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THE SYNTHESIS OF PROTEINS UPON RIBOSOMES.
J. D. WATSON.
The Biological Laboratories Harvard University.
The information conveyed by the sequence of the four main nucleotides in
genes (the genetic code) is used to order the sequence of the 20 different
amino acids in proteins. In this process the four letter nucleotide code is
translated into the 20 letter amino acid alphabet. Until several years ago,
there existed much confusion about the molecular basis of the reading of the
genetic code. Now, however, there instead exists the prevalent belief to
which I also subscribe that the main biochemical devices underlying the
transfer of genetic information to polypeptide chains are known. This happy
state of affairs is in large part due to the following experimental and
conceptual insights :
1) The clean demonstration that an RNA intermediate is used to convey
the genetic information of DNA to the sites of protein synthesis. Even
though this is an old idea, it became a hard fact when the in vitro enzymatic
synthesis of RNA molecules on DNA templates was demonstrated (WEISS
and NAKAMOTO, 1961 ; HURWITZ et al., 1960 ; STEVENS, 1960 ; CHAMBERLIN
and BERG, 1962). These experiments showed that the enzyme RNA
polymerase forms complementary copies of single DNA strands. Most
importantly, in vitro only one of the two complementary DNA strands is
copied (MARMUR et al., 1963). Thus the information needed to order amino
acid sequences is carried by single stranded RNA molecules.
2) The insight in 1956 by Francis CRICK (1958) that most unmodified
amino acids would not be attracted by RNA molecules, This led him to
predict that the amino acids would first be attached to specific adaptor
molecules, which in turn would bind to the RNA template. This prediction
was quickly verified when HOAGLAND and ZAMECNIK (1958) found that the
amino acids, prior to incorporation into protein, are attached to small RNA
molecules (commonly known as soluble RNA (sRNA)) (Figure 1). Subsequent
experiments showed that sRNA molecules are specific for a given amino acid
and that it is the sRNA component of the AA ­ sRNA complexes which
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selectively bind to specific regions of the RNA template. (CHAPEVILLE, LIPMANN,
von EHRENSTEIN, WEISBLOM, RAY and BENZER, 1962).
Diagrammatic View of Amino-acyl~sRNA Structure
FIG. 1. — Diagrammatic view of Amino­acyl~sRNA structure.
3) The realization that ribosomes are genetically unspecific. A given ribosome
can be used as the site of synthesis of any cellular protein. The genetic
information to order proteins is not present in the RNA component of the
ribosomes (ribosomal RNA, rRNA). Instead the genetic information is carried by
a third RNA form, messenger RNA mRNA, which attaches to ribosomes (GROS,
GILBERT, HIATT, KURLAND, RISEBROUGH and WATSON, 1961 ; BRENNER, JACOB
and MESELSON, 1961 ; JACOB and MONOD, 1961). Thus protein synthesis involves
the coordinated interaction of three forms of RNA (mRNA, sRNA and rRNA) only
one of which, mRNA, functions as a template.
4) Clear proof was provided that rRNA and sRNA are also synthesized on
DNA templates (YANKOFSKY and SPIEGELMAN, 1962 ; GIACOMONI and
SPIEGELMAN, 1962 ; GOODMAN and RICH, 1962). The synthesis of RNA upon DNA
templates is thus not limited to mRNA molecules, but instead includes all normal
cellular RNA.
5) The genetic experiments of CRICK and BRENNER (1961) which elegantly
used nucleotide insertion and deletion mutations to very strongly hint that the
words in the genetic code corresponding to single amino acids consist of three
nucleotides (codons).
6) The discovery that addition of mRNA to cell­free extracts containing
ribosomes promotes the synthesis of proteins whose amino acid sequences were
determined by the externally added mRNA. This was first shown by the dramatic
result of NIRENBERG and MATTHAI (1961) that Poly U directs the incorporation of
phenylalanine into polyphenylalanine. More recently use of other synthetic homo
and co­polyribonucleotides resulted in the specific incorporation of other amino
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acids (NIRENBERG, JONES, LEDER, CLARK, SLY and PESTKA, 1963 ; SPEYER,
LENGYEL, BASILIO, WAHBA, GARDNER and OCHOA, 1963). These results taken
together with the genetic experiments of CRICK and BRENNER has allowed the
tentative assignment of a number of three letter nucleotide words (codons) for all
the amino acids. Many amino acids are coded by more than one codon
(redundancy).
7) Structural analysis of ribosomes from many organisms which showed that they
are always constructed from two sub­units (Figure 2), one approximately twice
the size of the other (TISSIÈRES and WATSON,
FIG. 2. — Diagrammatic representation of the structure of the E. coli 70s ribosome.
1958 ; BOLTON, HOYER and RITTER, 1958). mRNA binds to the smaller of the sub­
units (OKAMOTO and TAKANAMI, 1963), while the growing polypeptide chain is
attached to the larger sub­unit (GILBERT, 1963).
8) The isolation of an enzyme fraction (the transfer fraction) which catalyzes
the formation of a peptide bond when the amino acid component of the AA~sRNA
precursor is transferred to the growing end of a polypeptide chain (NATHANS and
LIPMANN, 1960). In a still unknown way GTP is needed in this process. Recently
the transfer factor has been separated into two enzyme fractions, each of which is
necessary for sustained protein synthesis (NAKAMOTO, CONWAY, ALLENDE,
SPYRIDES and LIPMANN, 1963). Whether both are necessary to form the peptide
bond per se has not yet been ascertained.
9) The demonstration that polypeptide chains grow by stepwise addition of
single amino acids commencing with the amino terminal amino acid (Figure 3)
(BISHOP, LEAHY and SCHWEET, 1960 ; DINTZIS, 1961). This means that the amino
acid at the growing end of the poly­peptide is still connected to its sRNA adaptor.
This adaptor fits into a cavity (Figure 4) in the larger ribosomal sub­unit
(CANNON, KRUG and GILBERT, 1963). It is the binding of the terminal sRNA to the
ribosome which holds the growing polypeptide chain to its ribosomal site of
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synthesis. There is one binding site on each ribosome and so at a given time, each
ribosome can grow one polypeptide chain.
FIG. 3. — Stepwise growth of a polypeptide chain. Initiation begins at the free NH2
end. Thus the growing point is terminated by a sRNA molecule.
10) The finding that polypeptide chains begin to fold into their final three­
dimensional configuration (Figure 5) before they are released in a finished form
from the ribosomes (KIHARA, HU and HALVORSON, 1961, ZIPSER, 1963).
FIG. 4. — Diagrammatic representation of the binding of a growing poly­peptide
chain to a 70s ribosome. The COOH terminal sRNA fits in a cavity (protein
binding site) in the 50s ribosome. The incoming AA~sRNA molecule attaches to an
adjacent cavity (AA~sRNA binding site).
Often the more finished of these incomplete chains have enzymatic activity. We
thus now can understand many observations why a small fraction of many
enzymes is found firmly attached to ribosomes.
11) Recent experiments showing the single mRNA molecules simultaneously
function on several ribosomes (GILBERT, 1963 ; GIERER,1963 ; MARKS, BURKA and
SCHLESSINGER, 1962 ; WARNER, KNOPF and RICH, 1963 ; WETTSTEIN, STAEHLIN
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and NOLL, 1963). The ribosomes actively making protein are usually found in
groups (polyribosomes) held together by single mRNA molecules (Figure 6).
FIG. 5. — Schematic view of a ribosomal bound, nascent enzyme.
There is great variation in polyribosomal size reflecting corresponding variation
in the mRNA length. The discovery of the binding of single mRNA molecules to
Polyribosomes
a) messenger (template) RNA moves over the site of protein synthesis
b) each mRNA molecule has a unique length depending on the number
and molecular weights of its respective polypeptide products
c) hence corresponding variation in the average number of ribosomes
present on a given polyribosome
FIG. 6. — Diagrammatic view of the growth of
polypeptide chains on a polyribosome.
many ribosomes explains why cells need relatively little mRNA. Only about 2 p.
100 of the total RNA need be mRNA to allow maximum utilization of the
ribosomal factories. The finding of polyribosomes plus the existence of only a
single polypeptide binding site on a ribosome tells us that the ribosome and
mRNA do not remain in static orientation during protein synthesis. Instead the
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mRNA chain moves across the ribosome thus binding successive codon words to
the ribosomal site where they can select the corresponding AA~sRNA precursor.
12) In contrast to the metabolically stable mRNA and rRNA molecules, mRNA
chains are metabolically unstable in many types of cells. This is particularly true
of bacteria cells where the average life of a mRNA chain is about 1/20 to 1/10 the
generation time (LEVINTHAL, KEYNAN and HIGA, 1962). As a result the proportion
of specific bacterial protein templates can quickly alter to meet changes in the
external environment. mRNA chains are not particularly unstable in all cells. For
example, in reticulocytes which continuously synthesize hemoglobin, the mRNA
templates appear to be relatively stable. The enzymatic basis for differential
mRNA breakdown in those cells in which it is unstable has not yet been clarified.
It is nonetheless clear, however, that many cells, including the intensively
studied E. coli, are rich in mRNA destroying enzymes. These enzymes are active
in cell­free extracts under conditions where protein synthesis is usually studied
and cause extensive destruction of mRNA templates.
Most of the basic facts about protein synthesis can be brought together in the
diagram shown in Figure 7. It seems most unlikely that any of these general
Fig. 7. — Schematic view of protein synthesis.
relations will be found wrong. All have been thoroughly counterchecked. The
general pathways of the flow of genetic information are thus known. It is
therefore possible to more confidently explore the molecular details by which
these steps take place. Here shall focus attention on the involvement of the
ribosomal particles in the reading of the genetic code. First we must look at the
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state of our knowledge of ribosome structure itself. Then we shall see to what
extent we can correlate this structure with its protein synthesizing role.
Unfortunately, we cannot accurately describe at the chemical level how a
structure functions unless we know first its structure. Work with ribosomes has
an obvious parallel with experiments on enzyme function. Here also many people
work on how molecules act without knowing their exact chemical structure. In
both cases, it is the basic importance of the problem which generates this
seemingly premature behavior. Even though we badly want to know both how
proteins speed up the rate of chemical reactions and exactly how messenger RNA
molecules order amino acid sequences in proteins, neither goal will be easy to
attain. They demand elucidation of the relevant 3­D structures and present
difficulties of the first order of magnitude. This is especially true of the ribosomes
which have molecular weights about 3 x 10 6, a size 200 x larger than that of the
oxygen­carrying molecule myoglobin, the only protein whose 3­D structure has
yet been determined.
THE STRUCTURE OF RIBOSOMES.
Two important facts must always be considered when thinking about
ribosomes. The first is that they are chemically very complex. The second is that
they are always constructed from two dissociable sub­units, one approximately
twice the size of the other.
Over 30 different structural proteins are found in each 70s E. coli ribosomal
particle in addition to variable lengths of a nascent growing polypeptide chain.
Approximately 10 are found in the smaller (30s) sub­unit while the larger 50s
particle most likely contains about 20. Their great structural complexity was first
demonstrated by J. P. WALLER (1961) using the technique of starch gel
electrophoresis. Figure 8 shows one of his electrophoretic patterns which
compares the electrophoretic movement of proteins isolated from 30s ribosomes
with those found on 50s particles (WALLER, 1964). There is only moderate overlap
between the two patterns indicating that most if not all the proteins of the 30s
particle are different from the 50s proteins. Very recently, much finer resolution
of the different ribosomal proteins has been obtained by J. FLAKS (1964) using
disc electrophoresis. He has obtained very clear separation of 34 components (28
basic and 6 acidic) with even better indication that the 30s and 50s proteins are
chemically different. Amino terminal end group analysis of Dr. WALLER suggests
that the average molecular weight of the ribosomal proteins is about 30,000.
Since the total protein component of a complete 70s ribosome represents about
900,000 daltons, each ribosome probably contains one of each of its structural
components.
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J. D. WATSON.
FIG. 8. — Starch gel electrophoresis in 6 M urea of the structural proteins from 70s
ribosomes and their 30s and 50s sub­units. The upwards movement is toward the
anode, pH = 7.4 (from WALLER, 1964).
The RNA within each of the ribosomal sub­units exists as single stranded
molecules containing hairpin­like double helical regions held together by
hydrogen bonded base pairs (FRESCO, ALBERTS and DOTY, 1960). Approximately
75 p. 100 of the bases are hydrogen bonded to each other. The remainder are
available to hydrogen bond with free bases on other RNA molecules. Covalent
linkages appear to unite all the nucleotides in a given molecule. Arguments have
been given for the presence of smaller sub­units held together only by weak
secondary bonds (ARONSON and MCCARTHY, 1961). These claims most likely
reflect unintentional enzymatic degradation during ribosome purification. When
care is taken to avoid ribonuclease attack, no disaggregation occurs when the
secondary structure of rRNA molecules is temporarily destroyed by heating above
the melting temperature of its hydrogen bonds (MOLLER and BOEDTKER, 1962). It
is now also very clear, though we initially guessed the contrary, that the base
composition of the rRNA from the larger sub­unit may he very different from that
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of the smaller rRNA molecule. This is now an expected result, for if there was
close resemblance in base sequences, we might expect corresponding similarity in
their protein components.
As yet we have no idea why the ribosome structure is so complex. The fact,
however, that so many different proteins are used, strongly hints that virtually
all parts of the ribosome participate in protein synthesis. Eventually it should be
possible to test the functional role of the various proteins. This will be possible
when methods are found to reconstitute ribosomes from their RNA protein
constituents.
There also exists great need to devise a method to crystallyze ribosomes and
make possible x­ray crystallographic analysis of ribosome structure. Until this
happens, no one will really know what ribosomes look like at the molecular level.
One obvious complication in front of this goal is the presence of variable lengths
of a large variety of nascent proteins, bound through their terminal sRNA
molecules to 50s sub­units. It may be possible, however, to remove their chains
through use of hydroxylamine which specifically breaks covalent bonds of the
type uniting sRNA molecules to amino acids. We have also worried about the
complication that a fraction of the purified ribosomes seemed to be tightly bound
to degradative enzymes like ribonuclease and deoxyribonuclease (ELSON, 1958 ;
SPAHR and HOLLINGWORTH, 1961). Just recently, however, there are hints (NEU
and HEPPEL, 1964 ; HILMOE, 1964 ; FUKASAWA, 1964) that in vivo these enzymes
are not ribosomal bound but lie between the cell membrane and cell wall and
function to break down complex food sources. After cell rupture they are released
and free to stick on ribosomes. There is thus good reason to believe that these
enzymes are not necessary for ribosomal function. Hence it may be possible to
obtain E. coli mutants which lack these enzymes and to prepare ribosomes free of
these enzymes. Then may be a good time to return to the obviously devilishly
tricky goal of crystallization.
SIGNIFICANCE OF SUB­UNIT CONSTRUCTION.
As yet there exist not even bad clues about why all ribosomes contain two
dissociable sub­units. The reason clearly cannot be mere subdivision of function,
with the small sub­unit carrying on some duties and the larger one having other
tasks. Given the large variety of ribosomal proteins, there is no obvious reason
why all the ribosomal functions could not occur on a single particle. We are thus
led to the expectation that during synthesis some cycle must exist which
demands the coming apart of the two sub­units. It is thus very important to know
the proportion of sub­units which exist free during protein synthesis.
Unfortunately this is not an easy number to determine because the relative
proportion of the various particles is influenced by the surrounding ionic
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J. D. WATSON.
environment (Figure 9) (TISSIÈRES, WATSON, SCHLESSINGER and HOLLINGWORTH,
1959 ; COHEN AND LICHTENSTEIN, 1960 ; MARTIN AND AMES, 1962).
FIG. 9. — Diagrammatic view of the association properties of E. coli ribosomes.
The principal ions which affect the equilibrium are K +, Mg++ and the polyamine
spermidine. They work in the way shown in equations (1) and (2).
Initially we thought that cells actively synthesizing protein might contain a
large number of 100s dimers. This belief came from experiments in which in vitro
proportions of the various particles were studied in the absence of K + ions (Figure
10 a). Then, at the Mg++ concentration (10­2 M) most favorable for cell­free
synthesis using native mRNA, about 1/2 the particles are 70s and 1/2 100s. Now,
however, it is clear that growing cells accumulate K + ions and that the
intracellular concentration is at least 0.1 M (SOLOMON, 1962). Under these
conditions (Figure 10 b) the proportion of 100 s ribosomes is greatly reduced
(CAPECCHI, 1964), strongly suggesting that this particle is not involved in protein
synthesis. Now we do not yet possess accurate enough data to say with confidence
the proportion of 30 s, 50 s, and 70 s particles. Before this can be known, the ion
content of actively growing E. coli must be systematically worked out.
The fact that 70 s particles tend to break apart in low Mg ++ or polyamine
concentrations initially suggested that the 30 s and 50 s subunits are held
together in 70 s particles by Mg++ ions (or polyamine) forming ionic bridges
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between the negatively charged phosphate groups or ribosomal RNA. This
picture now looks oversimplified since if the 30 s and 50 s sub­units are first
lightly treated with CH 2O, they do not bind together. At this level of CH 20,
FIG. 10. — Relative molar concentrations of the various E. coli ribosomes as a
function of the external Mg++ ion concentration. a) in 5 x 10­3 M Tris, pH 7.4. b) in
5 x 10­3 M Tris, pH 7.4 + 0.1 M KCl. The molecule composition of particles
sedimenting at 85s is not known. It might represent the temporary association of a
70s and a 30s ribosome or 2 x 50s ribosomes (from CAPECCHI, 1964).
exposure the most likely groups to react are the free NH 2 groups of adenine,
guanine and cytosine. It is thus possible that the Mg ++ ions or polyamines are
needed chiefly to neutralize the charge on nearby phosphate groups and that the
30 s and 50 s are attached by specific hydrogen bonds between free nucleotide
bases on the 30 s and 50 s ribosomal RNA molecules.
BINDING OF Mg++ IONS RIBOSOMES AND RNA.
When purified ribosomes are exposed to increasing Mg ++ ion concentrations,
the number of bound Mg++ ions increases until a Mg++/P ratio of about 1/2 is
reached (EDELMAN, TSO and VINOGRAD, 1960 ; PETERMAN, 1958 ; GOLDBERG,
1963). This is shown in Figure 11. At low Mg ++ levels the amount of bound Mg++ is
lowered by the simultaneous presence of 10 ­1 M K+ ions. However, at higher Mg++
level there is relatively little competition reflecting the much higher affinity of
divalent ions like Mg++ for the binding sites. Virtually all of the bound Mg ++
attaches to the negatively charged RNA phosphate groups. This is seen by
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J. D. WATSON.
comparing the binding to ribosomes with that to purified ribosomal RNA.
FIG. 11. — The binding of Mg ++ ions to purified E. coli ribosomes dialyzed against 5 x
10­3 M Tris buffer, pH 7.4, and various concentrations of Mg ++ acetate (from
GOLDBERG, 1963).
Almost identical binding curves are found at lower Mg ++ levels (Figures 12 and
13). It has not been possible to compare their relative binding around
FIG. 12. — The binding of Mg ++ ions to purified rRNA and ribosomes. Samples were
measured after dialysis against 5 x 10 ­3 M Tris buffer, pH 7.4 various
concentrations of Mg++ acetate (from GOLDBERG, 1963).
10­2 M Mg++ since at this concentration precipitation of purified rRNA occurs.
Most of the RNA phosphate group in ribosomes can be thus neutralized by
external ionic groups. Very little neutralization occurs by positively charged
groups of the various basically charged E. coli ribosomal proteins. On the average
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each ribosomal protein molecule contains an excess of only four positive charges.
Altogether they could contribute some 120 positive charges, a very much smaller
number than the 5000 negatively charged groups in the rRNA component.
FIG. 13. — The binding of Mg++ ion to purified rRNA and ribosomes. Samples were
measured after dialysis against 5 x 10 ­3 M Tris buffer, pH 7.4, KCl at 10­2 M or 10­1
M and various concentrations of Mg++ acetate (from GOLDBERG, 1963).
Cell­free protein synthesis is generally studied in E. coli extracts containing
about 10­2 M Mg++. At this concentration virtually all the RNA phosphate groups
in the cell­free extracts are bound to Mg++ ions. This state of affairs, however, may
not exist within the living cell for there are hints (LUBIN and ENNIS, 1964) that
the internal Mg++ content (about 3 x 10­2 M) may be below the level needed for
phosphate charge neutralisation. We tend to forget how high the ribosome
concentration is within the rapidly dividing E. coli cell. Under optimal growth
conditions, about 30 p. 100 of the cell mass is ribosomes. This corresponds to a
RNA P molarity of ~ 0.12 M, a level that may be 2 x too large to be neutralized by
the internal Mg++. The only other cations present in sufficient quantity are K+
and Na+ ions and the polyamines putrescine and spermidine. Very likely, if
needed, the polyamines do the job since they have a very much stronger affinity
to RNA than any of the monovalent cations (FELSENFELD and HUANG, 1960).
BINDING OF mRNA TO RIBOSOMES.
mRNA specifically attaches to the 30 s sub­unit. This is seen by mixing Poly U
with either purified 30 s or 50 s particles in the presence of 10 ­2 M Mg++ and then
centrifuging the mixture through a sucrose gradient. Firm Poly U binding is seen
only to the 30 s particle (OKAMOTO and TAKANAMI, 1963). This attachment
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protects stretches of approximately 25 consecutive mRNA nucleotides from
ribonuclease digestion. This is shown by experiments of TAKANAMI and ZUBAY
(1964), who treated mixtures of 14C labelled Poly U and ribosomes with small
amounts of ribonuclease. Most of the labelled Poly U was quickly broken down to
free nucleotides. A finite Poly U fraction, however, could not be enzymatically
attacked because of binding to the ribosomes. When this resistant fraction was
chromatographed on sephadex, it was found to consist of chains between 25 and
30 nucleotides long.
The attachment of mRNA to E. coli ribosomes does not require energy but is
dependent upon the presence of Mg++ ions or polyamines. Roughly the same level
is required as is necessary for the binding together of the 30 s and 50 s ribosome
sub­units. Here also we initially guessed that the binding chiefly
Fig. 14. — The effect of CH2O on the binding of Poly U to ribosomes (from MOORE
and ASANO, 1964). a) Poly U binding to ribosomes treated for 100 minutes at
29°C in 2 p. 100 CH20, 10­2 M Mg++, 10­2 M triethanolamine buffer, pH 7.4. Run on
sucrose gradient in 10­2 Mg++, 0.005 M Tris, pH 7.4, in a SW 39 head at 38,000 rpm
for 1 1/2 hrs, 10°F setting. b) Poly U treated for 60 minutes at 67°C in 2 p. 100
CH2O, 0.1 M phosphate buffer, pH 7.4. Run on gradient in 10 ­2 M Mg++, 0.005 M
Tris, pH 7.4, with untreated ribosomes as in a).
involved ionic bridges holding together negatively charged phosphate groups on
different molecules. We liked this idea since it may afford a way for a mRNA
molecule with an irregular base sequence to regularly attach. Now, however,
some recent experiments (MOORE and ASANO, 1964) hint that the story may be
more complex. Again use was made of the ability of formaldehyde (CH 2O) to
specifically react with and block the free amino groups. Figure 14 shows the
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results of an experiment in which CH2O reacts either with free ribosomes or with
Poly U messenger molecules. No effect on binding is seen when the Poly U
templates are treated. This is expected since Poly U does not contain any NH 2
groups. However, a very mild CH2O exposure to the ribosomes destroys their
ability to bind Poly U. This loss of binding capacity does not reflect extensive
ribosome breakdown since ultracentrifugal examination of the treated ribosomes
shows intact 30 s and 50 s sub­units. CH 2O treated ribosomes also cannot firmly
bind Poly C molecules. In contrast, CH2O treatment of Poly C only slightly
reduces their ability to bind to ribosomes (Figure 15). There may, however, be an
almost qualitative difference in the behavior of Poly U or Poly C. Poly U binds
FIG. 15. — The effect of CH 2O upon the binding of Poly C to ribosomes (from MOORE
and ASANO, 1964). a) Poly C binding to ribosomes, CH 2O treated and run as in
Fig. 14 a. b) Poly C treated and run as the Poly U in Fig. 14 b.
very well to ribosomes while a significant Poly C binding to ribosomes is often
hard to observe (OKAMOTO and TAKANAMI, 1963).
These results show that the presence of certain free amino groups on the
ribosome influence the binding of messenger RNA molecules. Whether they form
hydrogen bonds is not yet clear. They clearly could bind to free keto groups of
Poly U. However, they do not bind the amino groups of Poly C molecules. It is
thus possible that hydrogen bonds involving bases are used in Poly U binding but
not that of Poly C. Instead the attachment of Poly C may only use binding of the
phosphate or ribose groups of Poly C. This would explain why Poly U binds so
much better than Poly C. On the other hand, we must be open to a completely
different type of explanation of the CH 2O effects. For example, formulation by
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J. D. WATSON.
somehow modifying the tertiary structure of ribosomes may sterically prevent
mRNA binding to ribosomes.
HYDROGEN BOND FORMATION BETWEEN mRNA AND rRNA.
When mRNA and purified rRNA molecules are mixed together in the presence
of high (10­2 M) Mg++ concentrations, specific hydrogen bonds form between free
amino (keto) groups of mRNA bases and corresponding keto (amino) groups on
rRNA. This conclusion comes from experiments studying the specific binding of
various mRNA molecules to purified rRNA chains. In Figure 16 the binding of
Poly U to rRNA is shown. Other experiments show a similar binding of
Fig. 16. — The binding of Poly U to rRNA as revealed by sucrose gradient
centrifugation (from MOORE and ASANO, 1964). a) Poly U and rRNA run on
sucrose gradient in 10­2 M Mg++, 0.005 M Tris, pH 7.4 (5­20 p. 100 linear gradient,
5 ml volume on SW39 head, 38,000 rpm, 4 1/2 hrs, 10°F setting). b) Same as 16a
except buffer is 10­3 M Mg++, 0.005 M Tris, pH 7.4, at 10­2 M Mg++.
Poly C to rRNA. Like the binding of mRNA to ribosomes, Mg ++ concentrations
above 5 x 10­3 M are necessary. Several sites on each rRNA molecule bind to
Poly U. This is shown in Figure 17 which tells us the amount of Poly U bound at
different ratios of Poly U to rRNA. In this experiment approximately 4 Poly U
molecules were bound per 1.6 x 106 daltons of rRNA (the complement of a 70 s
ribosome). These experiments also show that a single Poly U molecule can bind to
more than one molecule of rRNA, forming what might be called polyribosomal
RNA. This happens when the ratio of Poly U molecules /rRNA molecules is less
than one. The involvement of hydrogen bonds in the binding of mRNA to
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FIG. 17. — Saturation of rRNA binding sites with Poly U (from MOORE and ASANO,
1964). The amount of Poly U which will bind to 40  of rRNA at 10­2 M Mg++ is
shown as a function of the amount of Poly available/40  rRNA. Solid line
represents expected behavior if rRNA bound Poly U with 100 p. 100 efficiency up
to a saturating value of 2200 c.p.m. of Poly U (600 c.p.m./µg Poly U).
ribosomes is demonstrated by the specificity of CH 2O treatment. Light exposure
of rRNA to CH2O destroys its capacity to bind Poly U (keto) leaving untouched its
ability to bind Poly C (amino) (Figures 18 and 19).
FIG. 18. — The effect of CH2O on the binding of Poly U to rRNA (from MOORE and
ASANO, 1964). a) Poly U binding to rRNA treated with 0.5 p. 100 CH 20 in 10­2 M
Mg++, 10­2 M triethanolamine buffer, pH 7.4 for 70 hrs at 24°C. Gradient run in 10­
2
M Mg++ as in Fig. 16a. b) Poly U, treated for 1 hr at 67°C in 2 p. 100 CH 2O in 0.1
M phosphate buffer, pH 7.4, Run with rRNA at 10­2 M Mg++ as in Fig. 16a.
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
1416
J. D. WATSON.
Correspondingly CH2O treatment destroys the ability of Poly C (amino), but not
of Poly U (keto) to bind to untreated rRNA molecules.
The secondary structure of rRNA is uniquely suited among natural nucleic
acids to binding inRNA molecules. No interaction at 10 ­2 M Mg++ is seen between
Poly U and sRNA, or Poly U and phage R 17 RNA. Nor is there any interaction
between Poly U and double helical DNA or double helical Reovirus RNA. This
strict specificity at first hinted to us that the rRNA component of ribosomes is
involved in mRNA binding. Later, however, we found that Poly U and Poly C also
will bind to the synthetic copolymer Poly AGCU and thus realized that a unique
rRNA configuration was not necessary to bind messenger RNA.
FIG. 19. — The effect of CH 2O on the binding of Poly C to rRNA (from MOORE and
ASANO, 1964). a) Poly C binding to rRNA, CH 2O treated as in Fig. 18a, in 10 ­2 M
Mg++, run as in Fig. 16a. b) Poly C, treated in the manner of the Poly U in Fig.
18b, run with rRNA in 10­2 M Mg++ as in Fig. 16a.
Moreover, there are several important ways in which mRNA binding to rRNA
does not mimic its binding to ribosomes. Firstly, the binding of Poly U to
ribosomes competitively inhibits the binding of Poly C. This suggests that Poly U
and Poly C bind to the same ribosomal site. On the contrary, saturation of the
rRNA binding sites with Poly U does not prevent the simultaneous attachment of
Poly C­indicating that Poly U and Poly C attach to different rRNA groups.
Secondly, about 4 x as much Poly U binds to rRNA as to purified ribosomes.
Thirdly, only the 30 s ribosomes strongly bind poly U molecules while Poly U
attaches to both 16 s and 23 s rRNA components. Fourthly, the experiments
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
SYNTHESIS OF PROTEINS UPON RIBOSOMES.
1417
using CH2O argue that hydrogen bonds between base pairs hold mRNA and
rRNA chains together. In contrast, it appears that hydrogen bonds to the amino
group of cytosine are not involved in binding Poly C to ribosomes. We thus see no reason to believe that our above observed in­vitro binding of
mRNA to rRNA reflects the binding process by which mRNA attaches to
ribosomes. Instead it now seems more likely merely a reflection of the fact that
purified rRNA (and Poly AGCU) chains contain a large number of bases which
are not hydrogen bonded and which tend to spontaneously form hydrogen bonds
with other RNA molecules containing suitably oriented free bases. This
conclusion, however, is still incomplete for it completely avoids the fundamental
question of the functional role of rRNA and does not attempt to answer the
question why rRNA chains, unlike other natural RNA forms, have free bases
which can bind mRNA. Now I find it hard to avoid the speculation that despite
our essentially negative results some of the free bases of rRNA nonetheless will
be found eventually to physiologically interact in some yet undiscovered fashion
with other RNA molecules,
BINDING OF AA~sRNA TO mRNA~RIBOSOME COMPLEX.
This process also does not seem to require energy but does demand the
presence of K+ ions. The AA~sRNA precursors are reversibly held by hydrogen
bonds (and possibly Mg++ bridges) to a cavity (AA~sRNA binding site) jointly
formed by the ribosome and a mRNA codon. This is revealed by the specificity of
the reaction (SPYRIDES and LIPMANN, 1964). When Poly U is attached to E. coli
ribosomes, phenylalanine~sRNA is bound and correspondingly lysine~sRNA
binds to the Poly A ­ ribosome complex. The number of bound AA~sRNA
molecules per ribosome has not yet been accurately determined though there are
preliminary hints that only one is bound tight enough to be detected after
centrifugation through a sucrose gradient. The requirement for K + (NH4), though
not yet understood, is possibly the basis of Lubin's and Ennis' (1964) observation
that protein synthesis, but not DNA or RNA synthesis, stops in E. coli mutant
cells which are unable to concentrate large amounts of K+.
ATTACHMENT OF THE NASCENT PROTEIN CHAINS TO THE 50 S SUB­UNIT.
No covalent bond links the growing polypeptide chain to its ribosomal site of
synthesis. Instead the nascent chains are firmly bound to the ribosome through
the binding of their terminal sRNA molecule to a cavity in the 50 s ribosome
(GILBERT, 1963 a). This fact was shown by dialyzing ribosomal­bound nascent,
protein against low Mg++ (5 x 10­5 M) concentrations. As the Mg ++ level is lowered,
the bound mRNA first falls off the ribosomes. Further dialyses then dissociate
the ribosomes to their 30 s and 50 s sub­units. At this stage, virtually all the
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
1418
J. D. WATSON.
nascent protein is seen stuck to the 50 s particle. Removal of the nascent protein
does occur after more prolonged dialysis. This removal, however, need not be
irreversible since if the Mg++ level is raised, the nascent protein goes back on the
ribosomes by inserting its terminal sRNA molecule into the 50 s binding site
(SCHLESSINGER and GROS, 1963). The presence of a nascent protein upon a
ribosome frequently stabilizes the attachment of the 30s ribosomes to 50s
ribosomes at low Mg++ concentrations (the “stuck” ribosomes of Tissières,
Schlessinger and Gros, 1961). This probably reflects the fact that some nascent
chains temporarily form a variety of secondary bonds with portions of both the
30s and 50s sub­units. This is clearly, however, not the case with
polyphenylalanine since even in its presence 30s sub­units quickly dissociate
from their 50s partners.
The 50s site which binds the nascent chains we call the protein binding site.
It must be clearly different from the K + dependent (AA~sRNA binding site) which
holds the incoming AA~sRNA molecules prior to peptide bond formation. There
are thus at least two ribosomal sites which hold sRNA molecules. One of these is
very likely the binding site discovered by CANNON, KRUG and GILBERT (1963),
who found that one sRNA molecule became firmly attached to a 50s ribosome in
the presence of 10­2 M Mg++. This binding is reversible and no energy need be
supplied for attachment. Nor was there a requirement found for the
simultaneous presence of bound mRNA or for the sRNA to be charged with an
amino acid. Now there is no good way of deciding which of two possible sites
Cannon et al. looked at. The absence of requirements for mRNA or for amino acid
charged sRNA argues for equating it with the protein binding site. On the other
hand, its quickly reversible character argues for the AA~sRNA binding site.
THE MECHANISM OF CHAIN GROWTH REMAINS A PUZZLE.
As a polypeptide chain elongates, the AA~sRNA molecule bound to the
AA~sRNA binding site is transferred to the growing (carboxyl) end of the nascent
chain. Exactly how this happens is very unclear. Figure 20 shows a hypothetical
scheme by which this process might occur. We see that each growing chain is
normally terminated by a sRNA molecule fitting into the 50s protein binding site.
Each time a peptide bond is made, the terminal sRNA molecule is broken off and
ejected from the protein binding site. Simultaneously the new free carboxyl group
then forms a peptide bond with the NH 2 group of the AA~sRNA molecule found
in the AA~sRNA binding site. At this moment we hypothesize that the nascent
chain is bound to the ribosome through the AA~sRNA binding site. The process
then completes itself by the movement of the terminal sRNA molecule into the
protein binding site.
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
SYNTHESIS OF PROTEINS UPON RIBOSOMES.
1419
We further suggest that the terminal sRNA molecules remain firmly stuck to
their respective mRNA codons. This means that when they move from the
FIG. 20. — Diagrammatic hypothetical representation of the addition of an AA~sRNA
molecule to the COOH growing end of a growing polypeptide chain. The mRNA
molecule moves from the right to the left.
AA~sRNA site to the protein binding site that a corresponding movement of the
mRNA template will also take place and thus bring a new codon in the correct
position to select the next AA~sRNA precursor. Now there is no way to decide
whether energy must be supplied to operate this cycle. The fact that the amino
acyl linkage is of the high energy variety has hinted that new energy might not
be necessary to make the peptide bond. On the other hand, the requirement for
GTP tells us that energy must be supplied somewhere and it is tempting to
connect its need with a device for insuring mRNA movement across the ribosomal
surface.
WE DO NOT KNOW HOW CHAIN GROWTH IS INITIATED OR STOPPED.
There is very good evidence, especially from RNA viral systems, that some
mRNA molecules code for more than one protein. Signals, therefore must exist in
the genetic code to signify that at a given position synthesis should either start or
stop. Already there exist two clear experimental demonstrations that code signals
are used to end chain growth. The first comes from the observation that the
polyphenylalanine molecules synthesized under the directions of Poly U
templates remain attached to their terminal sRNA molecules and are not
normally released from the 50s protein binding site (GILBERT, 1963 b). It is thus
very clear that a protein product is not necessarily released when the terminal
end of a messenger template is reached. Furthermore, the nucleotide sequence
UUU... does not provide the information to cut the connection between the
terminal sRNA and a completed polypeptide chain. The second demonstration of
the involvement of code signals in chain release comes from study of the
polypeptide products of genes containing nonsense mutations. Brenner and his
collaborators (SARABHAI, STRETTON, BRENNER and BOLLE, 1964) show that
certain nonsense mutations in the gene coding for the T4 head protein produce
incomplete proteins. The length of the incomplete chains depends upon where the
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
1420
J. D. WATSON.
nonsense mutations occur, strongly suggesting that a specific nucleotide sequence
has a sense codon changed into one signaling chain termination. Now there are
no hints how termination is achieved at the molecular level, in particular
whether a specific sRNA molecule is required.
Almost no hints exist about chain initiation. The fact that Poly U, Poly C and
Poly A all act as templates tells us that in our currently employed in vitro
systems, chain initiation does not require a special codon and suggests that
perhaps any free end is capable of starting chain growth. On the other hand, the
existence of mRNA molecules coding for more than one protein indicates that
code messages which start chain growth do exist. We must thus consider the
possibility that Poly U, Poly C and Poly A all are capable of initiating growth only
when mistakes occur in the reading process. For example, the codon UUU
correctly read may never initiate growth. But if some accident happens, so that it
momentarily looks like for example UGU, then the signal would be given to start
synthesis. Until very recently we have tended to ignore the possibility that codon
reading errors might be important in cell­free experiments. But now, as the next
sections indicate, under certain conditions reading errors are very common.
MODIFICATION IN RIBOSOME STRUCTURE WHICH INDUCE CODE READING MISTAKES.
The first suggestion that mRNA messages could be misread in cell­free
systems was the observation that in the absence of phenylalanine, Poly U, (UUU)
stimulates incorporation of leucine (UUC, UUG, UUA). The significance of this
observation was at first obscure and the possibility was even considered that
ambiguity could exist in normal cell conditions how a codon was read. Very
recently, work of SZER and OCHOA (1964) has greatly clarified its significance.
They find that the amount of leucine incorporation increases with Mg ++
concentrations, and that at high Mg ++ levels the incorporation of still other amino
acids (tyrosine, isoleucine, serine) is also stimulated. Most importantly, virtually
no mistakes are made at relatively low Mg ++ levels. These levels, however, are
below the concentrations (~0.016 M) commonly employed which were chosen to
optimize phenylalanine incorporation. It thus appears that 0.016 M represents a
Mg++ concentration which distorts the normal configuration of the mRNA­
AA~sRNA ribosome complex and occasionally allows the insertion of the wrong
amino acid.
Virtually simultaneously, DAVIES, GILBERT and GORINI (1964) have found that
addition of streptomycin to an in vitro protein synthesizing system also causes
extensive misreading of the genetic code. This is dramatically seen in
experiments which follow the effect of streptomycin on Poly U stimulated
synthesis of polyphenylalanine. First it was thought that streptomycin inhibited
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
SYNTHESIS OF PROTEINS UPON RIBOSOMES.
1421
total protein synthesis. While phenylalanine incorporation is decreased, there is
a marked stimulation of isoleucine incorporation. Other experiments showed that
the incorporation of smaller amounts of several other amino acids (serine and
leucine) is also stimulated and that the rate of total amino acid incorporation into
protein is unchanged by these low amounts of streptomycin. Qualitatively the
same results are found at other Mg ++ levels ; in all cases streptomycin greatly
increases isoleucine incorporation in the presence of Poly U templates.
The action of streptomycin results from its binding to the 30s ribosome itself.
This is shown by experiments employing ribosomes from streptomycin resistant
E. coli mutant cells. When ribosomes from resistant bacteria are mixed with
supernatant factors from sensitive cells, streptomycin has no visible effect on
amino acid incorporation (SPEYER, LENGYEL and BASILIO, 1962 ; FLAKS, COX,
WITTING and WHITE, 1962). On the contrary, if the supernatant factors are from
resistant cells and the ribosomes from sensitive cells, marked inhibition occurs.
This shows that the ribosome is the primary site of streptomycin action as
postulated by SPOTTS and STANIER (1961). Furthermore, when the ribosomes
contain a 30s sub­unit from resistant cells and a sensitive 50s component, they
are not effected by streptomycin (DAVIES, 1964, COX, WHITE and FLAKS, 1964). In
contrast, a 70s ribosome composed of a “sensitive” 30s component and a resistant
50s is inhibited.
FIG. 21. — Diagrammatic view of the streptomycin induced distortion of the
Poly U­ribosome complex. The isoleucine binding is arbitrarily represented as
mimicking attachment to a UUA codon.
DAVIES, GILBERT and GORINI (1964) interpret these results in the following
way (Figure 21) ; When streptomycin binds to a sensitive 30s ribosome, it distorts
the normal Poly U­ribosome complex and allows the occasional insertion of the
wrong AA~sRNA molecule against the (UUU) codon. The distortion of the Poly U
is very specific, since it greatly increases the probability of one specific mistake,
phenylalanine  isoleucine (UUA ?). The mutation to streptomycin resistance
changes the configuration of the 30s ribosome so that, even though it still binds
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
1422
J. D. WATSON.
streptomycin (personal communication from J. DAVIES), it no longer seriously
distorts the mRNA­ribosome conformation to an extent allowing frequent
mistakes. There are hints, however, that the binding of streptomycin to resistant
ribosomes still slightly upsets the genetic code. The evidence comes from
experiments of GORINI and KATAJA (1964) which reveal the phenomenon of
streptomycin activated suppression. Certain non­functional mutant genes
produce a small fraction of normal protein products if they are located in
streptomycin resistant cells growing in the presence of streptomycin. GORINI and
KATAJA interpret this result by postulating that streptomycin bound to resistant
ribosomes causes an increased number of translation mistakes. Most of these
mistakes occur in the translation of functional genes to produce a small fraction
of nonfunctional molecules amongst very much larger fractions of functional
molecules. Other mistakes, however, occur when mutant genes are being
translated and occasionally copy a missense or nonsense codon as a sense codon.
In this way a few good protein copies can be made from genetically “bad”
templates.
DISCUSSION AND SUMMARY.
We could end here as we started with the statement that the structure of
ribosomes is woefully unclear and there does not appear on the horizon any
obvious shortcuts to its determination. Even today only a handful of protein
chemists are interested in the separation and study of their structural proteins.
Thus we cannot expect any rapid answers to the molecular details of how the
ribosomes are uniquely adjusted to correctly bringing together of the mRNA
codons and the appropriate AA~sRNA molecules. Nonetheless, there is no reason
to be unduly pessimistic about the fact that some of our answers might best be
described as semi­molecular. Even at this level, several important new insights
are emerging. They are briefly (and not necessarily in order of their basic
importance) the following :
1) The realization that in many cases the Mg++ level used in cell­free protein
synthetic systems was higher than the “physiological normal concentration”
provides a strong incentive to recheck codon assignments under conditions where
codon reading mistakes are minimalized.
2) The discovery of streptomycin conditional suppressors opens up the
possibility that the molecular mistakes responsible for many examples of
intergenic suppression occur on the ribosome. Previously, thoughts had
emphasized errors which caused amino acids to become attached to the wrong
sRNA adaptor. This suggestion is strengthened by the observation of MESELSON
(1963, personal communication) that when E. coli strain C600 (a commonly
employed suppressor strain) becomes streptomycin resistant, it often loses the
ability to suppress certain sus mutations in phage  However, if streptomycin is
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
SYNTHESIS OF PROTEINS UPON RIBOSOMES.
1423
present, it regains the ability to suppress these mutations. This suggests that the
suppressor ability of C600 is due to its possession of a mutant ribosomal protein
slightly different from the corresponding protein in non­suppressing strains.
When, however, C600 mutates to become streptomycin resistant, its ribosome
configuration may be changed so that it no longer distorts mRNA­AA~sRNA
interaction unless streptomycin is present. Thus by studying the genetics of
either streptomycin resistance or of intergenic suppression, we should be able to
attack the genetic control of many of the ribosomal proteins.
3) The finding that several, supposedly ribosomal bound degradative enzymes
actively are located between the cell wall and cell membrane destroys the
argument that they are involved in ribosome function and immediately opens the
possibility that mutant cells, unable to make these enzymes, should be isolatable.
Mutants unable to synthesize ribonuclease or proteases could prove to be of
inestimable value in preparing good cell­free systems for protein synthesis.
Because RNase I binds very tightly in an enzymatically inactive state to 30s
ribosomes (ELSON, 1958 ; SPAHR and HOLLINGWORTH, 1961), we have previously
believed that it should cause no important damage to cell­free systems. Now,
however, it is clear that it may cause much RNA degradation during the interval
between its release from the membrane­cell wall complex and its attachment to
30s ribosomes.
I thus believe it makes sense to conclude with the assertion that slowly but
definitely we are beginning to get an order of magnitude feeling for the
complexities involved in the making of a protein. They are indeed many, and
provide many potential pitfalls for those of us who either silently or openly dream
that we shall be the first to achieve usefully large scale synthesis of specific
enzymes. On the other hand, this task no longer appears Heraclean and with
persistance may be accomplished much sooner than we now guess. Certainly all
of us hope for this success to happen soon for until it comes, the molecular
understanding of repressors and corepressors and their rote in the control of
protein synthesis is likely to remain very incomplete.
ACKNOWLEDGMENTS.
I have talked much about protein synthesis with Dr. Walter GILBERT. Most
of the experiments demonstrating the involvement of hydrogen bonds in the
binding of mRNA to rRNA have been very capably conceived and carried out by
Mr. Peter Moore. The continued support of the Public Health Service (GM 09541­
03) and the National Science Foundation (GB 1254) is gratefully acknowledged.
BULL. SOC. CHIM. BIOL.,
1964, 46, N° 12.
1424
J. D. WATSON.
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