138

                  PROTEIN SYNTHESIS             139
Once the central and unique role of proteins is admitted there seems little
point in genes doing anything else. Although proteins can act in so many
different ways, the way in which they are synthesized is probably uniform
and rather simple, and this fits in with the modern view that gene action,
being based upon the nucleic acids, is also likely to be uniform and rather
simple.

Biologists should not deceive themselves with the thought that some new
class of biological molecules, of comparable importance to the proteins,
remains to be discovered. This seems highly unlikely. In the protein
molecule Nature has devised a unique instrument in which an underlying
simplicity is used to express great subtlety and versatility; it is impossible
to see molecular biology in proper perspective until this peculiar combi-
nation of virtues has been clearly grasped.

ON PROTEIN SYNTHESIS

BY F. H. c. [CRICK

Medical Research Council Unit for the Study of Molecular Biology,
       Cavendish Laboratory, Cambridge

I. INTRODUCTION

Protein synthesis is a large subject in a state of rapid development. To cover
it completely in this article would be impossible. I have therefore deliber-
ately limited myself here to presenting a broad general view of the problem,
emphasizing in particular well-established facts which require explanation,
and only selecting from recent work those experiments whose implications
seem likely to be of lasting significance. Much very recent work, often of
great interest, has been omitted because its implications are not clear.
I have also tried to relate the problem to the other central problems of
molecular biology-those of gene action and nucleic acid synthesis. In
short, I have written for the biologist rather than the biochemist, the
general reader rather than the specialist. More technical reviews have
appeared recently by Borsook (1956), Spiegelman (1957), and Sin&in &
Work (19576 and this Symposium),

The importance of proteins

It is an essential feature of my argument that in biology proteins are
uniquely important. They are not to be classed with polysaccharides, for
example, which by comparison play a very minor role. Their nearest rivals
are the nucleic acids. Watson said to me, a few years ago, `The most
significant thing about' the nucleic acids is that we don't know what they
do.' By contrast the most significant thing about proteins is that they can
do almost anything. In animals proteins are used for structural purposes,
but this is not their main role, and indeed in plants this job is usually done
by polysaccharides. The main function of proteins is to act as enzymes.
Almost all chemical reactions in living systems are catalysed by enzymes,
and all known enzymes are proteins. It is at first sight paradoxical that it is
probably easier for an organism to produce a new protein than to produce
a new small molecule, since to produce a new small molecule one or more
new proteins will be required in any case to catalyse the reactions.

I shall also argue that the main function of the genetic material is to
control (not necessarily directly) the synthesis of proteins. There is a little
direct evidence to support this, but to my mind the psychological drive
behind this hypothesis is at the moment independent of such evidence.

II. THE PROBLEM

Elementary facts about proteins

(I) Composition. Simple (unconjugated) proteins break down on hydro-
lysis to amino acids. There is good evidence that in a native protein the
amino acids are condensed into long polypeptide chains. A typical protein,
of molecular weight about 25,000, will contain some 230 residues joined
end-to-end to form a single polypeptide chain.

Two points are important. First, the actual chemical step required to
form the covalent bonds of the protein is always the same, irrespective of
the amino acid concerned, namely the formation of the peptide link with
the elimination of water. Apart from minor exceptions (such as S-S links
and, sometimes, the attachment of a prosthetic group) all the covalent links
within a protein are formed in this way. Covalently, therefore, a protein is to
a large extent a linear molecule (in the topological sense) and there is little
evidence that the backbone is ever branched. From this point of view the
cross-linking by S-S bridges is looked upon as a secondary process.

The second important point-and I am surprised that it is not remarked
more often-is that only about twenty different kinds of amino acids occur
in proteins, and that these same twenty occur, broadly speaking, in all
proteins, of whatever origin-animal, plant or micro-organism. Of course
not every protein contains every amino acid-the amino acid tryptophan,
which is one of the rarer ones, does not occur in insulin, for example-
but the majority of proteins contain at least one of each of the twenty
amino acids. In addition all these twenty amino acids (apart from glycine)
have the L configuration when they occur in genuine proteins.

There are a few proteins which contain amino acids not found else-


                  PROTEIN SYNTHESIS             `4'
fully extended but is thrown into folds and superfolds, maintained by weak
physical bonds, and in some cases by covalent -S-S- links and possibly
some others. This folding is also thought to be at least broadly the same for
each copy of a particular protein, since many proteins can be crystallized,
though the evidence for perfect homogeneity of folding is perhaps rather
weak.* As is well known, if this folding is destroyed by heat or other
methods the protein is said to be `denatured'. The biological properties
of most proteins, especially the catalytic action of enzymes, must depend
on the exact spatial arrangement of certain side-groups on the surface of
the protein, and altering this arrangement by unfolding the polypeptide
chains will destroy the biological specificity of the proteins.           `,
(4) Amino acid requirements. If one of the twenty amino acids is supplied
to a cell it can be incorporated into proteins; amino acids are certainly protein
precursors. The only exceptions are amino acids like hydroxyproline, which
are not among the magic twenty. The utilization of peptides is controversial
but the balance of evidence is against the occurrence of peptide inter-
mediates. (See the discussion by Simkin & Work, this Symposium.)  I:
If, for some reason, one of the twenty amino acids is not available to the

organism, protein synthesis stops. Moreover, the continued synthesis of
those parts of the protein molecules which do not contain that amino acid
appears not to take place. This can be demonstrated particularly clearly in
bacteria, but it is also true of higher animals. If a meal is provided that
lacks an essential amino acid it is no use trying to make up for this deficiency
by providing it a few hours later.

Very little is known about the accuracy with which the amino acids are
selected. One would certainly expect, for example, that the mechanism
would occasionally put a valine into an isoleucine site, but exactly how often
this occurs is not known. The impression one gets from the rather meagre
facts at present available is that mistakes occur rather infrequently.

In recent years it has been possible to introduce amino acid analogues
into proteins by supplying the analogue under circumstances in which the
amino acid itself is not easily available (see the review by Kamin & Handler,
1957). For example in Escherichia co& fluorophenylalanine has been in-
corporated in place of phenylalanine and tyrosine (Munier & Cohen, 1956)
and it has even proved possible to replace completely the sulphur-containing
amino acid methionine by its selenium analogue (Cohen & Cowie, 1957).
Of the enzymes produced by the cell in these various ways some were
active and some were inactive, as might have been expected.
(5) Contrast with polysacchavides. It is useful at this point to contrast
proteins with polysaccharides to underline the differences between them.
               * See previous footnote.

140                 PROTEIN SYNTHESIS
where-the hydroxyproline of collagen is a good example--but in all such
cases it is possible to argue that their presence is due to a modification of
the protein after it has been synthesized or to some other abnormality. In
Table I, I have listed the standard twenty amino acids believed to be of
universal occurrence and also, in the last column, some of the exceptional
ones. The assignment given in Table I might not be agreed by everyone,

Table I

The magic twenty amino acids found
   universally in proteins
,           ,           -I
Glycine
Alanine     g,;y*z
Valine        Aspartic acid
Leucine         Glutamic acid
Isoleucine
Proline*        Arginine

Phenylalanine     Lysine
             Hi&dine
z$gehe       Tryptophan

Threonine

Cysteinet
Methionine

Other amino acids found in proteins
Hydroxyproline'
Hydroxylysine
Phosphoserine
Diaminopimelic `kid
Thyroxine and related molecules

Cystinet

o These are,.of course, imino acids. This distinction is not made in the text.
t This classification implies that all the cystine found in proteins is formed by the

joining together of two cyst&e molecules.

as the evidence is incomplete, but more agreement could be found for this
version than for any other. Curiously enough this point is slurred over by
almost all biochemical textbooks, the authors of which give the impression
that they are trying to include as many amino acids in their lists as they
can, without bothering to distinguish between the magic twenty and the
others. (But see a recent detailed review by Synge, 1957.)

(2) Homogeneity. Not only is the composition of a given protein fixed,
. but we have every reason to believe that the exact order of the amino acid
residues along the polypeptide chains is also rigidly determined: that each
molecule of haemoglobin in your blood, for example, has exactly the same
sequence of amino acids as every other one. This is clearly an overstate-
ment ; the mechanism must make mistakes sometimes, and, as we shall see,
there are also interesting exceptions which are under genetic control.
Moreover, it is quite easy, in extracting a protein, to modify some of the
molecules slightly without affecting the others, so that the `pure' protein
may appear heterogeneous. The exact amount of `microheterogeneity' of
proteins is controversial (see the review by Steinberg & Mihaiyi, 1957), but
this should not blind one to the astonishing degree of homogeneity of most
proteins.*

(3) Structure. In a native globular protein the polypeptide chain is not

* The y-globulins and other antibody molecules are exceptions to these generalizations.
They are probably heterogeneous in folding and possibly to some extent in composition.


`42                 PROTEIN SYNTHESIS
(I do not include nucleic acids among the polysaccharides.) - Polysac-
charides, too, are polymers, but each one is constructed from one, or at the
most only about half-a-dozen kinds of monomer. Nevertheless many
different monomers are found throughout Nature, some occurring here,
some there. There is no standard set of monomers which is always used, as
there is for proteins. Then polysaccharides are polydisperse-at least so
far no monodisperse one has been found-and the order of their monomers
is unlikely to be rigidly controlled, except in some very simple manner.
Finally in those cases which have been carefully studied, such as starch,
glycogen and hyaluronic acid, it has been found that the polymerization is
carried out in a straightforward way by enzymes.
(6) The genetics and taxonomy of proteins. It is instructive to compare
your own haemoglobin with that of a horse. Both molecules are indis-
tinguishable in size. Both have similar amino acid compo8itions; similar
but not identical. They differ a little electrophoretically, form different
crystals, and have slightly different ends to their polypeptide chains. All
these fact8 are compatible with their polypeptide chains having similar
amino acid sequences, but with just a few changes here and there.
This `family likeness' between the `same' protein moleculesfromdifferent
species is the rule rather than the exception. It has been found in almost
every case in which it ha8 been looked for. One of the best-studied examples
is that of insulin, by Sanger and his co-workers (Brown, Sanger & Kitai,
1955; Harris, Sanger 8z Naughton, 1956), who have worked out the
complete amino acid sequence8 for five different species, only two of which
(pig and whale) are the same. Interestingly enough the differences are all
located in one small segment of one of the two chains.
Biologists should realize that before long we shall have a subject which
might be called `protein taxonomy `-the study of the amino acid sequences
of the proteins of an organism and the comparison of them between species.
It can be argued that these sequences are the most delicate expression
possible of the phenotype of an organism and that vast amounts of
evolutionary information may be hidden away within them.
There is, however, nothing in the evidence presented so far to prove that
these differences between species are under the control of Mendelian genes.
It could be argued that they were transmitted cytoplasmically through the
egg. On the other hand, there is much evidence that genes do affect enzymes,
especially from work on micro-organisms such as Neurospora (see Wagner
& Mitchell, 1955). The famous `one gene-one enzyme' hypothesis
(Beadle, 1945) expresses this fact, although its truth is controversial
(personally I believe it to be largely correct). However, in none of these
cases has the protein (the enzyme, that is) ever been obtained pure.

                PROTEIN SYNTHESIS             `43
There are a few cases where a Mendelian gene has been shown un-

ambiguously to alter a protein, the most famous being that of human sickle-
cell-anaemia haemoglobin, which differs electrophoretically from normal
adult haemoglobin, as was discovered by Pauling and his co-workers (1949).
Until recently it could have been argued that this was perhaps not due to a
change in amino acid sequence, but only to a change in the folding. That
the gene does in fact alter the amino acid sequence has now been con-
clusively shown by my colleague, Dr Vernon Ingram. The difference is due
to a valine residue occurring in the place of a glutamic acid one, and
Ingram has suggestive evidence that this is the onZy change (Ingram,
1956, 1957). It may surprise the reader that the alteration of one amino
acid out of a total of about 300 can produce a molecule which (when
homozygous) is usually lethal before adult life but, for my part, Ingram's
result is just what I expected.                           ., ,,
                                                    I
                                                 t i'

The nature of protein synthesk

1 :

The basic dilemma of protein synthesis ha8 been realized by many people;
but it has been particularly aptly expressed by Dr A. L. Dounce (1956);

My interest in templatea, and the conviction of their necessity, originated from
a question asked me on my Ph.D. oral examination by Professor J. B. Sumner.
He enquired how I thought protein8 might be synthesized. I gave what seemed
the obvious answer, namely, that enzymes must be responsible. Professor
Sumner then asked me the chemical nature of enzymea, and when 1 answered
.that enzymes were protein8 or contained proteins 88 essential components, he
asked whether these enzyme protein8 were synthesized by other enaymes and 80
on ad injim'tum.

The dilemma remained in my mind, causing me to look for possible solutions
that would be acceptable, at least from the standpoint of logic. The dilemma, of
course, involve8 the specificity of the protein molecule, which doubtless depends to
a considerable degree on the sequence of amino acid8 in the peptide chains of the
protein. The problem is to find a reasonably simple mechanism that could account
for specific sequence8 without demanding the presence of an ever-increasing
number of new specific enzyme8 for the synthesis of each new protein molecule.

It is thus clear that the synthesis of proteins must be radically different
from the synthesis of polysaccharides, lipids, co-enzymes and other small
molecules; that it must be relatively simple, and to a considerable extent
uniform throughout Nature; that it must be highly specific, making few
mistakes; and that in all probability it must be controlled at not too many
removes by the genetic material of the organism.

The essence of the problem

A systematic discussion of our present knowledge of protein synthesis
could usefully be set out under three headings, each dealing with a flux:


`44                 PROTEIN SYNTHESIS
the flow of energy, the flow of matter, and the flow of information. I shall
not discuss the first of these here. I shall have something to say about the
second, but I shall particularly emphasize the third-the flow of information.

By information I mean the specification of the amino acid sequence of
the protein. It is conventional at the moment to consider separately the
synthesis of the polypeptide chain and its folding. It is of course possible
that there is a special mechanism for folding up the chain, but the more
likely hypothesis is that the fokfing is simply a function of the order of the
amino acids, provided it takes place as the newly formed chain come8 off
the template, I think myself that this latter idea may well be correct,
though I would not be surprised if exceptions existed, especially the
y-globulins and the adaptive enzymes.

Our basic handicap at the moment is that we have no easy and precise
technique with which to study how proteins are folded, whereas we can at
least make some experimental approach to amino acid sequences. For this
reason, if for no other, I shall ignore folding in what follows and concentrate
on the determination of sequences. It is as well to realize, however, that
the idea that the two processes can be considered separately is in itself an
assumption.

The actual chemical step by which any two amino acids (or activated
amino acids) are joined together is probably always the same, and may well
not differ significantly from any other biological condensation. The unique
feature of protein synthesis is that only a single standard set of twenty
amino acids can be incorporated, and that for any particular protein the
amino acids must be joined up in the right order. It is this problem, the
problem of ` sequentialization', which is the crux of the matter, though it is
obviously important to discover the exact chemical steps which lead up to
and permit the crucial act of sequentialization.

As in even a small bacterial cell there are probably a thousand different
kinds of protein, each containing some hundreds of amino acids in its own
rigidly determined sequence, the amount of hereditary information required
for sequentialization is quite considerable.

III. RECENT EXPERIMENTAL WORK

The role of the nucleic acids

It is widely believed (though not by every one) that the nucleic acids are
in some way responsible for the control of protein synthesis, either directly
or indirectly. The actual evidence for this is rather meagre. In the case of
deoxyribonucleic acid (DNA) it rests partly on the T-even bacteriophages,
since it has been shown, mainly by Hershey and his colleagues, that

                  PROTEIN SYNTHESIS            `45
whereas the DNA of the infecting phage penetrates into the bacterial cell
almost all the protein remains outside (see the review by Hershey, 1956);
and also on Transforming Factor, which appears to be pure DNA, and
which in at least one case, that of the enzyme mannitol phosphate dehydro-
genase, controls the synthesis of a protein (Marmur & Hotchkiss, 1955).
There is also the indirect evidence that DNA is the most constant part of
the genetic material, and that genes control proteins. Finally there is the
very recent evidence, mainly due to the work of Benzer on the rI1 locus of
bacteriophage, that the functional gene-the ' cistron' of Benzer's termind-
logy-consists of many sites arranged strictly in a linear order (Benzer, 1g57)
as one might expect if a gene controls the order of the amino acids in some
particular protein.                                     :.,,;
As is well known, the correlation between ribonucleic acid (RNA) tihd

protein synthesis was originally pointed out by Brachet and by Caspersson.
Is there any more direct evidence for this connexion? In particular is there
anything to support the idea that the sequentialization of the amino acids
is controlled by the RNA?                              ,!`. : `,
The most telling evidence is the recent work on tobacco mosaic virus.

A number of strains of the virus are known, and it is not difficult to show
(since the protein sub-unit of the virus is small) that they differ in amino
acid composition. Some strains, for example, have histidine in their protein,
whereas others have none. Two very significant experiments have been
carried out. In one, as first shown by Gierer & Schramm (rg56), the RNA
of the virus alone, although completely free of protein, appears tp be
infective, though the infectivity is low. In the other, first done by Fraenkel-
Conrat, it has proved possible to separate the RNA from the protein of the
virus and then recombine them to produce virus again. In this case the
infectivity is comparatively high, though some of it is usually lost. If a
recombined virus is made using the RNA of one strain and the protein of
another, and then used to infect the plant, the new virus produced in the
plant resembles very closely the strain from which the RNA was taken.  If
this strain had a protein which contained no histidine then the offspring
will have no histidine either, although the plant had never been in contact
with this particular protein before but only with the RNA from that strain.
In other words the viral RNA appears to carry at least part of the informa-
tion which determines the composition of the viralprotein. Moreover the viral
protein which was used to infect the cell was not copied to any appreciable
extent (Fraenkel-Conrat, 1956).

It has so far not proved possible to carry out this experiment-a model
of its kind-in any other system, although very recently it has been claimed
that for two animal viruses the RNA alone appears to be infective.
   IO                                              E II s XII


146               PROTEIN SYNTHESIS

Turnover experiments have shown that while the labelling of DNA is
homogeneous that of RNA is not. The RNA of the cell is partly in the
nucleus, partly in particles in the cytoplasm and partly as the `soluble'
RNA of the cell sap; many workers have shown that all these three fractions
turn over differently. It is very important to realize in any discussion of the
role of RNA in the cell that it is very inhomogeneous metabolically, and
probably of more than one type.

The site of protein synthesis

There is no known case in Nature in which protein synthesis proper (as
opposed to protein modification) occurs outside cells, though, as we shall
see later, a certain amount of protein can probably be synthesized using
broken cells and cell fragments. The first question to ask, therefore, is
whether protein synthesis can take place in the nucleus, in the cytoplasm,
or in both.

It is almost certain that protein synthesis can take place in the cytoplasm
without the presence of the nucleus, and it is probable that it can take place
to some extent in the nucleus by itself (see the review by Brachet &
Chantrenne, 1956). Mirsky and his colleagues (see the review by Mirsky,
Osawa & Allfrey, 1956) have produced evidence that some protein synthesis
can occur in isolated nuclei, but the subject is technically difficult and in
this review I shall quite arbitrarily restrict myself to protein synthesis in
the cytoplasm.

In recent years our knowledge of the structure of the cytoplasm has
enormously increased, due mainly to the technique of cutting thin sections
for the electron microscope. The cytoplasm of many cells contains an
` endoplasmic reticulum' of double membranes, consisting mainly of
protein and lipid (see the review of Palade, x956). On one side of each
membrane appear small electron-dense particles (Palade, 1955). Bio-
chemical studies (Palade & Siekevitz, 1956; among others) have shown
that these particles, which are about IOO-zoo A. in diameter, consist almost
entirely of protein and RNA, in about equal quantities. Moreover the
major part of the RNA of the cell is found in these particles.

When such a cell is broken open and the contents fractionated by
centrifugation, the particles, together with fragments of the endoplasmic
reticulum, are found in the ' microsome' fraction, and for this reason I shall
refer to them as microsomal particles.

These microsomal particles are found in almost all cells. They are
particularly common in cells which are actively synthesizing protein,
whereas the endoplasmic reticulum is most conspicuously present in
(mammalian) cells which are secreting very actively. Thus both the cells

                   PROTEIN SYNTHESIS             I47

.  of the pancreas and those of an ascites tumour contain large quantities of
microsomal particles, but the tumour has little endoplasmic reticulum,
whereas the pancreas has a lot. Moreover, there is no endoplasmic reti-
culum in bacteria.

On the other hand particles of this general description have been found
in plant cells (Ts'o, Bonner & Vinograd, rg56), in yeast, and in various
bacteria (Schachman, Pardee 8z Stanier, 1953); in fact in all cells which.
have been examined for them.
These particles have been isolated from various cells and examined in the

ultra-centrifuge (Petermann, Mizen & Hamilton, 1952; Schachmari et al.
1953 ; among others). The remarkable fact has emerged that they do not
have a continuous distribution of sedimentation constants, but usually fall
into several well-defined groups. Moreover some of the particles are prob-
ably simple aggregates of the others (Petermann & Hamilton, t957). This
uniformity suggests immediately that the particles, which have `molecular
weights' of a few million, have a definite structure. They are; in fact,.
reminiscent of the small spherical RNA-containing viruses, `and Watson
and I have suggested that they may have a similar type of substructure
(Crick & Watson, 1956).

Biologists should contrast the older concept of microsomes with the more
recent and significant one of microsomal particles. Microsomes came in all
sizes, and were irregular in composition; microsomal particles occur in a
few sizes only, have a more fixed composition and a much higher pro-
portion of RNA. It was hard to identify microsomes in all cells, whereas
RNA-rich particles appear to occur in almost every kind of cell. In short,
microsomes were rather a mess, whereas microsomal particles appeal
immediately to one's imagination. It will be surprising if they do not prove
to be of fundamental importance.

It should be noted, however, that Simpson and his colleagues (Simpson
& McLean, 1955 ; Simpson, McLean, Cohn & Brandt, 1957) have reported
that protein synthesis can take place in mitochondria. It is known that .
mitochondria contain RNA, and it would be of great interest to know
whether this RNA is in some kind of particle. Mitochondria are, of course,
very widely distributed but they do not occur in lower forms such as
bacteria. Similar remarks about RNA apply to the reported incorporation
in chloroplasts (Stephenson, Thimann & Zamecnik, 1956).

Microsomal particles and protein synthesis
It has been shown by the use of radioactive amino acids that during
protein synthesis the amino acids appear to flow through the microsomal
particles. The most striking experiments are those of Zamecnik and his

                                                    10-2


148                PROTEIN SYNTHESIS                                              PROTEIN SYNTHESIS             `49
co-workers on the livers of growing rats (see the review by Zamecnik et aZ.
IgsO                           in all cells engaged in protein synthesis. Recently Cole, Coote & Work( 1957)
                              have reported their presence in a variety of tissues from a number of animals.

Two variations of the experiment were made. In the first the rat was
given a rather large intravenous dose of a radioactive amino acid. After a
predetermined time the animal was sacrificed, the liver extracted, its cells
homogenized and the contents fractionated. It was found that the micro-
somal particle fraction was very rapidly labelled to a constant level.

In the second a very small shot of the radioactive amino acid was given,
so that the liver received only a pulse of labelled amino acid, since this small
amount was quickly used up. In this case the radioactivity of the micro-
somal particles rose very quickly and then fed away. Making plausible
assumptions Zamecnik and his colleagues have shown that this behaviour
is what one would expect if most of the protein of the microsomal particles
was metabolically inert, but I or 2% was turning over very rapidly, say
within a minute or so.

So far good evidence has been found for this reaction for about half the
standard twenty amino acids, but it is believed that further research will
reveal the full set. Meanwhile Davie, Koningsberger & Lipmann (1956)
have purified the tryptophan-activating enzyme. It is specific for trypto-
phan (and certain tryptophan analogues) and will only handle the L-isomer.
Isolation of the tyrosine enzyme has also been briefly reported (Konings-
berger, van de Ven & Overbeck, 1957; Schweet, 1957).

Very similar results have been obtained by Rabinovitz & Olson (1956,
1957) using intact mammalian cells, in this case rabbit reticulocytes. They
have also been able to show that the label passed into a well-defined globular
protein, namely haemoglobin. Experiments along the same general lines
have also been reported for liver by Simkin & Work (1957 a).
We thus have direct experimental evidence that the microsomal particles
are associated with protein synthesis, though the precise role they play is

The properties of these enzymes are obviously of the greatest interest,
and much work along these lines may be expected in the near future. For
example, it has been shown that the tryptophan-activating enzyme contaIni
what is probably a derivative of guanine (perhaps GMP) very tightly
bound. It is possible to remove it, however, and to show that its presence
is not necessary for the primary activation step. Since the enzyme is
probably involved in the next step in protein synthesis it is naturally
suspected that the guanine derivative is also required for this reaction,
whatever it may be.                      . `!      ": ?`...* s,i;

In vitro incorporation

,,   .(  ,.
       .,
`,

In order to study $he relationship between the activating enzymes and
the microsomal particles it has proved necessary to break open the cells and
work with certain partly purified fractions. Unfortunately it is rare to
obtain substantial net protein synthesis from such systems, and there is a
very real danger that the incorporation of the radioactivity does not
represent true synthesis but is some kind of partial synthesis or exchange
reaction. This distinction haa been clearly brought out by Gale (1953).
The work to be described, therefore, has to be accepted with reservations.
(See the remarks of Sin&in & Work, this Symposium.) It has been shown,
however, in the work described below, that the amino acid is incorporated
into true peptide linkage.

not clear.

Activating enzymes

  It now seems very likely that the first step in protein synthesis is the
.  activation of each amino acid by means of its special `activating enzyme',
The activation requires ATP, and the evidence suggests that the reaction is
    amino acid + ATP = AMP -amino acid + pyrophosphate.
The activated amino acid, which is probably a mixed anhydride of the form

NH,
d-d 0
      0-P-0-RiboseAdenine
$ \\o d

in which the carboxyl group of the amino acid is phosphorylated, appears
to be tightly bound to its enzyme and is not found free in solution.

These enzymes were first discovered in the cell-sap fraction of rat liver
cells by Hoagland (Hoagland, 1955; Hoagland, Keller & Zamecnik, 1956)
and in yeast by Berg (1956). They have been shown by DeMoss & Novelli
(rg56)to bewidel y d t b t d
           is ri u e in bacteria, and it is surmised that they occur

Again the significant results were first obtained by Zamecnik and his co-
workers (reviewed in Zamecnik et al. 1956). The requirements so far known
appear to fall into two parts:

(I) The activation of the amino acids for which, in addition to the
labelled amino acid, one requires the ` pH 5 ' fraction, containing the
activating enzymes, ATP and (usually) an ATP-generating system. There
appears to be no requirement for any of the pyrimidine or guanine
nucleotides.

(2) The transfer to the microsomal particles. For this one requires the
previous system plus GTP or GDP (Keller & Zamecnik, 1956) and of


,

150                PROTEIN SYNTHESIS
course the microsoma! particles; the endoplasmic reticulum does not
appear to be necessary (Littlefield & Keller, 1957).

Hultin & Beskow (1956) have reported an experiment which shows clearly
that the amino acids become bound in some way. They first incubate the
mixture described in (I) above. They then add a great excess of unlabelled
amino acid before adding the microsomal particles. Nevertheless some of
the labelled amino acid is incorporated into protein, showing that it was in
some place where it could not readily be diluted.

Very recently an intermediate reaction has been suggested by the work
of Hoagland, Zamecnik & Stephenson (1957), who have discovered that in
the first step the `soluble' RNA contained in the `pH 5' fraction became
labelled with the radioactive amino acid. The bond between the amino
acid and the RNA appears to be a covalent one. This labelled RNA can be
extracted, purified, and then added to the microsomal fraction. In the
presence of GTP the labelled amino acid is transferred from the soluble
RNA to microsomal protein. This very exciting lead is being actively
pursued.

Many other experiments have been carried out on cell-free systems, in
particular by Gale & Folkes (1955) and by Spiegelman (see his review,
1957), but I shall not describe them here as their interpretation is difficult.
It should be mentioned that Gale (reviewed in Gale, 1956) hai isolated
from hydrolysates of commercial-yeast RNA a series of fractions which
greatly increase amino acid incorporation. One of them, the so-called
`glycine incorporation factor' has been purified considerably, and an
attempt is being made to discover its structure.

.  RNA turnover and protein synthesis
  From many points of view it seems highly likely that the presence of
RNA is essential for cytoplasmic protein synthesis, or at least for specific
protein synthesis. It is by no means clear, however, that the #urnover of
RNA is required.

In discussing this a strong distinction must be made between cells which
are growing, and therefore producing new microsomal particles, and cells
which are synthesizing without growth, and in which few new microsomal
particles are being produced.

This is a difficult aspect of the subject as the evidence is to some extent
conflicting. It appears reasonably certain that not all the RNA in the
cytoplasm is turning over very rapidly-this has been shown, for example,
by the Hokins (1954) working on amy!ase synthesis in slices of pigeon
pancreas, though in the light of the recent work of Straub (this Symposium)
the choice of amylase was unfortunate. On the other hand Pardee (1954)

                  PROTEIN SYNTHESIS             151
has demonstrated that mutants of Escherichia coli which require uracil or
adenine cannot synthesize /3-galactosidase unless the missing base is
provided.

Can RNA be synthesized without protein being synthesized? This can
be brought about by the use of chloramphenicol. In bacterial systems
chloramphenicol stops protein synthesis dead, but allows `RNA' synthesis
to continue. A very interesting phenomenon has been uncovered in E. coli
by Pardee & Prestidge (1956), and by Gros & Gros (1956). If a mutant is
used which requires, say, leucine, then when the external supply of leucine
is exhausted both protein and RNA synthesis cease. If now chloram;
phenicol is added there is no effect, but if in addition the cells are given a.
small amount of leucine then rapid RNA synthesis takes place. If the
chloramphenicol is removed, so that protein synthesis restarts, then this
leucine is built into proteins and then, once again, the synthesis of both
protein and RNA is prevented. In other words it appears as if ff'ree';
leucine (i.e. not bound into proteins) is required for RNA synthesis. %`hiq
effect is not peculiar to leucine and has already been found for s&era1
amino acids and in several different organisms (Y&s & Brawerman, 1955):
As a number of people have pointed out, the most likely ipterpretation
of these results is that protein and RNA require common intermediates for
their synthesis, consisting in part of amino acids and in part of RNA
components such as nucleotides. This is a most valuable idea; it explains
a number of otherwise puzzling facts and there is some hope of getting
close to it experimentally.

For completeness it should be stated that Anfinsen and his co-workers
have some evidence that proteins are not produced from (activated) amino
acids in a single step (see the review by Steinberg, Vaughan & Anfinsen,
1956), since they find unequal labelling between the same amino acid at
different points on the polypeptide chain, but this interpretation of their
results is not accepted by all workers in the field. This is discussed more
fully by Sin&in & Work (this Symposium).

Summary of experimental work

Both DNA and RNA have been shown to carry some of the specificity
for protein synthesis. The RNA of almost all types of cell is found mainly
in rather uniform, spherical, virus-like particles in the cytoplasm, known
as microsomal particles. Most of their protein and RNA is metabolically
rather inert. Amino acids, on their way into protein, have been shown to
pass rapidly through these particles.

An enzyme has been isolated which, when supplied with tryptophan and
ATP, appears to form an activated tryptophan. There is evidence that


152                PROTEIN SYNTHESIS
there exist similar enzymes for most of the other amino acids. These
enzymes are widely distributed in Nature.

Work on cell fractions is difficult to interpret but suggests that the first
step in protein synthesis involves these enzymes, and that the subsequent
transfer of the activated amino acids to the microsomal particles requires
GTP. The soluble RNA also appears to be involved in this process.
Whereas the presence of RNA is probably required for true protein
synthesis its rapid turnover does not appear to be necessary, at least not for
all the RNA. There is suggestive evidence that common intermediates,
containing both amino acids and nucleotides, occur in protein synthesis.

IV. IDEAS ABOUT PROTEIN SYNTHESIS

It is an extremely difficult matter to present current ideas about protein
synthesis in a stimulating form. Many of the general ideas on the subject
have become rather stale, and an extended discussion of the more detailed
theories is not suitable in a paper for non-specialists. I shall therefore
restrict myself to an outline sketch of my own ideas on cytoplasmic protein
synthesis, some of which have not been published before. Finally I shall
deal briefly with the problem of `coding'.

General p&ciples

My own thinking (and that of many of my colleagues) is based on two
general principles, which I shall call the Sequence Hypothesis and the
Central Dogma. The direct evidence for both of them is negligible, but
I have found them to be of great help in getting to grips with these very
complex problems. I present them here in the hope that others can make
similar use of them. Their speculative nature is emphasized by their
names. It is an instructive exercise to attempt to build a useful theory
without using them. One generally ends in the wilderness.

The Sequence Hypothesis

This has already been referred to a number of times. In its simplest
form it assumes that the specificity of a piece of nucleic acid is expressed
solely by the sequence of its bases, and that this sequence is a (simple)
code for the amino acid sequence of a particular protein.

This hypothesis appears to be rather widely held. Its virtue is that it
unites several remarkable pairs of generalizations : the central biochemical
importance of proteins and the dominating biological role of genes, and in
particular of their nucleic acid ; thelinearity of protein molecules (considered
covalently) and the genetic linearity within the functional gene, as shown by
the work of Benzer (1957) and Pontecorvo (this Symposium); the simplicity

              PROTEIh SYNTHESIS            I53
of the composition of protein molecules and the simplicity of the nucleic
acids. Work is actively proceeding in several laboratories, including our
own, in an attempt to provide more direct evidence for this hypothesis.

The Central Dogma

  This states that once `information' has passed into protein it cannot get
i  out again. In more detail, the transfer of information from nucleic acid
to nucleic acid, or from nucleic acid to protein may be possible, but
transfer from protein to protein, or from protein to nucleic acid is im-
possible. Information means here the precise determination of sequence,
either of bases in the nucleic acid or of amino acid residues in the protein.
  This is by no means universally held-Sir Macfarlane Burnet, for
example, does not subscribe to it-but many workers now think along these
lines. As far as I know it has not been expkitly stated before.        :

Some ideas on cytopla.vnic protein synthesis

From our assumptions it follows that there must be an RNA template in
the cytoplasm. The obvious place to locate this is in the microsomal
particles, because their uniformity of size suggests that they have a regular
structure. It also follows that the synthesis of at least some of the micro-
somal RNA must be under the control of the DNA of the nucleus. This is
because the amino acid sequence of the human haemoglobins, for example,
is controlled at least in part by a Mendelian gene, and because spermatozoa
contain no RNA. Therefore, granted our hypotheses, the information
must be carried by DNA.

What can we guess about the structure of the microsomal particle? On
our assumptions the protein component of the particles can have no
significant role in determining the amino acid sequence of the proteins
which the particles are producing. We therefore assume that their main
function is a structural one, though the possibility of some enzyme activity
is not excluded. The simplest model then becomes one in which each
particle is made of the same protein, or proteins, as every other one in the
cell, and has the same basic arrangement of the RNA, but that different
particles have, in general, different base-sequences in their RNA, and
therefore produce different proteins. This is exactly the type of structure
found in tobacco mosaic virus, where the interaction between RNA and
protein does not depend upon the sequence of bases of the RNA (Hart &
Smith, 1956). In addition Watson and I have suggested (Crick & Watson,
1956), by analogy with the spherical viruses, that the protein of microsomal
particles is probably made of many identical sub-units arranged with cubic
symmetry.


I54                 PROTEIN SYNTHESIS
On this oversimplified picture, therefore, the microsomal particles in a

cell are all the same (except for the base-sequence of their RNA) and are
metabolically rather inert. The RNA forms the template and the protein
supports and protects the RNA.

This idea is in sharp contrast to what one would naturally assume at first
glance, namely that the protein of the microsomal particles consists entirely
of protein being synthesized. The surmise that most of the protein is
structural was derived from considerations about the structure of virus
particles and about coding; it was independent of the direct experimental
evidence of Zamecnik and his colleagues that only a small fraction of the
protein turns over rapidly, so that this agreement between theory and
experiment is significant, as far as it goes.

It is obviously of the first importance to know how the RNA of the
particles is arranged. It is a natural deduction from the Sequence Hypo-
thesis that the RNA backbone will follow as far as possible a spatially
regular path, in this case a helix, essentially because the fundamental
operation of making the peptide link is always the same, and we therefore
expect any template to be spatially regular.

  Although we do not yet know the structure of isolated RNA (which may
be an artifact) we do know that a pair of RNA-like molecules can under
some circumstances form a double-helical structure, somewhat similar to
DNA, because Rich & Davies (1956) have shown that when the two
polyribotides, polyadenylic acid and polyuridylic acid (which have the
same backbone as RNA) are mixed together they wind round one another
to form a double helix, presumably with their bases paired. It would not
be surprising, therefore, if the RNA backbone took up a helical configura-
tion similar to that found for DNA.
_
  This suggestion is in contrast to the idea that the RNA and protein

interact in a complicated, irregular way to form a `nucleoprotein'. As far
as I know there is at the moment no direct experimental evidence to decide
between these two points of view.

However, even if it turns out that the RNA is (mainly) helical and that the
structural protein is made of sub-units arranged with cubic symmetry it is
not at all obvious how the two could fit together. In abstract terms the
problem is how to arrange a long fibrous object inside a regular poly-
hedron. It is for this reason that the structure of the spherical viruses is of
great interest in this context, since we suspect that the same situation occurs
there; moreover they are at the moment more amenable to experimental
attack. A possible arrangement, for example, is one in which the axes of
the RNA helices run radially and clustered in groups of five, though it is
always possible that the arrangement of the RNA is irregular.

                PROTEIN SYNTHESIS             I55
It would at least be of some help if the approximate location of the RNA

in the microsomal particles could be discovered. Is it on the outside or the
inside of the particles, for example, or even both? Is the microsomal
particle a rather open structure, like a sponge, and if it is what size of
molecule can diffuse in and out of it? Some of these points are now ripe
for a direct experimental attack.

The adaptor hypothesis

Granted that the RNA of the microsomal particles, regularly arranged,
is the template, how does it direct the amino acids into the correct order?
One's first naive idea is that the RNA will take up.a configuration capable
of forming twenty different `cavities', one for the side-chain of each of the
twenty amino acids. If this were so one might expect to be able to play the
problem backwards-that is, to find the configuration of RNA by trying
to form such cavities. All attempts to do this have failed, and on physical-,
chemical grounds the idea does not seem in the least plausible (Crick,
1957 a). Apart from the phosphate-sugar backbone, which we have assumed
to be regular and perhaps linked to the structural protein of the particles,
RNA presents mainly a `sequence of sites where hydrogen bonding could
occur. One would expect, therefore, that whatever went on to the tem-
plate in a specific way did so by forming hydrogen bonds. It is therefore
a natural hypothesis that the amino acid is carried to the template by an
`adaptor' molecule, and that the adaptor is the part which actually fits on
to the RNA. In its simplest form one would require twenty adaptors, one
for each amino acid.

What sort of molecules such adaptors might be is anybody's guess. They
might, for example, be proteins, as suggested by Dounce (1952) and by
the Hokins (1954) though personally I think that proteins, being rather
large molecules, would take up too much space. They might be quite
unsuspected molecules, such as amino sugars. But there is one possibility
which seems inherently more likely than any other-that they might
contain nucleotides. This would enable them to join on to the RNA
template by the same `pairing' of bases as is found in DNA, or in
polynucleotides.

If the adaptors were small molecules one would imagine that a separate
enzyme would be required to join each adaptor to its own amino acid and
that the specificity required to distinguish between, say, leucine, iso-
leucine and valine would be provided by these enzyme molecules instead
of by cavities in the RNA. Enzymes, being made of protein, can probably
make such distinctions more easily than can nucleic acid.
An outline picture of the early stages of protein synthesis might be as


156               PROTEIN SYNTHESIS
follows: the template would consist of perhaps a single chain of RNA.
(As far as we know a single isolated RNA backbone has no regular con-
figuration (Crick, x9573) and one has to assume that the backbone is
supported in a helix of the usual type by the structural protein of the
microsomal particles.) Alternatively the template might consist of a pair
of chains. Each adaptor molecule containing, say, a di- or trinucleotide
would each be joined to its own amino acid by a special enzyme. These
molecules would then diffuse to the microsomal particles and attach to
the proper place on the bases of the RNA by base-pairing, so that they
would then be in a position for polymerization to take place.
It will be seen that we have arrived at the idea of common intermediates
without using the direct experimental evidence in their favour; but there
is one important qualification, namely that the nucleotide part of the inter-
mediates must be specific for each amino acid, at least to some extent. It is
not suflicient, from this point of view, merely to join adenylic acid to each
of the twenty amino acids. Thus one is led to suppose that after the acti-
vating step, discovered by Hoagland and described earlier, some other more
specific step is needed before the amino acid can reach the template.

The soluble RNA   -
If trinucleotides, say, do in fact play the role suggested here their

synthesis presents a puzzle, since one would not wish to invoke too many
enzymes to do the job. It seems to me plausible, therefore, that the
twenty different adaptors may be synthesized by the break- of RNA,
probably the `soluble' RNA. Whether this is in fact the same action which
the `activating enzymes' carry out (presumably using GTP in the process)
remains to be seen.

From this point of view the RNA with amino acids attached reported
recently by Hoagland, Zamecnik & Stephenson (rg57), would be a half-
way step in this process of breaking the RNA down to trinucleotides and
joining on the amino acids. Of course alternative interpretations are
possible. For example, one might surmise that numerous amino acids
become attached to this RNA and then proceed to polymerize, perhaps
inside the microsomal particles. I do not like these ideas, because the
supernatant RNA appears to be too short to code for a complete poly-
peptide chain, and yet too long to join on to template RNA (in the micro-
somal particles) by base-pairing, since it would take too great a time for a
piece of RNA twenty-five nucleotides long, say, to diffuse to the correct
place in the correct particles. If it were only a trinucleotide on the other
hand, there would be many different `correct' places for it to go to (where-
ever a valine was required, say), and there would be no undue delay.

               PROTEIN SYNTHESIS             157
Leaving theories on one side, it is obviously of the greatest interest to

know what molecules actually pass from the `pH 5 enzymes' to the
microsomal particles. Are they small molecules, free in solution; or are
they bound to protein? Can they be isolated? This seems at the moment
to be one of the most fruitful points at which to attack the problem.

                                                      i :
Subseque?at steps

What happens after, the common, intermediates have entered the
microsomal particles is quite obscure, Two views are possible, , which
might be called the Parallel Path and the Alternative Path theories. .In the
first an intermediate is used to produce both protein and RNA at about the .
same time. In the second it is used to produce either protein, or RNAi but
not both. If we knew the exact nature of the intermediates we could piob-
ably decide which of the two was more likely.- At the moment there seems
little reason to prefer one theory to the other,         : p.   +?`I.$'
The details of the polymerization step are also quite unk.novvii~;\ One

tentative theory, of the Parallel Path type, suggests that the intermediates
first polymerize to give an RNA molecule with amino acids attached. This
process removes it from the template and it diffuses outside the microsomal
particle. There the RNA folds to a new configuration, and the amino acids
become polymerized to form a polypeptide chain, which folds up as it is
made to produce the finished protein. The RNA, now free of amino acids,
is then broken down to produce fresh intermediates, A great variety of
theories along these lines can be constructed. I shall not discuss these
further here, nor shall I describe the various speculations about the actual
details of the chemical steps involved.

Two types of RNA

It is an essential feature of these ideas that there should be at least two
tyges ojRNA in the cytoplasm. The first, which we may call `template RNA'
is located inside the microsomal particles. It is probably synthesized in the
nucleus (Goldstein & Plaut, 1955) under the direction of DNA, and carries
the information for sequentialization. It is metabolically inert during
protein synthesis, though naturally it may show turnover whenever micro-
somal particles are being synthesized (as in growing cells), or breaking
down (as in certain starved cells).

The other postulated type of RNA, which we may call `metabolic RNA', is
probably synthesized (from common intermediates) in the microsomal par-
ticles, where its sequence is determined by base-pairing with the template
RNA. Once outside the microsomal particles it becomes `soluble RNA'
and is constantly being broken down to form the common intermediates


158                PROTEIN SYNTHESIS
with the amino acids. It is also possible that some of the soluble RNA
may be synthesized in a random manner in the cytoplasm; perhaps, in
bacteria, by the enzyme system of Grunberg-Manago & Ochoa (1955).
One might expect that there would also be metabolic RNA in the
nucleus. The existence of these different kinds of RNA may well explain
the rather conflicting data on RNA turnover.

The coding problem
So much for biochemical ideas. Can anything about protein synthesis be
discovered by more abstract arguments? If, as we have assumed, the
sequence of bases along the nucleic acid determines the sequence of amino
acids of the protein being synthesized, it is not unreasonable to suppose
that this inter-relationship is a simple one, and to invent abstract descrip-
tions of.it. This problem of how, in outline, the sequence of four bases
`codes' the sequence of the twenty amino acids is known as the coding
problem. It is regarded as being independent of the biochemical steps
involved, and deals only with the transfer of information.
This aspect of protein synthesis appeals mainly to those with a background
in the more sophisticated sciences. Most biochemists, in spite of being
rather fascinated by the problem, dislike arguments of this kind. It seems
to them unfair to construct theories without adequate experimental facts.
Cosmologists, on the other hand, appear to lack such inhibitions.
The first scheme of this kind was put forward by Gamow (1954). It was
supposedly based on some features of the structure of DNA, but these are
irrelevant. The essential features of Gamow's scheme were as follows:
(a) Three bases coded one amino acid.
(b) Adjacent triplets of bases overlapped, See Fig. I.
(c) More than one triplet of bases stood for a particular amino acid
(degeneracy).

In other words it was an overlapping degenerate triplet code. Such a
code imposes severe restrictions on the amino acid sequences it can produce.
It is quite easy to disprove Gamow's code from a study of known sequences-
even the sequences of the insulin molecule are sufficient. However, there
are a very large number of codes of this general type. It might be thought
almost impossible to disprove them all without enumerating them, but
this has recently been done by Brenner (Ig57), using a neat argument.
He has shown that the reliable amino acid sequences already known are
enough to make all codes of this type impossible.

Attempts have been made to discover whether there are any obvious
restrictions on the allowed amino acid sequences, although the sequence
data available are very meagre (see the review by Gamow, Rich & YEas,

                 PROTEIN SYNTHESIS             I59
1955). So far none has been found, and the present feeling is that it may
well be that none exists, and that any sequence whatsoever can be produced.
This is very far from being established, however, and for all we know there
may be quite severe restrictions on the neighbours of the rarer amino acids,
such as tryptophan.

Jf there is indeed a relatively simple code, then one of the most important
biological constants is what Watson and I have called `the coding ratio'
(Crick & Watson, 1956). If B consecutive bases.are required to code A
consecutive amino acids, the coding ratio is the number B/A, when B and
A are large. Thus in Gamow's code its value is unity; since' a stiing of
moo bases, for example, could code 998 amino acids. (Noti& that when
the coding ratio is greater than unity stereochemiciil problems arise, .
since a polypeptide chaiii has 2 distance bf only about 34 A; between
its residues, which is about thi: minimum distance between `sitctessive
bases in nucleic acid. kowever, it has been pointed but bi Bienner
(personal communication), that this difficulty may not be serious if the
polypeptide chain leaves the template as it is being synthesized.)

B,CACDDABABDC

               BCA
Overlapping code         CAC
                   ACD
                      CDD

      I BCA
Partial overlapping code       ACD
      1          DDA
                             ABA

      f BCA
Non-overlapping code           CDD
      1               A B A
                                     BDC

Fig. I. The letters A, B, C, and D stand for the four bases of the four common nucleotides.
The top row of letters represents an imaginary sequence of them. In the codes illustrated
here each set of three letters represents an amino acid. The diagram shows how the first
foti amino acids of a sequence are coded in the three classes of codes.

If the code were of the non-overlapping type (see Fig. I) one would still
reauire a triplet of bases to code for each amino acid, since pairs of bases

wiuld only allow 4 x 4= 16 permutations, though a possible but not very
likely way round this has been suggested by Dounce, Morrison & Monty
(1955). The use of triplets raises two difficulties. First, why are there not
4 x 4 x 4= 64 different amino acids? Second, how does one know which
of the triplets to read (assuming that one doesn't start at an end)? For
example, if the sequence of bases is . . . . ABA, CDB, BCA, ACC, . . . .
where A, B, C and D represent the four bases, and where ABA is supposed
to code one amino acid, CDB another one, and so on, how could one read
it correctly if the commas were removed?


160               PROTEIN SYNTHESIS
Very recently Griffith, Orgel and I have suggested an answer to both

these di%culties which is of some interest because it predicts that there
should be only twenty kinds of amino acid in protein (Crick, GrifEth &
Orgel, 1957). Gamow & Y&s (1955) had previously put forward a code
with this property, known as the `combination code' but the physical
assumptions underlying their code lack plausibility, We assumed that some
of the triplets (like ABA in the example above) correspond to an amino
acid-make `sense ' as we would say-and some (such aa BAC and ACD,
etc., above) do not so correspond, or as we would say, make `nonsense'.

We asked ourselves how many amino acids we could code if we allowed
all possible sequences of amino acids, and yet never accidentally got `sense'
when reading the wrong triplets, that is those which included the imaginary
commas. We proved that the upper limit is twenty, and moreover we could
write down several codes which did in fact code twenty things. One such
code of twenty triplets, written compactly is

AB;

where A B B A means that two of the allowed triplets are ABA and ABB,
etc. The example given a little further back haa been constructed using this
code. You will see that ABA, CDB, BCA and ACC are among the allowed
triplets, whereas the false overlapping ones in that example, such as BAC,
ACD and DBB, etc., are not. The reader can easily satisfy himself that no
sequence of these allowed triplets will ever give one of the allowed triplets
in, a false position. There are many possible mechanisms of protein synthesis
for which this would be an advantage. One of them is described in our
*  paper (Crick et al. 1957).

Thus we have deduced the magic number, twenty, in an entirely natural
way from the magic number four. Nevertheless, I must confess that I find
it impossible to form any considered judgment of this idea. It may be
complete nonsense, or it may be the heart of the matter. Only time will
show.

V. CONCLUSIONS

I hope I have been able to persuade you that protein synthesis is a central
problem for the whole of biology, and that it is in all probability closely
related to gene action. What are one's overall impressions of the present
state of the subject? Two things strike me particularly. First, the existence
of general ideas covering wide aspects of the problem. It is remarkable
that one can formulate principles such as the Sequence Hypothesis and the

PROTEIN SYNTHESIS

x61

Central Dogma, which explain many striking facts and yet for which firrdof
is completely lacking. This gap between theory and experiment is ai great
stimulm to the imagination. Second, the extremely active state of the
subject experimentally both on the genetical side and the biochemical side.
At the moment new and sign&ant results are being reported evei$`few
months, and there seems to be no sign of work coming to a standstill
because experimental techniquea are inadequate. For both these reasons
I shall be surprised if the main features of protein synthesis a&, not
discovered within the next tqn years.

It is a pleasure to thank Dr Sydney Brenner, not enly for `many
interesting discussions, but also for much help in redrafting this `$aper. i
                                                  *...

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