Download polyribosomes

POLYRIBOSOMES
These collections of the particles called ribosomes appear
be
10
the assembly lines on which the living cell manufactures protein
lTIolecules. How the assembly line ,yorks is just no\v becolTIing
clear
by Alexander Hich
A
typical mammalian cell contains
and "The Genetic Code: II," by Mar­
instructions
shall VV. Nirenberg
for
making
many
thousands of different proteins
CAK,
[SCIENTIFIC AMERI­
amino acid. It has also been shown that
DNA does not take part directly in pro­
March]. The genetic code of the
tein synthesiS. Instead the genetic code
and has the capacity to turn out thou­
cell, which constitutes the instructions
in the long double-chain molecule of
sands of protein molecules every minute.
for the synthesis of the cell's proteins, is
DNA is transcribed into shorter single
To a very large extent the living cell is an
embodied in a double-chain molecular
chains of RNA, which carry away the
expression of the particular kinds of pro­
helix of deoxyribonucleic acid (DNA).
information
teins it manufactures. It has been known
The code itself consists of sequences of
kind of polypeptide chain, or perhaps in
for several years that the site of protein
four different kinds of subunit called
some cases several chains. Because these
synthesis within the cell is the particle
bases. The DNA of a bacterium may con­
molecules of RNA bear the genetic code
needed
to
construct
one
called the ribosome. Visible only in the
tain some five million pairs of bases,
to the site of protein synthesis they are
electron microscope, ribosomes are ap­
which are needed to specify several
called messenger RNA.
proximately spherical and can be seen
thousand
different proteins. The DNA
How do the amino acid molecules get
throughout the substance of all living
of a mammalian cell may contain nearly
to the site of synthesis and find thcir
cells. Although the internal structure of
100 times as many base pairs, which
proper place in the polypeptide chain?
these particles is obscure, it has been
specify many more proteins.
established that they are composed of
As a first step they must be "activated,"
Proteins consist of linear chains of
Short chains or
a task performed by the energy-rich sub­
protein and ribonucleic acid (RNA) in
amino acid subunits.
about equal amounts.
chains that lack full protein activity are
So activated, they can be "recognized"
by still smaller RNA molecules, contaiJl­
stance adenosine triphosphate
(ATP).
18 months experi­
called polypeptides. Polypeptide chains
ments in our laboratory at the Massachu­
can be folded into a specific three­
ing about 70 base subunits, called trans­
setts Institute of Technology and else­
fer, or soluble, RNA. There is a different
where have led to the hypothesis that
dimensional configuration, and they of­
·
ten combine to form complex proteins.
the protein "factories" of the cell are
For example, the protein hemoglobin,
acid. The transfer HNA and amino aCJd
not single ribosomes working in isola­
which carries oxygen in the blood, is
are jOined by a specific enzyme, a pro­
tein with catalytic activity. The transfc r
Within the past
kind of transfer HNA for each amino
tion but collections of ribosomes working
composed of four polypeptide chains,
together in orderly fashion as if they
each of which contains about 150 amino
RNA then acts as an adapter for deposit­
were machines on an assembly line. We
acid subunits. Protein chains are built
ing a given amino acid at a position in
have called such collections polyribo­
up from about 20 different kinds of
the polypeptide chain specified by mes­
somes, or simply polysomes. As we shall
amino acid. Each chain must have the
senger RNA. Presumably the ultimate
see, the polyribosome is not the usual
right sequence of amino acid subunits to
selection of an amino acid is determined
kind of assembly line. In such an assem­
make sense, just as a sentence must con­
by weak chemical bonds between
bly line the product moves down the
sist of the right sequence of letters,
quence of bases in messenger HNA and a
a
se­
line and component parts are added to it.
spaces and punctuation. It is evident that
complementary
In the polyribosome assembly line the
an enormous number of different poly­
RNA. By this mechanism, through the
sequence
in
transfer
ribosomes move down the line and each
peptide chains can be constructed from
agency of the ribosome, the information
one makes a complete product. There is
20 different amino acids, just as an
coded in messenger RNA is translated
much evidence that the ribosomes are
enormous number of different sentences
into a polypeptide chain.
all alike, or at least interchangeable.
can be composed from the 26 letters of
They can move from one assembly line
our alphabet.
�
to another, making whatever protein a
given line happens to call for.
The
kernel
of
the
genetic
coding
problem was to discover how a sequence
out a year and a half ago my colleagues and I began puzzling about
one geometrical aspect of this system.
How this specification of a protein
of four different bases in DNA could
Consider for a moment the problem of
takes place has been fully described in
specify a sequence of 20 different amino
synthesizing
these
acids in a protein. It now appears that a
chains of hemoglobin, which contains
Crick
triplet code is employed: a sequence of
about 150 amino acid subunits. If each
October, 1962]
three bases is needed to specify each
subunit is specified by a triplet code, the
pages,
Genetic
most
Code,"
by
recently
F.
[SCIENTIFIC AMERICAN,
H.
in
C.
"The
44
© 1963 SCIENTIFIC AMERICAN, INC
one
of
the
polypeptide
messenger RNA must contain 450 bases
merely to specify the sequence of sub­
units in one chain. In most RNA mole­
cules the bases are stacked on top of one
another more or less like a pile of pen­
nies. Since the bases have a thickness of
3.4 angstrom units, the messenger RNA
strand for the hemoglobin polypeptide
chains should have a molecular length
of at least 1,500 angstroms. In other pos­
sible arrangements of the RNA molecule
the length might be almost twice as
great.
By comparison,
the individual
ribosome has a diameter of only about
230 angstroms, and so we wondered
how the long messenger molecule inter­
acted with such a small particle to manu­
facture a polypeptide chain. Some inves­
tigators thought that the RNA chain
might be wrapped around the outside of
the ribosome, but it was hard to visualize
how intimate contact between the two
could
maintained.
be
The
wrapping
problem would be still more difficult for
RNA
chains
20,000
angstroms
long,
which are found in many viruses. Alter­
native suggestions that the messenger
RNA might somehow be coiled inside
the ribosome seemed to present even
more formidable topological problems.
It occurred to us that proteins might
actually be made on groups of ribosomes,
linked together somehow by messenger
RNA. There was already a little evidence
pointing in this direction. Walter Gil­
bert of Harvard University, as well as
other investigators, had found that when
a synthetic RNA was added to a cell­
free system of bacterial ribosomes, the
ribosomes would tend to clump together.
(In such experiments, initiated by Mar­
shall Nirenberg at the National Institutes
of Health, the ribosomes make synthetic
polypeptides in accordance with instruc­
tions coded in the synthetic RNA. By
comparing the base composition of the
RNA with the amino acid composition
of the polypeptide it is possible to com­
pile a genetic code "dictionary.")
In some of the initial experiments in
our laboratory Jonathan R. Warner, then
a graduate student, tried to find in bac­
terial cells structures larger than single
ribosomes. He was initially unsuccessful
because, as we later realized, the vigor­
ous grinding needed to break open the
bacteria also destroys the delicate poly­
ribosome structure.
At the same time Paul M. Knopf, a
post-doctoral fellow in our group, was
working
that
with
make
reticulocytes-the
hemoglobin-from
cells
rabbits.
Since the cells were readily available, we
began looking for multiple ribosomal
structure in them. The reticulocyte is
a
cell that has lost its nucleus but retains
LARGE POLYRIBOSOMES obtained from a culture of human tumor cells are enlar ged
100,000 diameters in this electron micrograph made by the author and his colleagues. Indi·
vidual globular units in the clusters are ribosomes, believed t o b e held t o gether by strands
of messenger RNA (ribonucleic acid) . Polyrib o s o m e s are the site of protein synthesis.
45
© 1963 SCIENTIFIC AMERICAN, INC
CELL COMPONENT
STRUCTURE
DNA
A polymer molecule in the form of a double-strand
helix containing many thousands of subunits.
(DEOXYRIBONUCLEIC ACID)
MESSENGER RNA
(A FORM OF RIBONUCLEIC ACID)
TRANSFER, OR SOLUBLE, RNA
(A FORM OF RIBONUCLEIC ACID)
RIBOSOME
POLYRIBOSOME OR POLYSOME
FUNCTION
A single-strand polymer molecule containing hundreds of subunits.
A single-strand polymer molecule containing about
70
subunits. May be folded into a double helix in some
regions.
A globular structure conSisting of
and 60 per cent RNA.
40 per cent protein
Contains genetic information coded in sequences o'
subunits called bases.
Transcribes from DNA the information needed to
make a protein molecule and ca rries it to site of pro
tein synthesis.
Conveys specific amino acids to site of protein syn
thesis. Each amino acid has its own type of transfel
RNA.
Collaborates with messenger RNA to link togethel
amino acids delivered by transfer RNA, thereby creat
ing proteins.
Strings of ribosomes temporarily held together by
messenger RNA.
Provides actual mechanism of protein synthesis.
GLOSSARY OF CELL COMPONENTS required for protein syn­
the collaboration among these components is t o produce protein
thesis describes their structure, function and size. The end result of
molec ules whose composition has been specified by the genetic code
the molecular apparatus for producing
polypeptide chain and proceeding to the
on individual ribosomes
hemoglobin molecule s . It is also a highly
other.
some clusters. At about the same time
but
on ribo­
specialized cell: hemoglobin is virtually
The choice of reticulocytes for our
Alfred Gierer, working independently at
the only protein it manufactures. For this
search proved fortunate because they
the Max Planck Institute in Tiibingen,
reason the reticulocyte offers many ad­
can be broken open by gentle m e thod s .
made similar observations with rabbit
vantages for studying protein synthesis .
T h e cells a r e suspended in a medium
reticulocytes . A short time later F . O.
Using this cell, for example, Howard M .
whose salt concentration is lower than
Wettstein, Theophil Staehelin and Hans
Dintzis was a b l e to s h o w at M .LT. that
that within the cells. Water flows into
Noll
the polypeptides in hemoglobin are as­
the cell, m aking it swell until it burst s .
found ribosome clusters in liver tissues.
sembled by the sequential addition of
A series of experiments demonstrated
The basic technique we used in our
amino acids, starting at one end of the
that protein synthesis is carried out not
work was sucrose-gradient centrifuga-
1
of
3
the
University
of Pittsburgh
4
15
(2) and spun in a centrifuge (3). Separated frae·
(5) and analyzed. Ribosomes reveal
SUCROSE·GRADIENT TECHNIQUE provides a simple way t o
sucrose gradient
sep a rate cell components t h a t sediment at different rates when
ti ons are removed in sequence
centrifuged. The gradient consists of ordinary su g a r dissolved in a
test tube
(1). In a typical experiment rabbit reticulo cytes (red·
their presence by strongly absorbing ultravi o , l et radiation at 2,600
angstrom units
(6). A radiation counter determines the presence of
(7).
blo o d cells) are incubated 45 seconds with amino a cids containing
newly synthesized p olypeptide chains containing carbon 14
radioa ctive carbon 14. Ribosomes from the cells are layered on the
These chains turn out t o be in the faster sedimenting fra ctions.
46
© 1963 SCIENTIFIC AMERICAN, INC
taining molecules of different sizes, is
SIZE
Diameter: 20 angstrom units
Length: several thousand angstroms
up to several millimeters
Diameter:
Length:
Length:
10 to 15 angstroms
1,000 to several thousand angstroms
250 angstroms unfolded
Diameter: about
230 angstroms
sensitive method for determining their
the
presence . In addition the radioactivity
sugar
solution;
the
tube
is
then
placed in a centrifuge with a swinging­
of the various fractions was measured.
bucket rotor. The sucrose gradien t is pre­
This
served during the centrifugation and is
presence of amino acids containing car­
still maintained after the run by gravity.
bon 14, told us which fractions con­
RNA holding ribosomes together
measurement,
by indicating the
During the run molecules that sediment
tained polypeptide chains that were still
at different speeds travel different dis­
growing.
tances and remain separated when the
The results are shown in the top il­
run is ended. The plastic tube is removed
lustration o n the next p age. It can be seen
from the centrifuge and its bottom is
that
punctured to allow the collection of a
have migrated from the top of the tube.
two
ultraviolet-absorbing
The first, or slow-moving, peak is typical
top. These fractions can now be analyzed
of the peak for single ribosomes.
in various ways.
speed of movement is represented by the
sedimentation
constant 74.
Its
The fast­
periment. A suspension of rabbit retic­
moving peak has traveled about two and
ulocytes
a h alf times farther and is much broader.
was incubated in a n utrient
medium and then fed for 45 seconds
Furthermore,
contained in DNA. Proteins are built u p
with amino acids containing the radio­
growing hemoglobin chains was associ­
f r o m ab out
active isotope carbon 14. The time was
ated with the fast-moving peak and not
kept short because we were interested in
with the peak containing single ribo­
20
varieties
of
amino
acid.
the
radioactivity in the
looking at the early stages of protein syn­
somes. This clearly suggested that the
tion, which enables one to separate ma­
thesis . After 45 seconds the cells were
fast-movin g peak rather than the single­
terial s that sediment at different speeds
chilled to stop further metabolic activitv,
ribosome peak was the site of protein
in a strong gravitational field. In this
gently broken open
synthesis.
technique a plastic centrifuge tube is
sucrose
centrifugation
We then set about analyzing the fast­
filled with a sugar solution that varies
the fractions collected from the sucrose
moving peak. It seemed plausible that it
smoothly from a concentration of 30 per
gradient were treated in two ways. The
might contain clusters of ribosomes held
optical density, or amount of absorption,
together by one or more strands of mes­
cent at the bottom of the tube to 15 per
cent at the top. The gradient is obtained
simply by slowly filling the tube from
two reservoirs containing 15 and 30 per
cent sucrose. The sample material , con-
gradient.
and placed on a
After
was read in the ultraviolet region a t a
senger RNA. If this were so, it should be
wavelength of 2,600 angstroms, where
possible to free the ribosomes by sub­
nucleic
jectin g the cell-free medium to ribo­
acids
strongly
absorb
radia­
tion. Because ribosomes contain l arge
5
6
OPTICAL
DENSITY
z
Q
I0.0:::
nuclease,
an
enzyme
that
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RADIOACTIVITY
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peaks
sequence of fractions from bottom to
We designed the following simple ex­
Length: varies with length of messenger
amounts of ribonucleic acid, this is a
carefully deposited in a layer on top of
,
If
be'
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FRACTION NUMBER
FRACTION NUMBER
47
© 1963 SCIENTIFIC AMERICAN, INC
.7
POLYRIBOSOMES
breaks RNA chains. In f lct , when a very
, )
SINGLE RIBOSOMES
small amount of ribonuclease was added
to the medium before centrifugation, the
.
fast-moving peak did not appear. Both
6
its optical den sity and its radioactivity
were transferred to the peak containing
single ribosomes
.5
on
500
z
.4
400
>
«
cr:
I....J
=>
w
I=>
z
2
Iw
....J
Q
[see bottom illustration
page].
This
confirmed
the
hypothesis that the fast-moving peak
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200
represented ribosomes held together by
RNA.
Further
experiments
told
us
more
ahout this fast-m oving component . We
learned, for example, that it is fairly
fragil e . When we subjected the gently
opened
cells
to a modest amount of
shearing in a homogenizer, the sucrose­
gradient p attern ch anged dramatically
[see illustration on opposite page]. The
broad peak containing the fast-moving
component
disappeared
and
was
re­
placed by a series of peaks. The sedimen­
tation pattern again told us the slow­
.1 r-----�----��----r-----���
_1------+_�--_1 100
moving first peak contained single ribo­
somes . We speculated that the second
peak might contain pairs of ribosomes,
o
5
10
15
20
the third peak clusters of three and so on.
30
25
This tentative hypothesis was readily
FRACTION NUMBER
confirmed
NORMAL RIBOSOME DISTRIBUTION in rabbit reticulocytes consists of a fast-sediment­
illg fra,·tion of polyribosomes and a slow-sediment i n g fract i on of single ribosomes_ Hi gh
radi"'H·tivity (c%
r) indicates tbat pol)Tibowmes contain newly synthesized po lypeptides.
by
an
electron-microscope
examination conducted in the laboratory
of Cecil E. Hall at M.LT . A sample taken
from the first peak showed single ribo­
somes. A sample from the third peak
showed mainly clusters of three ribo­
1.4
somes, and the fifth peak showed m ainly
1,400
SINGLE RIBOSOMES
clusters of five . These initial observations
showed us that hemoglobin synth esis is
actually carried out on a group of ribo­
1,200
1.2
somes, which we named the polyribo­
�
some, or polysome .
urther analysis showed that hemo-
1,000
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(J)
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10
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30
a
polysome containing five units , as
shown clearly in electron micrograph s .
T h e micrographs also show, however, a
fair number of four-unit and six-unit
polysomes
[see top illustration on page
50]. We were quite sure that these were
not artifacts and that they must reflect
the mechanism of protein synthesis.
subjected to mechanical forces , as well
as its sensitivity to small amounts of
ribonuclease, suggested that the ribo­
I
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25
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=>
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u
in
The fragility of the polysome when
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2
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Fgl obin synthesis takes place primarily
somes are held together by a single
200
strand of R N A.
This impression was
strongly reinforced by more specialized
electron micrographs made by Henr y S .
o
35
FRACTION NUMBER
AFTER ADDITION OF RIBONUCLEASE, an enzyme that breaks RNA chains, the ribo­
Slayter o f M.LT. The technique called
negative staining shows that the ribo­
somes in a polysome are separated by
gaps of 50 to 150 angstrom s . Positive
staining with uranyl acetate reveals that
somes from reticulocytes no longer exhibit a fast-sedimenting fract i on_ This implies that
the ribosomes are connected by a thin
polyri bosomes are held together by RNA, which, on breaking, releases single ribosomes.
thread 10 to 15 angstroms in diameter,
48
© 1963 SCIENTIFIC AMERICAN, INC
vvhich is about the thickness of a single
the following picture of how the polysome
Let us now imagine that the messen­
strand of RNA. From the size of the
functions. The fact that the ribosomes
ger R N A for hemoglobin contains not
gap between ribosomes one can compute
are separated by a considerable distance
just one ribosome but five , all moving ,
tbat the over-all length of a five-unit
makes it seem unlikely that they co­
s a y , f r o m left to right
polysome is near 1,500 angstroms. (The
operate in synthesizing a single p olypep­
[see bottom illus­
tration on next two pages J. The ribosome
five ribosomes h ave a total diameter of
tide chain. Furthermore, if a ribosome
at the extreme left has just attached itself
5 X 230, or about 1, 150 angstroms , and
is to have access to all the information
to the strand and has started synthesizing
there are five inter-ribosomal gaps of 50
coded in messenger R N A , it must "read"
a
to 150 angstroms each.) These measure­
the strand from one end to the other. As
ribosomes
along in the synthesis process and the
polypeptide
are
chain.
The
other
proportionately
four
further
ments of total polysome length are thus
it travels it must build up a polypeptide
near the length that we concluded must
chain, adding one amino acid after an­
one at the extreme right has almost com­
be needed to specify the information in
other according to instructions. A simila r
pleted a polypeptide chain. Presumably
a hemoglobin polypeptide chain of 150
conclusion was reached by Gilbert after
the ribosomes are carried along by a
amino acid subunits. In other words, the
he studied how transfer RNA is bound
ratchet-like mechanism that does not
messenger RNA for a hemoglobin poly­
to the ribosome. The conclusion is also
allow them to go backward. At each sta­
peptide chain is about the right length
consistent with Dintzis' observation that
tion
to hold together a five-unit polysome.
hemoglobin synthesis proceeds in se­
amino acid, borne by transfer RNA, is
quence .
selected from the cellular milieu and
These various observations led us to
along
the
way
the
appropriate
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800
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600
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FRACTION NUMBER
EFFECT OF GENTLE GRINDING is t o produce a series of sedi.
(top) were made of samples obtained from the first, third and
mentation peaks, indi cating that the p o lyribosomes from reticulo·
fifth peaks from the left. They contained respectively five·unit
cytes have been b roken up into sma ller units. Electron mi cro graphs
p o lyribosomes, three· unit p olyribosomes and single ribosomes.
49
© 1963 SCIENTIFIC AMERICAN, INC
RETICULOCYTE POLYRIBOSOMES are shown at left shadowed with platinum and ma g·
been po sitively stained with uranyl acetate
nified 100,000 diameters in the electron mi<To scope. Reticulyte polyribos omes at ri ght have
and ma gnified 400,000 diameters by Hen ry
added to the growing polypeptide chain.
tein-synthesis time may be as little as 10
somes we see in some pictures. In other
When
seconds.
the synthesis
is
complete,
the
cases a ribosome may attach at one end
before the fifth is released at the other
ribosome liberates the polypeptide chain
In the reticulocyte the five-unit poly­
and itself drops off the messenger strand.
some is the most common species. The
end, thereby giving rise to a six-unit
At about the same time another ribosome
gaps between ribosomes vary somewhat,
polysome.
has found its way onto the messenger at
however, suggesting that the movement
operation would account for the distri­
the other end. The time needed for a
of ribosomes along the messenger strand
bution of polysome sizes observed in the
single ribosome to traverse the m essen­
has a statistical character. In some cases
reticulocyte.
ger strand and produce a hemoglobin
a ribosome will detach at one end before
Detroit might well envy the efficiency
polypeptide chain has been estimated
a new ribosome is attached at the other;
of the cell's protein factories. It is evident
at one minute. In a bacterial cell the pro-
this could account for the four-unit poly-
that protein synthesis is not really an
S uch a
statistical
mode of
>
>
>
POLYRIBOSOME MECHANISM, as now visualized, consists of a
themselves temporarily. As each ribosome travels along the strand
long strand of messenger RNA to which single ribosomes attach
it "reads" the information needed t o synthe size a complete p olypep·
50
© 1963 SCIENTIFIC AMERICAN, INC
easily be fed through a battery of identi­
peptide ch,tins being synthesized, but
cal tools. The living cell evidently makes
this mav not be the only interpretation .
one tape serve for many tools because
Some of the long messenger R N A strands
this is an efficient way to do the job.
associated with polysomes that contain
.N the concept of the polysome became
mation for making mor e than one kind
clearer we were naturally anxious to
20 or more ribosomes may contain infor·
of polypeptide chain .
It
This is almost certainly true of poly­
seemed likely that a variety of messenger
somes consisting of 50 to 70 ribosomes,
look for polysomes
in
other cells .
lengths and polysome sizes would be
which are found in cells infected by the
found . This has turned out to be the case.
virus of poliomyelitis . The long chain of
A human tumor cell known as the
R N A that bears the genetic code of this
HeLa cell is widely grown in tissue cul­
virus evidently serves as a strand of mes­
ture and provides a convenient example
senger RNA when it enters a mammalian
of a mammalian cell that produces many
host cell . Experiments by Penman, Dar­
kinds of protein. Polvsomes from the
nell, Becker and Klaus S cherrer have
HeLa cell were prepared at M.I . T . by
shown that the polysomes that occur
Sheldon Penman, Yachiel Becker and
normally in a mammalian cell in tissue
James E. Darnell . When we subjected
culture decrease sharply when the cell
these polysomes to sucrose-gradient cen­
has been infected by polio viru s . The
trifugation,
rate of disappearance of the polysomes
we
obtained
the
curves
plotted in the illustration at the top of the
can be hastened by feeding the cells
next
actinomycin D , an antibiotic that pre­
page.
The
electron
microscope
shows that the most common polysome
vents
species is one containing five or six ribo­
R N A. Thus about three hours after poliO­
the
manufacture
of
messenger
much
virus infection and treatment with acti­
broader than that in the reticulocyte .
nomycin D few polysomes can be found
S. Slayter of the Ma ssa chusetts Institute of
Some of the He La polysomes contain 30
in the cell. Half an hour later, however,
Techn o l o gy. Note the connect i n g threads.
or 40 ribosomes.
a new class of polysomes appears . The
somes,
but
the
distribution
is
I t is not surprising that the distrib u­
proteins synthesized on these polysomes
tion of polysomes from another kind of
are
assembly line process as it is normally
mammalian cell is much broader than
rather than of the mammalian cell . These
characteristic
of
understood . It would be more appro­
that found in the reticulocyte. The retic­
virus-induced polysomes are among the
priate to compare protein synthesis with
ulocyte is highly specialized and pre­
largest we have seen in the electron
the operation of a tape-controlled ma­
dominantly makes a single protein . Oth­
microscop e . They undoubtedly manufac­
chine tool. The tool will turn out an ob­
er mammalian cells make a great variety
ture more than one kind of protein mole­
ject of any shape within its range of
of protein molecules to conduct a variety
cule; hence some additional features may
capabilities, in response to information
of metabolic activitie s . A broad distribu­
have to be added to the simple polysome
coded on the input tape. In factories
tion of polysome sizes implies that a cell
model I h ave described.
where such tools a re used each tool is
contains messenger RNA of many differ­
I shall mention brieRy a few of the
provided with its own tape, but if it
ent length s . Presumably their length is
experiments we have deSigned to test
served any purpose a single tape could
proportional to tbe lengths of the poly-
our polysome
model.
the
The
polio
virus
model
as-
>
>
tide chain. The ami n o a cids (black dots) for the cha i n a re deliv·
here contains 150 amino acid subunits, the number found in one
ered by tran sfer RNA (oblong shapes). The p o lypeptide shown
chain of hemoglobin. The complete protein contains four chains.
51
© 1963 SCIENTIFIC AMERICAN, INC
2
sumes, for example, that an individual
' ,v,)[1
2-3
5-6
8-12
14-22
30-40
ribosome should be able to synthesize a
polypeptide chain even though it nor­
mally works side by side with
1.6
ing
z
Q
ID0::
0
(/)
w
I::::J
1,2
600
(1)
<t:
other
ribosomes . This can be tested by saturat­
800
a
reticulocyte extract with
large
amounts of external messenger R N A,
such as the synthetic messenger R N A
z
polyuridylic acid. T h i s substance, which
0::
w
mally found in messenger R N A, pro­
�
contains only one of the four bases nor­
I-
D-
Q
>
I-
only one kind of amino acid: p henylala­
::::J
0
U
nine. By adding enough of the syntheti c
w
-'
<t:
0::
I-'
::::J
(/)
.8
400
z
duces a synthetic polypeptide containing
messenger to a reticulocyte extract one
can obtain an extract in which there are
A
as many messenger m olecules as ribo­
200
somes . In this case most of the ribosomes
should pair off with a messen ger, leav­
ing few ribosomes to form polysomes .
o
L-____�__������_______L__�__L_____
�
10
20
30
40
50
The polysomes already in solution should
O
be un affected by the introduction of new
60
messen gers . Experiments of this type
FRACTION NUMBER
performed in our laboratory, as well as
MAMMALIAN POLYRIBOSOMES appear as a very broad peak of fa st-sediment i n g mate­
rial when ana lyzed by sucro se-gradient centrifu gation_ Electron micr o g raphs show that one
peak o f the ult ravi olet absorption coi ncides with polyribosomes conta inin g five or six units.
by Gierer in Tiibingen, have shown that
single ribosomes actively m ake the syn­
thetic
polypeptide
polyphenylalanine
when polyuridylic acid is added but that
the polysomes themselves arc inactive.
Hence it is clear that individual ribo­
7
somes
90
40
MINUTES
messen ger
Q
8
J
some could attach itself anywhere, chaos
would result . In our simple model there
JI
should be only one attachment site on a
If
!'H IiI
MINUTES
"f
'f \
5 MINUTES
3
that a ribosome can attach itself to only
one end of a messenger stran d . If a ribo­
polysome, whether it contains five ribo­
I
I
I
4
2
some to a polysome. We have postulated
J
,,..,,
15 MINUTES
z
:;
single
Our model also suggests that it should
"
J I
J I
J
J
MINUTES
5
Iw
-'
0
to
be possible to attach an additional ribo­
6
IDo::
0
(/)
(1)
<t:
attached
strands can produce a polypeptide.
r.
MINUTES
I
<t:
0::
I-'
::::J
somes or 10. I n two fractions containing
I
equal numbers of ribosomes, however,
I
there should be twice as many attach­
II
ment sites in a fraction composed of five­
unit polysomes as in a fraction composed
of 10-unit polysomes.
I
Experiments to test this assumption
j
were performed in our laboratory by
Howard M. Goodman, a graduate stu­
dent. Using a culture of HeLa cells, he
produced sin gle ribosomes labeled with
2
� ...
::::t:::?
:.--
:.2::--
o
�
�
----
5
-----
10
--
-- --
the radioactive isotope hydrogen 3, or
.h
tritium . These single ribosomes were ex­
tracted and added to an unlabeled HeLa
extract that contained a normal distribu­
'Y'//1
�
tion of sin gle ribosomes and polysomes.
�
After a short period of incubation the ex­
tract was subjected to sucrose-gradient
-
15
20
centrifugation .
25
30
FRACTION NUMBER
RELEASE OF SINGLE RIBOSOMES is demonstrated by incubatin g cell extracts with
amino acids and an energy supply_ Sucro se-gradient experiment s show that the b r o a d initial
peak o f polyribosomes gradually disappears and that the single-rihos ome peak rises steadily_
52
© 1963 SCIENTIFIC AMERICAN, INC
A
test for radioactivity
showed that some of the tritium-labeled
single ribosomes had indeed become at­
tached to polysomes . Moreover, in ac­
cordance with the prediction, twice as
many s ingle ribosomes were attached to
five-unit polysomes as to 10-unit poly-
somes when the total number of ribo­
This suspension was incubated under
plies that on the average there is almost
and
half of a complete polypeptide chain on
predictions
radioactivity was measured as a function
each ribosome in the polysome region .
about events at the terminal end of the
of time in the polysome fraction as well
This is consistent with
messenger stran d . I t indicates that both
as in the soluble-protein fraction Roating
mechanism, which suggests that there is
protein-synthesizing
somes in each fraction was equal.
Our
model
also
makes
condition s,
our
proposed
ribosomes and polypeptide chains should
at the top of the sucrose gradient. As
one growing p olypeptide chain for each
be released from polysomes that are in­
incubation proceeded, the radioactivity
ribosome in the polysom e .
cubated under protein-synthesizing con­
decreased in the former fraction and
Whether or not polysomes exist i n all
ditions. This can readily be tested in
rose in the latter, showing that most of
living cells is still to b e determined . To
cell-free extracts because the extracts do
the labeled amino acids, originally held
date polysomes have been isolated from
not fully reproduce the functions of the
in the polysomes, were ultimately re­
several species
intact cell. I n particular, the messenger
leased as soluble protein. In sum, these
primitive plantlike organisms known as
of bacteria,
from
the
R N A is not replaced and other sub­
three groups of experiments show that it
slime molds, from unicellular protozoa
stances are destroyed, so that in the
is possible to attach ribosomes to poly­
and from much more complex cells, in­
course of 90 minutes to two hours the
somes, to detach ribosomes from poly­
cluding those of m a n . Therefore I be­
cell extract gradually loses its ability
somes and to liberate p olypeptide chains
lieve that polyribosomes may be the
to initiate the synthesis of protein . We
under the conditions
are still, however, able to test for the
thesis.
release of ribosomes
and polypeptide
To determine if single ribosomes ar3
from
syn­
polysomes
as
protein
general method used by nature for as­
sembling amino acids into most protein s,
I shall mention just one more experi­
ment that supports our polysome model.
chains from polysomes.
released
of protein
and that protein synthesis does not usual­
ly occur on single ribosome s .
This experiment, performed in our lab­
T h e discovery of polysomes represents
oratory by Warner, established the aver­
the latest addition to the rapidly grow­
synthesis proceeds we incubated cell
age length of the incomplete polypep­
ing body of knowledge that describes at
extracts for varying periods before sub­
tide chains in the polysomes of the re­
the molecular level how genetic informa­
jecting them to sucrose-gradient centrif­
ticulocyte .
In a complete polypeptide
tion coded in DNA is eventually ex­
ugatio n . The results are plotted in the
chain found in hemoglobin there are 1 7
pressed in terms of active proteins that
govern the metabolism and structur e of
bottom illustration on the opposite page.
subunits o f the amino acid leucin e . War­
At the beginning there is a large poly­
ner incubated intact reticulocytes with
the cell . One of the key problems still to
somal peak and a modest peak of single
carbon- 1 4-labeled
deter­
be explained i s how complicated globu­
leucine
and
ribosomes, representing the normal dis­
mined the number of leucine subunits in
lar proteins are put together to form a
tribution in the mammalian HeLa cell. As
the
biologically active molecul e .
incubation proceeds there is a gradual
number of ribosomes per polysome, he
these proteins have more than one poly­
polysome
fraction.
Knowing
the
Some of
decrease in the number of polysomes
could easily calculate the number of
peptide chain, and it may be that the
and a decrease in their size. At the same
leucine subunits per ribosome. He found
polysomes play an active role in this next
time there is an increase in the number
the average number was 7 . 4 . This im-
step of protein synthesis.
of single ribosome s . At the end of 90
minutes of incubation most of the poly­
somes have disappeared, having been
converted
into
single
ribosomes.
\Ve
have established that this release of sin­
gle ribosomes takes place only if the
energy necessary for protein synthesis is
added to the reaction mixture . In other
words, the system is not degraded simply
by the passage of time.
To determine if polypeptides are re­
leased as incubation proceeds we devi sed
the following experimen t . A suspension
of living mammalian cells was incubated
for a minute and a half with carbon - 1 4 labeled amino acids a n d then t h e cells
were chilled to halt protein synthesi s .
T h i s process l o a d e d t h e polysomes with
labeled amino acids that were linked
into still unfinished polypeptide chain s .
N o w t h e cells were broken open a n d the
ribosomes and polysomes were isolated
by centrifuging them into a pellet. The
liquid on top of the pellet was poured
off in order to get rid of the labeled
amino acids Roating around in the cell
extract . The ribosomes and polysomes
were then resuspended in a fresh cell ex­
tract identical with that removed except
that it contained normal rather than
POLIO VIRUS POLYRIBOSOMES, the largest yet observed in the electron microscope,
radioactive amino acids .
contain at least 50 individual ribosomes. These have been enlarged 115,000 diameters.
53
© 1963 SCIENTIFIC AMERICAN, INC