Adv. Space Res. V o l . 4 , No.12, p p . 1 4 3 - 1 5 1 , 1984 0273-1177/84 $0.00 + .

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POLYNUCLEOTIDE EVOLUTION,
HYPERCYCLES AND THE ORIGIN OF
THE GENETIC CODE
P Schuster

lnstitut far Theoretische Chemie und Strahlencheraie Universitiit

Wien, Wahringerstrasse 17, A 1090 Wien, Austria

ABSTRACT

E x p e r i m e n t s on p o l y n u c l e o t i d e r e p l i c a t i o n are d e s c r i b e d w i t h i n the frame of

a k i n e t i c theory of m o l e c u l a r evolution. Four p r i n c i p l e s of early e v o l u t i o n
are d i s c u s s e d and i l l u s t r a t e d by m e a n s of a m o d e l for the o r i g i n of trans-
lation.

INTRODUCTION

P o l y n u c l e o t i d e s , in p a r t i c u l a r the RNA from simple b a c t e r i o p h a g e s , r e p r e s e n t

the only p r e s e n t l y known m o l e c u l e s w h i c h can be used for e x p e r i m e n t a l studies
on early evolution. By "evolution" we m e a n here a p r o c e s s of d e v e l o p m e n t in
the sense of D a r w i n ' s theory of b i o l o g i c a l evolution. This is an o p t i m i -
zation process of excess p r o d u c t i o n based on m u t a t i o n and selection. In re-
p l i c a t i n g p o l y n u c l e o t i d e s the excess p r o d u c t i o n is given by the d i f f e r e n c e in
rate c o n s t a n t s of r e p l i c a t i o n and degradation. D u r i n g the last two decades
e x t e n s i v e i n v e s t i g a t i o n s were c a r r i e d out on the Q~ r e p l i c a s e system /I-3/.
This is an e x p e r i m e n t a l system for "in vitro" RNA r e p l i c a t i o n w h i c h c o n s i s t s
of an RNA d e p e n d e n t RNA p o l y m e r a s e - the c o m m o n l y called QB r e p l i c a s e -,
a c t i v a t e d m o n o n u c l e o t i d e s , the t r i p h o s p h a t e s GTP, ATP, CTP and UTP as w e l l as
s u i t a b l e t e m p l a t e for replication. Such a suitable t e m p l a t e is either the
natural RNA of the phage Q8 or any other RNA w h i c h carries the r e c o g n i t i o n
site for the r e p l i c a s e and w h i c h has a s u f f i c i e n t l y rich s e c o n d a r y s t r u c t u r e
in order to avoid c o m p l e t e double strand a s s o c i a t i o n d u r i n g replication. The
s p e c i f i c r e c o g n i t i o n site c o n s i s t s of two CCC t r i p l e t s at a rather flexible
d i s t a n c e of a few to about 15 bases /4/. Recently, an attempt to attach the
recognition sites onto a n o n - v i r a l m e s s e n g e r RNA was s u c c e s s f u l /5/. This
RNA can be a m p l i f i e d now by the Q8 system, and thereby an i m p o r t a n t step
forward towards a g e n e t i c e n g i n e e r i n g on the RNA level has been accomplished.

E f f i c i e n t r e p l i c a t i o n r e q u i r e s r e c o g n i t i o n sites on b o t h the plus and the

minus strands of the RNA. The r e s u l t are "inverted p a l i n d r o m e s " on the 3'
and 5' ends of the p o l y n u c l e o t i d e (Figure I). The e x i s t e n c e of r e c o g n i t i o n
sites on both strands as a p r e r e q u i s i t e of RNA r e p l i c a t i o n has b e e n found
also w i t h other RNA viruses, e.g. w i t h i n f l u e n z a A virus /6/. The other re-
q u i r e m e n t of RNA r e p l i c a t i o n by QB r e p l i c a s e is a s u f f i c i e n t l y rich secon-
dary structure. R e p l i c a t i o n p r o c e e d s first t h r o u g h d o u b l e strand formation,
but then the enzyme stops at certain positions, the s o - c a l l e d pause sites
/7/. Then, the s t r u c t u r e of the t e m p l a t e and the newly s y n t h e s i z e d strand
are r e f o l d e d under s e p a r a t i o n of the d o u b l e - h e l i c a l region. W h e n the RNA
does not form a s u f f i c i e n t l y rich s e c o n d a r y structure, the enzyme synthe-
sizes a double strand w h i c h is not a c c e p t e d as t e m p l a t e for further RNA
synthesis. An i l l u s t r a t i v e example is p r o v i d e d by p o l y C: the h o m o p o l y m e r
binds to the enzyme since it carries the r e c o g n i t i o n site (CCC .... CCC). Then
the c o m p l e m e t a r y strand is s y n t h e s i z e d and a double h e l i x poly C . p o l y G is
formed. This is the final p r o d u c t since the poly C ' p o l y G strands do not
separate.

The a v o i d a n c e of double strand f o r m a t i o n is one of the m o s t d i f f i c u l t pro-

blems for e n z y m e - f r e e RNA replication. In their e l e g a n t series of e x p e r i m e n t s
L e s l i e Orgel and c o w o r k e r s s u c c e e d e d to find the c o n d i t i o n s for e f f i c i e n t
t e m p l a t e induced e n z y m e - f r e e RNA s y n t h e s i s /8/. T h e y have not been able so
far to s e p a r a t e the double strands formed under the c o n d i t i o n s of the

143
144 P. Schuster

Plus strand Binding sites

5'pppGGGGACCCCCCGGAAGGGGGG~---- ACC[~GAAGGGGGGUUC~-~ 3'

Minus strand
5'pppGGGGAACCCCCCUUCGGGGGU~ cccr6~UUCCGGGGGGUC[~C" ] 3'

Fig.1. The 3'- and 5 ' - t e r m i n a l sequences of an RNA w h i c h is r e c o g n i z e d

as template by Q B - r e p l i c a s e /4/. Note, that the sequences of plus- and
m i n u s - s t r a n d r e f l e c t the r e q u i r e m e n t that both have to be r e c o g n i z e d by
the enzyme in order to m a k e r e p l i c a t i o n possible. This fact, in turn,
creates a r e l a t i o n b e t w e e n the 3'- and the 5' terminus.

p o l y m e r i z a t i o n reaction. RNA replication, however, requires single strand

templates.

S e p a r a t i o n of the p r i m a r y d o u b l e helix formed during template induced RNA-

synthesis into two single strands is one reason for the use of an enzyme in
the test tube e v o l u t i o n experiments. A n o t h e r reason is the time r e q u i r e d for
such studies. E n z y m e free RNA p o l y m e r i z a t i o n is simply too slow in order to
allow for a s u f f i c i e n t l y large n u m b e r of g e n e r a t i o n s w h i c h are n e c e s s a r y to
be able to observe e v o l u t i o n a r y phenomena. Low fidelity of enzyme free repli-
cation p r e s u m a b l y r e p r e s e n t s an i m p o r t a n t o b s t a c l e for e f f i c i e n t r e p l i c a t i o n
as well.

EVOLUTIONARY OPTIMIZATION "IN VITRO"

In this section we p r e s e n t b r i e f l y the results of e x p e r i m e n t s on D a r w i n i a n

e v o l u t i o n of RNA m o l e c u l e s w h i c h w e r e u n d e r t a k e n in order to prove and support
a kinetic theory of m o l e c u l a r e v o l u t i o n f o r m u l a t e d in the early s e v e n t i e s by
M a n f r e d Eigen /9/ and d e v e l o p e d further by himself and others i n c l u d i n g the
author /10/.

E v o l u t i o n can be v i s u a l i z e d as an o p t i m i z a t i o n process of r e p l i c a t i o n rates

w h i c h is based on m u t a t i o n and selection. It is, thus, using c o u n t e r a c t i n g
m e c h a n i s m s w h i c h create v a r i a t i o n or uniformity, respectively. In the primi-
tive systems we c o n s i d e r now we are d e a l i n g w i t h e x c l u s i v e l y c o m p e t i n g repli-
cating elements, the RNA molecules. Then we have e s s e n t i a l l y two m e c h a n i s m s
leading to a u n i f o r m population: (I) a d a p t i v e s e l e c t i o n and (2) r a n d o m se-
lection. V a r i a t i o n is c r e a t e d by point m u t a t i o n s and other r e a r r a n g e m e n t s of
p o l y n u c l e o t i d e s e q u e n c e s such as d e l e t i o n s of parts of the sequence or in-
sertions by partial r e p e t i t i o n s (Figure 2).

E v o l u t i o n a r y o p t i m i z a t i o n has been o b s e r v e d e x p e r i m e n t a l l y w i t h RNA in the QB

system. The e x p e r i m e n t a l t e c h n i q u e o r i g i n a l l y applied by Sol S p i e g e l m a n /I/
m i m i c s an open system far from e q u i l i b r i u m by "serial transfer" (Figure 3).
During the first few t r a n s f e r s the rate of RNA synthesis i n c r e a s e s spontane-
ously, the p r o p e r t y to infect b a c t e r i a l cells is lost, and the m o l e c u l a r
w e i g h t decreases. A f t e r about 100 transfers the r e p l i c a t i n g system a p p r o a c h e s
an optimum. Later on an i m p o r t a n t d i s c o v e r y was made by Sumper and Luce /11/
w h i c h was c o n f i r m e d and a n a l y s e d in detail by B i e b r i c h e r et ai./12-14/: in
the absence of t e m p l a t e the enzyme QB r e p l i c a s e synthesizes RNA "de nova".
O p t i m i z a t i o n of r e p l i c a t i o n rates leads to e l o n g a t i o n of the RNA m o l e c u l e s in
this case and finally an o p t i m u m RNA is selected. The results of these test
tube e x p e r i m e n t s on the e v o l u t i o n of RNA m o l e c u l e s are s u m m a r i z e d in Figure 4.

Recently, Hill and B l u m e n t h a l /15/ q u e s t i o n e d the "de nova" e x p e r i m e n t s with

QS replicase. They tried to d i s p r o v e the onset of RNA synthesis w i t h o u t tem-
plate by using h i g h l y p u r i f i e d enzyme fractions. U n f o r t u n a t e l y , these experi-
ments were c a r r i e d out under c o n d i t i o n s under w h i c h "de nova" s y n t h e s i s was
also not d e t e c t a b l e w i t h o t h e r enzyme p r e p a r a t i o n s (Biebricher, p e r s o n a l
communication). Thus, this c r i t i c i s m of "de nova" synthesis is off the point.
For the d e t a i l e d a r g u m e n t s on w h i c h the o c c u r e n c e of "de nova" RNA synthesis
is based we refer to the l i t e r a t u r e /13,14/.
 Polynucleotide Replication and the Origin of the Code 145

[MECHANISMS CREATINGI

I'~NIFORMITYI • I vAR'A"'uTY I

(1)ADAPTIVE SELECTION (1) POINTMUTATION

(2}RANDOM SELECTION ~ (2) DELETION
"" (3)INSERTION

~" ~ (GENEDUPLICATION)

(3)CO-OPERATION [&)RECOMBtNATION
(HYPERCYCLES) (COMPARTMENTS)

Fig. 2. M e c h a n i s m s c r e a t i n g u n i f o r m i t y and v a r i a t i o n in e v o l v i n g
populations.

I A NALYSER I

Q/I-REPLI CASE
tO t2 GTP. ATP
CTP,UTP
At ~l At At At ....... • 1 EXCESS

t, to . At
t2 to • 2at
t3 : tO • 3 ~
t~ : t o • /.At
ts : t o • sat

Fig. 3. The technique of serial t r a n s f e r e x p e r i m e n t s /I/. RNA of the

b a c t e r i o p h a g e Q8 grows in a m e d i u m w h i c h c o n t a i n s the enzyme QB repli-
case as w e l l as the four n u c l e o s i d e t r i p h o s p h a t e s ATP, UTP, GTP and CTP
in excess. A f t e r a time At a sample is t a k e n out of the test tube. Part
of it is analyzed, part of it is t r a n f e r r e d into a new medium. The pro-
cedure is r e p e a t e d after time i n t e r v a l s of At.

A D A P T I V E AND R A N D O M S E L E C T I O N

P r o v i d e d several c o n d i t i o n s are f u l f i l l e d we o b s e r v e s e l e c t i o n in an e n s e m b l e
of r e p l i c a t i n g p o l y n u c l e o t i d e s /16/. For the p u r p o s e of i l l u s t r a t i o n we
assume the f o l l o w i n g m e c h a n i s m s of r e p l i c a t i o n and d e g r a d a t i o n of RNA m o l e -
cules:
fk
(A)+Ik~ * 2I k (I)
f,
k
dk
Ik4 * (B) (2)

w i t h k=1,2,...,n. By I k we denote an i n d i v i d u a l RNA sequence, A stands for

the a c t i v a t e d m o n o m e r s GTP, ATP, CTP and UTP, B for the d e g r a d a t i o n products
GMP, AMP, CMP and UMP. In our simple e x a m p l e the c o n c e n t r a t i o n s of A and B
are a s s u m e d to be constant.
146 P. Schuster

DE6RADATION THROUGH
ReARRANGENeNTS
VIRAL-RNA ]

v=/.500 ~ v-300-500

.se~,A, TRANSfeR I ~T,~AL-R,A I

v =220

v=1 "'DE NOVO" SYNTHESIS

I MONO_NUCLEOTIDES] CHAIN eLONGATION

GTP ATP.CTP.UTP
SMALL OLIGO-NUCLEOTIDES?
v =2.3.,'.....
Fig. 4. The results of e v o l u t i o n a r y o p t i m i z a t i o n in the test tube
(schematically)

The occurrence of s e l e c t i o n is bound to four p r e r e q u i s i t e s :

(I) an energy or m a t e r i a l flux w h i c h keeps the s y s t e m far from equilibrium,
(2) a d e g r a d a t i o n p r o c e s s w h i c h is i r r e v e r s i b l e for p r a c t i c a l p u r p o s e s , d ~ < < d k
or d~=O r e s p e c t i v e l y (k=1,2 ..... n),
(3) a r e p l i c a t i o n process w h i c h is i r r e v e r s i b l e for p r a c t i c a l p u r p o s e s , f ~ < < f k
or f~=O r e s p e c t i v e l y (k=1,2 ..... n) and
(4) a r e p l i c a t i o n process w h i c h is a u t o c a t a l y t i c in first order as e.g.
reaction (I). Higher order a u t o c a t a l y t i c processes, these are r e a c t i o n s w h e r e
more than one r e p l i c a t i n g m o l e c u l e c a t a l y s e s the r e p l i c a t i o n p r o c e s s (see be-
low), can have m u l t i p l e steady states and, hence, there is no u n i q u e l y de-
fined e v o l u t i o n a r y optimum.

The first two p r e r e q u i s i t e s are "conditiones sine quibus non" w h e r e a s the

latter two, (3) and (4), are n e c e s s a r y for "global selection". By this we
mean selection for all values of rate c o n s t a n t s and external parameters. If
(3) and (4) are not f u l f i l l e d we observe s e l e c t i o n only for c e r t a i n ranges of
p a r a m e t e r values.

Let us assume that we are d e a l i n g w i t h an e n s e m b l e of n p o l y n u c l e o t i d e s with

d i f f e r e n t values of the rate c o n s t a n t s f~ and d~ (k=I,2, .... n). Then, we have
an example for a d a p t i v e selection. The RNA m o l e c u l e w i t h the m a x i m u m net rate
of synthesis
Im: fm-dm = max{ (fk-dk); k=I,2 ..... n} (3)

is selected. During a d a p t i v e s e l e c t i o n we observe a m o n o t o n o u s increase of the

total rate of RNA synthesis in the system. A d a p t i v e s e l e c t i o n is s u m m a r i z e d
in Figure 5.
Now, we consider the a l t e r n a t i v e extreme, all rate c o n s t a n t s are equal: fl =
=fg=...=f =f and d ~ = d o = . . . = d =d. D e s p i t e this k i n e t i c degeneracy, s e l e c t i d n
do~s occug. In thi~ c~se, however, it is the result of a s t o c h a s t i c process.
The sequence which is u l t i m a t e l y s e l e c t e d is chosen at r a n d o m and we charac-
terize the process as " r a n d o m selection". The m a t h e m a t i c a l a n a l y s i s of the
c o r r e s p o n d i n g equations of s t o c h a s t i c k i n e t i c s can be found in/17, 18/.One
c h a r a c t e r i s t i c feature of r a n d o m s e l e c t i o n concerns the total rate of RNA
synthesis: it remains e s s e n t i a l l y c o n s t a n t (Figure 5). A d o m i n a t i n g s e q u e n c e
nevertheless, can be r e p l a c e d by a neutral mutant. This p r o c e s s is the analo-
gon to "neutral evolution" w i t h higher o r g a n i s m s as it is r e v e a l e d by the re-
sults of m o l e c u l a r e v o l u t i o n (For a recent c o l l e c t i v e volume on the "neutral
theory" see e.g. /19/).

A d a p t i v e selection d o m i n a t e s when the rate c o n s t a n t s of newly formed a d v a n t a -

geous m u t a n t s are s u f f i c i e n t l y d i f f e r e n t from thoseof the s e q u e n c e s p r e s e n t
in the population. This is the case in systems far from the e v o l u t i o n a r y op-
timum. We may call this phase as "early evolution". R a n d o m selection, in con-
trary, d o m i n a t e s the "late" phase of e v o l u t i o n w h e n the rate of RNA synthesis
is already very close to its o p t i m u m value.

ERROR T H R E S H O L D AND LIMITS OF GENOME LENGTHS

In order to describe RNA r e p l i c a t i o n a p p r o p r i a t e l y we have to extend the

simple m e c h a n i s m (1) and (2) by the e x p l i c i t c o n s i d e r a t i o n of mutations. The
 Polynucleotide Replication and the Origin of the Code 147

tl--d I

"--------""--e

® J
'.-'. i I Jt

l Ill . . . . . . . . . . . . . .

t.- 1. L i

I ,O~.T~ Ii,'T,O,, I l
TOTAL RATE 0~ R N & ' ~ Y N T I S ~ I " i , l
! .,~ ,,lli-II,i --.,~ ~A X

I EV~UT~O~RV a"Ti,,ZAT,O.I

f--d L

f--d e
NFUTI#AL FVoLurIox ILATF XHASF;
I
f--d I

f~l I

'I ................................

f~d .5 X, t; I,

I RAM00M ~ELECTJDNI

TOTAL PATE OF NNA'SYNTW~S

• CONSY

Fig. 5. A c o m p a r i s o n of adaptive and random selection as they occur

during the early and late phase of e v o l u t i o n (In the latter case neutral,
i.e. kineticly d e g e n e r a t e m u t a n t s are indicated by "prime", "double
prime" Ii I~ I~ etc.)

c o r r e s p o n d i n g kinetic systems have been d i s c u s s e d e x t e n s i v e l y in the past

(see e.g. /9,10,20/). We d i s p e n s e here from all details and summarize only
the most important results of these investigations.

Mutations have two important c o n s e q u e n c e s on selection:

(I) They create a m u t a n t d i s t r i b u t i o n w h i c h a c c o m p a n i e s the selected "master
sequence". The stationary d i s t r i b u t i o n has been called "quasispecies" in ana-
logy to the notion of a species in b i o l o g y /10/.
(2) The formation of a q u a s i s p e c i e s is c o n s t r a i n e d to an accuracy of replica-
tion which is above a p r e c i s e l y defined m i n i m u m a c c u r a c y Q~4~" B e l o w this
m i n i m u m the m e c h a n i s m of inheritance breaks down. Genetic T ~ T o r m a t i o n is no
longer conserved in a given population.
The m i n i m u m accuracy of r e p l i c a t i o n is e q u i v a l e n t to a m a x i m u m chain length
of the p o l y n u c l e o t i d e to be r e p l i c a t e d p r o v i d e d there exists a mean single
digit accuracy of the r e p l i c a t i o n process. This m e a n single digit accuracy,
is a measure for the correct i n c o r p o r a t i o n of the c o m p l e m e n t a r y base into the
growing p o l y n u c l e o t i d e chain:
-~max
Qmin = q (4)
Equation (4), thus, predicts a limit to the length of genomes for a given
m e c h a n i s m of r e p l i c a t i o n which is c h a r a c t e r i z e d by its m e a n single digit
accuracy, q. Relation (4) has been checked for simple RNA viruses /10/. In
these systems we found that the actual genome lengths come very close to the
error threshold. In case of p r e b i o t i c e v o l u t i o n we cannot assume the existence
of o p t i m i z e d enzymes which can operate at a p r e c i s i o n w h i c h is comparable to
that of present day virus specific replicases. Hence, the lengths of prebio-
tic p o l y n u c l e o t i d e s were m u c h shorter, at least w h e n they acted as informa-
tion carriers. Using the results of Orgel and coworkers /8/ we estimate
]48 P. Schuster

m a x i m u m lengths in the range of 1OO bases. Roughly speaking, RNA m o l e c u l e s of

the same lengths as p r e s e n t day t-RNA's may have been p o s s i b l e w i t h o u t the
help of specific enzymes.

C O - O P E R A T I O N AND THE D Y N A M I C S OF COMPLEX SYSTEMS

In order to overcome the limits of error propagation, more complex m o l e c u l a r

systems have to form. Let us now ask what the m o s t simple nucleus for such a
more complex system m i g h t look like. First of all it would have to g u a r a n t e e
c o - o p e r a t i o n of i n f o r m a t i o n carriers. Since these i n f o r m a t i o n c a r r i e r s are
p o l y n u c l e o t i d e s and, thus, i n h e r e n t competitors, c o - o p e r a t i o n p r i m a r i l y m e a n s
s u p p r e s s i o n of competition.

The simplest d y n a m i c a l system w h i c h i n t r o d u c e s c o - o p e r a t i o n into an e n s e m b l e

of c o m p e t i t o r s makes use of second order autocatalysis. Then, the r e a c t i o n
in e q u a t i o n (I) has to be r e p l a c e d by

(A) + I k + Ij fk~5 ) 2I k + I 3 (5).

P o l y n u c l e o t i d e s have two functions: they act as templates for r e p l i c a t i o n

(Ik) and have catalytic a c t i v i t y (Ij). C o - o p e r a t i o n occurs in an e n s e m b l e of
second order a u t o c a t a l y s i s if m u t u a l e n h a n c e m e n t of the rates of synthesis
prevails over self-enhancement. For two c o m p e t i t o r s this simply m e a n s
f21 > f11 and f12 > f22 (6).
E q u a t i o n (6) is always f u l f i l l e d if s e l f - e n h a n c e m e n t can be e x c l u d e d (f,1=O,
f22=O). C o - o p e r a t i v e systems of this type have been called " h y p e r c y c l e s ~
/9,10/. Their dynamics has been a n a l y z e d in great detail (For a c o m p l e t e
p r e s e n t a t i o n of the kinetic a n a l y s i s see the two reviews /21,22/).
Simple second order a u t o c a t a l y s i s is not the only way to i n t r o d u c e c a t a l y t i c
a c t i v i t y into an e n s e m b l e of p o l y n u c l e o t i d e s . Other m e c h a n i s m s are complex
formation or t r a n s l a t i o n into p r o t e i n /10/. In general, one will o b t a i n a
rather involved d y n a m i c a l system w h i c h can be r e d u c e d to that of a "hyper-
cycle" or to some c o m p e t i t i v e system under suitable conditions. Despite se-
veral attempts to d e v e l o p a general theory of n o n l i n e a r a u t o c a t a l y t i c systems
in order to be able to m a k e p r e d i c t i o n s on c o m p l i c a t e d r e l a t i o n n e t w o r k s no
such theory is a v a i l a b l e yet. Predictions, therefore, can be made only for
special and simple enough systems (A recent a p p r o a c h to c o m p l e x r e a c t i o n net-
works has been p r e s e n t e d by Clarke /23/).

C o - o p e r a t i v e systems, once formed, are not easily r e p l a c e d by f a v o u r a b l e

mutants. The dynamics of these systems imposes a v e r y strong c o n s t r a i n t on
the o p t i m i z a t i o n process. We can v i s u a l i z e h y p e r c y c l e s as an a d d i t i o n a l
p r i n c i p l e c r e a t i n g u n i f o r m i t y (Figure 2).

A M O D E L FOR EARLY E V O L U T I O N

Based on the results of test tube e v o l u t i o n and on the general c o n s i d e r a t i o n s

about complex a u t o c a t a l y t i c r e a c t i o n n e t w o r k s a m o d e l for p r e b i o t i c e v o l u t i o n
has been w o r k e d out in some detail /10,24,25/. We do not intend to repeat
here w h a t has been said elsewhere. Instead, we give a summary of the sequence
of steps w h i c h lead from one level of o r g a n i z a t i o n to the next higher level
(Figure 6).

Let us assume that a d y n a m i c a l l y o r g a n i z e d system has been formed by evolu-

tionary o p t i m i z a t i o n of r e p l i c a t i o n rates and i n t e g r a t i o n of c o m p e t i t o r s into
a c o - o p e r a t i v e unit. Then, c o m p a r t m e n t f o r m a t i o n and i n d i v i d u a l i z a t i o n appear
to be the next logically r e q u i r e d steps /25/. The f o r m a t i o n of a c o - o p e r a t i v e
unit imposes a c o n s t r a i n t on e v o l u t i o n a r y optimization. The system is forced
to d e v e l o p a high degree of u n i f o r m i t y w h i c h later looks like a "frozen
accident". P h y s i c a l isolation of the c o - o p e r a t i v e unit in a c o m p a r t m e n t re-
stores e v o l u t i o n a r y optimization. The e v a l u a t i o n with r e s p e c t to r e p l i c a t i o n
and p r o l i f e r a t i o n is now laid on the level of entire compartments. We can in-
terpret c o m p a r t m e n t f o r m a t i o n or i n d i v i d u a l i z a t i o n as a p r i n c i p l e leading to
v a r i a b i l i t y (Figure 2).

An inspection of b i o l o g i c a l e v o l u t i o n suggests that the four p r i n c i p l e s of

e v o l u t i o n apply also to later stages, w h e n e u c a r y o t i c cells were formed from
p r i m i t i v e precursors, when m u l t i c e l l u l a r o r g a n i s m s o r i g i n a t e d from u n i c e l l u -
lar precursors, etc.
 Polynucleotide Replication and the Origin of the Code 149

IFOURP., ;C,P ES OF voLu ,o ;I

,II REPUCAT,ON IsELEcT'°NI POtYNUCL.EOT,OESI
OPTIMIZATIONOF )
(2} MUTATION REPLICATIONRATES

ERROR PROPAGATION:LIMITATIONOF GENOME LENGTHS

CO-OPERATION I INTEGRATION OF L...~REPLICATION-TRANSLATIONI

131
"HYPERCYCLES-[ COMPETITORS I I UNIT. GENETIC CODE
I
CONSTRAINED OPTIMIZATION. "FROZEN ACCIDENTS"

[ OPTIMIZATIONOF
INDIVIDUALIZATIONI
It,) |REPLICATION RATES PROTOCELLS I
"COMPARTMENTS]OF COMPARTMENTS

Fig. 6. Four p r i n c i p l e s of e v o l u t i o n w h i c h lead from a lower towards a

higher h i e r a r c h i c a l level of o r g a n i z a t i o n /24/.

A P R I M I T I V E G E N E T I C CODE

As i n d i c a t e d in F i g u r e 6 the m o d e l for e a r l y e v o l u t i o n has been applied to

the o r i g i n of g e n e t i c translation. Thereby, m o d e l c o n s i d e r a t i o n s were com-
bined w i t h c o m p a r a t i v e sequence studies of t-RNA's /26-28/ as well as with
the results of o l i g o n u c l e o t i d e i n t e r a c t i o n k i n e t i c s /29/. The e s s e n t i a l fea-
tures of our m o d e l are several p r e d i c t i o n s w h i c h appear to be m e a n i n g f u l in
the context of p r e b i o t i c c h e m i s t r y and w h i c h e v e n t u a l l y will lead to conclu-
sive e x p e r i m e n t s in the future:
(1) In order to g u a r a n t e e s u f f i c i e n t s t a b i l i t y of m e s s e n g e r - R N A - t-RNA
c o m p l e x e s the p r i m i t i v e codons and a n t i c o d o n s w o u l d have to be rich in G and
C.
(2) In order to a v o i d sliding of the t - R N A along the m e s s e n g e r d u r i n g ribo-
s o m e - f r e e t r a n s l a t i o n one needs some r e p e t i t i v e p a t t e r n of p e r i o d three as
p o s t u l a t e d by C r i c k et al. /30/. Actually, a p a t t e r n of the type -Purine.N.
P y r i m i d i n e - (N stands for either purine or pyrimidine) has not yet been com-
p l e t e l y r e m o v e d by m u t a t i o n s and does still e x i s t as a kind of b a c k g r o u n d
noise in the s e q u e n c e s of p r e s e n t day RNA's and DNA's /31/.
(3) C o m b i n i n g (I) and (2) in a s t r a i g h t f o r w a r d way we o b t a i n four codons
w h i c h are p r e s e n t l y used for the amino acids
glycine = GGC, alanine = GCC
a s p a r t i c acid = GAC and v a l i n e = GUC.
These four amino acids are the at the same time the m o s t a b u n d a n t amino
acids in p r e b i o t i c s i m u l a t i o n experiments.
(4) N u c l e o t i d e b i n d i n g p r o b a b l y was one of the m o s t a n c i e n t p r o p e r t i e s of en-
coded p o l y p e p t i d e s . R o s s m a n n et al. /32/ m a d e an a l i g n m e n t study of v a r i o u s
d e h y d r o g e n a s e s and other n u c l e o t i d e b i n d i n g p r o t e i n s in order to r e c o n s t r u c t
a p o s s i b l e c o m m o n a n c e s t o r protein. W a l k e r /33/ a n a l y z e d the 21-site amino
acid s e q u e n c e s of the n u c l e o t i d e - b i n d i n g surface and tried to i d e n t i f y the
p r e c u r s o r amino acids coded into the a n c e s t r a l surface w h i c h is assumed to be
about 3.109 years old. He c o n c l u d e s that v a l i n e and one or b o t h of the
group a s p a r t i c acid - g l u t a m i c acid w o u l d be likely p r e c u r s o r s w i t h v a l i n e
p r e d o m i n a t i n g . Both c o n s t i t u e n t s of the a n c i e n t r e c o g n i t i o n site are m e m b e r s
of the group of the first four amino acids.

Our k n o w l e d g e on the physics of p o l y n u c l e o t i d e - p o l y p e p t i d e i n t e r a c t i o n s is

f r a g m e n t a r y only. Therefore, all the d e t a i l s of the p r o c e s s and the proper-
ties w h i c h m i g h t have played major roles in e a r l y t r a n s l a t i o n are not known
yet. So, we have to w a i t for the results of e x p e r i m e n t a l r e s e a r c h in this
field before we can c o n c l u s i v e l y c o n t i n u e the s p e c u l a t i o n s on the origin of
the g e n e t i c code.
A c k n o w l e d g e m e n t s : This w o r k was s u p p o r t e d f i n a n c i a l l y by the "Austrian Fonds
zur F 6 r d e r u n g der W i s s e n s c h a f t l i c h e n F o r s c h u n g (Project no 5286). T e c h n i c a l
a s s i s t a n c e in p r e p a r i n g the m a n u s c r i p t by M r s . J . J a k u b e t z and M r . J . K ~ n i g is
gratefully acknowledged.
150 P. Schuster

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