ac id synthes ized
i n t h e p r e s e n c e o f d i a d e n y l i c a c i d a s p r i m e r13 , 16
. A s s h o w n i n t h e m i d d l e
p o r t i o n o f F i g . 7, r i b o n u c l ea s e w o u l d r e l e a s e a t r i n u c le o t i d e ( p A p A p U p )
f r o m t h e o r i g in , o n e m o l e cu l e o f u r i d i n e f r o m t h e e n d , a n d a n u m b e r o f
u r i d i n e 3 ’- m o n o p h o s p h a t e r e s id u e s f r o m t h e r e m a i n d e r o f t h e c h a in . T h e
t r i n u c l e o t i d e w a s i s o l a t e d b y c h r o m a t o g r a p h y a n d d i g e s t e d w i t h p o t a s s i u m
h y d r o x i d e . A s s h o w n i n t h e l o w e r s e c t i o n o f F i g . 7 , t h i s s h o u l d y i e l d e q u i -
m o l a r a m o u n t s o f a d e n o s i n e 5 ’ , 3 ’ - d i p h o s p h a t e , a d e n o s i n e 3 ’ - m o n o p h o s p h a t e
a n d u r i d i n e 3 ’- m o n o p h o s p h a t e . Th e f i g u r e a l so s h o w s t h a t t h e a m o u n t s
. a c t u a l l y r e c o v e r e d w e r e i n g o o d a g r e e m e n t w i t h t h e t h e o r y .
I t appears jus t i f ied to conclude tha t polynucleot ide phosphorylase may be
u n a b l e t o s t a r t t h e s y n t h e s i s o f a p o l y n u c l e o t i d e c h a i n f r o m n u c l e o s i d e d i -
phosphates as the only reactants and tha t the presence of an ol igonucleot ideto serve as nucleus for growth of new polynucleot ide chains i s probably in-
d i s p e n s a b l e . I f t h e e n z y m e c o u l d b e o b t a i n e d c o m p l e t e l y f r e e o f o l i g o n u -
c l e o t i d e p r i m e r m a t e r i a l , i t m i g h t p r o v e t o b e c o m p l e t e l y i n a c t i v e i n t h e
a b s e n c e o f a d d e d o l i g o n u c l e o t i d e s .
Biological sign if ican ce - P o l y n u c l e o t i d e p h o s p h o r y l a s e i s w i d e l y d i s t r i b u t e d
i n b a c t e r i a . T h e e n z y m e h a s b e e n p a r t i a l l y p u r i f i e d f r o m m i c r o o r g a n i s m s
o t h e r t h a n A z ot obacter v in elaan di i1 7 - 1 9
and polyr ibonucleot ides have been syn-
thes ized wi th these enzyme prepara t ions . Indica t ions have a lso been obta ined
f o r t h e p r e s e n c e o f t h e e n z y m e i n g r e e n l e a v e s17
. O n t h e o t h e r h a n d , i t h a s
b e e n d i f f i c u l t t o d e t e c t t h e e n z y m e i n a n i m a l t i s s u e s . R e c e n t l y , h o w e v e r ,
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656 1 9 5 9 S . O C H O A
Hilmoe and Heppel20
have reported the presence of polynucleotide phos-
phorylase in preparations from mammalian liver nuclei.
The occurrence of polynucleotide phosphorylase in nature appears to be
widespread enough to warrant the assumption that this enzyme may be
generally involved in the biosynthesis of RNA. This possibility appears to
be strengthened by recent studies with ribonucleoside diphosphates con-
taining different analogues of the naturally occurring bases. Thus j-bro-
mou ridine d iphosph ate wh ich contains 5-bromouracil, an analogue of uracil
or thymine which in experiments with intact bacterial cells is incorporated
into DNA but not into RNA, is not a substrate of polynucleotide phos-
phorylase, while thiouridine diphosphate containing the uracil analogue
thiouracil, which in similar experiments is incorporated into RNA but not
into DNA, is a substrate for phosphorylase. In line with these observations
is the fact that azauridine diphosphate containing the uracil analogue aza-
uracil which is not incorporated in vivo into RNA, does not react with poly-
nucleotide phosphorylase2 1. How ever, in spite of the fact that polynucleotide
ph osphorylase can bring abou t the synthesis of an RNA of the same nu cleo-
tide composition an d molecular w eight as that isolated from Azotobacter, an d
despite the intriguing specificity of priming by polyribonucleotides, there is
so far no evidence that the enzym e is able to rep licate the p rimer m olecules
as is the case with Kornberg’s DNA polymerase. Since there must be mech-
anisms in the cell capable of synthesizing individual ribonucleic acid mole-
cules with a determined nucleotide sequence, it is likely that enzymes capable
of performing this function are still to be discovered.
Enzymes catalyzing the add ition of a few nu cleotide un its to a p reexisting
RNA chain have recently been described from various laboratories22-24
.
These enzymes catalyze the transfer of cytidylic and adenylic acid residues
from the corresponding nucleoside triphosphates to the end of polynucleo-
tide chains with release of pyrop hosph ate. There are no ind ications that theycan bring about a net synthesis of RNA. Other investigators
25,26have de-
scribed the incorporation of ribonucleotides in the interior of RNA chains
by particulate cell fractions from animal tissues. In a recent report26
a frac-
tion from rat liver nuclei brought about this incorporation optimally from
a mixture of all four ribonucleoside triphosphates of adenosine, guanosine,
uridine and cytidine, and the reaction was markedly decreased after treat-
ment with ribonuclease suggesting requirement for an RNA primer. These
experiments su ggest that an enzyme similar to Kornberg’s DNA polymerase
might be involved.
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E N Z Y M A T I C S Y N T H E S I S O F R I B O N U C L E I C A C I D 657
Fig. 8. Electrophoresis and sedimentation patterns of poly A, poly U andpoly A + U
27
.
Physical-chemical studies on a variety of synthetic polyribonud eotides h ave
thrown much light on their macromolecular structure and may greatly fur-
ther our understanding of the biological properties of RNA and DNA.
Warner, in our laboratory, found that polyadenylic and polyuridylic acids
interact in solution to form a stable complex27. At suitable pH values this
comp lex migrates on electrophoresis with a sharp single boundary of mobil-
ity intermediate between that of poly A and poly U. On ultracentrifugation,
it has a higher sedimentation constant than that of the parent polynucleotides
(Fig. 8). Warner further observed that formation of the complex is accom-panied by a marked decrease of the absorption of ultraviolet light (Fig. 9).
Formation of the complex can in fact be observed visually through the
marked increase in viscosity that takes place on mixing solutions of poly A
and poly U.
X-ray diffraction studies of Rich and collaborators28
showed that fibers
made from the poly A + U complex give rise to a crystalline pattern not
unlike that of DNA29, indicating that this complex has a double-stranded
helical structure. Further studies30 demonstrated that the strands are held to-
gether by hydrogen bonds between the complementary pairs of bases ade-nine and uracil. These observations provided the first experimental dem-
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658 1 9 5 9 S . O C H O A
Fig. 9. Absorption spectrum of an equimolar mixture of poly A and poly U. Theupper curve refers to the mononucleotides obtained by alkaline hydrolysis of the mix-ture. The middle curve is that calculated for the separately measured spectra of theindividual polymers. The lower curve is the measured curve for the mixture of pol-
ymers 27.
onstration that polynucleotides can interact to form double-stranded helical
structures similar to that proposed by Watson and Crick for DNA and,
moreover, that the same configuration may be assumed by RNA.
The decrease of ultraviolet light absorption accompanying polynucleotide
complex format ion has facilitated an extensive stud y of interactions betw een
different polynucleotides by Rich and collaborators31-33
. These investigators
mad e the further importan t observation that triple-stranded h elical polyribo-
nu cleotide stru ctures can also be formed . As illustrated in Fig. 10, a complex
consisting of one poly A and two poly U molecules is form ed in the presence
of Mg++
. The figure shows the optical density at 259 mu on adding in-
creasing am oun ts of a solut ion of poly U to one of poly A. In the absence of
magnesium the absorbancy goes through a minimum when equimolecular
amounts of the two polynucleotides are present; in the presence of magne-
sium, minimum absorbancy is reached when the solution contains 2 molesof poly U per mole of poly A. Fig. 11 illustrates the type of hydrogen bond-
ing postulated by Rich for the poly A + U and the poly A + U + U
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E N Z Y M A T I C S YN T H E S I S O F R I BO N U C L E I C A C I D 659
Fig. 10. Optical density (x 103) of various mixtures of poly A and poly U (From G.Felsenfeld, D. R. Davies, and A. Rich, J. A m. Chem. Soc., 79 (1957) 2023.
complexes. A double-stranded helical complex between poly A and poly-
ribo-thymidylic acid has more recently been obtained by Rich and collab-
orators. Its formation is illustrated d iagrammatically in Fig. 12. I am greatly
indebted to Dr . Rich for permission to use these illustrations. Double-strand-
ed helical structures can be formed by poly-adenylic acid in solution. The
e l e g a n t experiments of Doty and collaborators34 have shown that, above
Table 5. Double- and triple-stranded polynucleotide complexes.
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1 9 5 9 S . O C H O A
Fig.11. Hydrogen-bonding system in poly A + U and poly A + U + U. The hy-drogen bonds are represented by dashed lines (Courtesy of A. Rich).
neutral pH, poly A exists in solution as a random coil but the chains are
joined to form a helica l com plex at pH values below neu trality. This tran s-
formation is reversible and has a sharp transition point. The various types
of polyribonucleotide complexes thus far obtained are listed in Table 5.
The above stud ies may be of importance for a better understand ing of the
physical-chemical interactions underlying the role of DNA in cell division.These interactions may also play a role in the biological behavior of RNA.
Since there are good indications that the genetic information stored in DNA
is first transmitted to RNA, it is believed that DNA may function as a tem-
plate for RNA replication. Oncoming ribonucleotide residues could be
linked into an organized p olynucleotide chain growing in the h elical groove
of the native, double-stranded DNA molecule to form a triple-stranded
helix. Alternatively, a single-stranded DNA template might be used to give
a double-stranded DNA-RNA helix35-37
.
The work of Kornberg and his collaborators38 has given us deep insight
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E N Z Y M A T I C S Y N T H E S I S O F R I BO N U C L E I C A C I D 661
Fig. 12. Diagram showing the combination of two random coil molecules of poly-adenylic and polyribo-thymidylic acid to form a double-stranded helix. The bases are
represented by short rods (Courtesy of A. Rich).
into the mode of replication of DNA and may lead in the not too distant
futu re to the synthesis of genetic material in the test tube. Since RNA is the
genetic ma terial of some viruses, the work reviewed in th is lecture may h elp
to pave the way for the artificial synthesis of biologically active viral RNA
and the synthesis of viruses. These particles are at the threshold of life and
app ear to hold the clue to a better u nd erstanding of some of its most fund a-
mental principles.
1. M. Grunberg-Manago and S. Ochoa, J. A m. Chem. Soc., 77 (1955) 3165.2. M. Grunberg-Manago, P. J. Ortiz, and S. Ochoa, Science, 122 (1955) 907.3. S. Ochoa, Federation Proc., 15 (1956) 832.4. M. Grunberg-Manago, P. J. Ortiz, and S. Ochoa, Biochim. Biophys. Acta, 20 (1956)
269.5. B. E. Griffin, A. Todd, and A. Rich, Proc. Natl. Acad. Sci. U.S., 44 (1958) 1123.
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662 1 9 5 9 S . O C H O A
6. S. Ochoa and L. A. Heppel, in The Chemical Basis Of Heredity, W. D. McElroy andB. Glass (Eds.), Johns Hopkins Press, Baltimore, 1957, p. 615; S. Ochoa, in Cellular
Biology, N ucleic Acids, and V iruses, N.Y. Acad. of Sci., Spec. Publ., 5 (1957) 191.7. L. A. Heppel, P. J. Ortiz, and S. Ochoa, J. Biol. Chem., 229 (1957) 679.8. L. A. Heppel, P. J. Ortiz, and S. Ochoa, J. Biol. Chem., 229 (1957) 695.9. P. J. Ortiz and S. Ochoa, J. Biol. Chem., 234 (1959) 1208.
10. K. Tanaka, F. Egami, T. Hayashi, J. E. Winter, A. W. Bemheimer, S. Mii, P. J.Ortiz, and S. Ochoa, Biochim. Biophys. Acta, 25 (1957) 663.
11. A. Tiselius, S. Hjertén, and Ö. Levin, A rch. Biochem. Biophys., 65 (1956) 132.12. S. Ochoa, X I Conseil de Chimie Solvay, Bruxelles, June, 1959.13. M. F. Singer, L. A. Heppel, and R. J. Hilmoe, Biochim. Biophys. Acta, 26 (1957) 447.14. S. Mii and S. Ochoa, Biochim. Biophys. A cta, 26 (1957) 445; S. Ochoa, S. Mii, and
M. C. Schneider, Proc. Intern. Symp. Enzyme Chem., Tokyo Kyoto, 2 (1957) 44.15. L. A. Heppel, P. J. Ortiz, and S. Ochoa, Science, 123 (1956) 415.16. M. F. Singer, L. A. Heppel, and R. J. Hilmoe, J. Biol. Chem., 235 (1960) 738.17. D. O. Brummond, M. Staehelin, and S. Ochoa, J. Biol. Chem., 229 (1957) 835;
S. Ochoa, in Recent Progress in Microbiology, Symp. 7th Intern. Congr. Microbial.,
Stockholm, 2 (1958) 122.18. U. Z. Littauer and A. Komberg, J. Biol. Chem., 226 (1957) 1077.19. R. F. Beers, Jr., N ature, 177 (1956) 790.20. R. J. Hilmoe and L. A. Heppel, J. Am. Chem. Soc, 79 (1957) 4810.21. J. Skoda, J. Kara, A. Sormova, and F. Šorm, Biochim. Biophys. Acta, 33 (1959) 579.22. L. I. Hecht, P. C. Zamecnik, M. L. Stephenson, and J. F. Scott, J. Biol. Chem., 233
(1958) 954.23. E. S. Canellakis, Biochim. Biophys. A cta, 25 (1957) 217.24. J. Hu rwitz, A. Bresler, and A. Kaye, Biochem. Biophys. Res. Commun., 1(1959) 3.25. E. Goldwasser, J. A m. Chem. Soc., 77 (1955) 6083.26. S. B. Weiss and L. Gladstone, J. A m. Chem. Soc., 81 (1959) 4118.27. R. C. Warner, Federation Proc., 15 (1956) 379; J. Biol. Chem., 229 (1957) 711.28. A. Rich and D. R. Davies, J. Am. Chem. Soc., 78 (1956) 3548; A. Rich , in The
Chemical Basis of Heredity, W. D. McElroy and B. Glass (Eds.), Johns HopkinsPress, Baltimore, 1957, p. 557.
29. J. D. Watson and F. H. C. Crick, N ature, 171 (1953) 737.
30. R. C. Warner and E.Breslow, Symp. 4th Intern. Congr. Biochem., Vienna, 9(1958) 157.31. G. Felsenfeld and A. Rich, Biochim. Biophys. A cta, 26. (1957) 457; A. Rich, inCellular Biology, Nucleic Acids, and Viruses, N.Y. Academy of Sciences, Spec. Publ.,
5 (1957) 186.32. A. Rich, Biochim. Biophys. A cta, 29 (1958) 502.33. A. Rich, N ature, 181 (1958) 521.34. J. R. Fresco and P. Doty, J. A m. Chem. Soc, 79 (1957) 3928; J. R. Fresco and E.
Klemperer, A nn. N .Y . A cad. Sri., 81 (1959) 730.35. G. Stent, A dvan. V irus Res., 5 (1958) 138.36. G. Zubay, Nature, 182 (1958) 1290.37. A. Rich, A nn . N .Y . A cad. Sci., 81 (1959) 709.38. A. Kornberg, This Volume, pp. 665-680.