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Honors Projects
Biology
1964
Nucleic acids and protein synthesis
Sheldon Nicol
Illinois Wesleyan University
Recommended Citation
Nicol, Sheldon, "Nucleic acids and protein synthesis" (1964). Honors Projects. Paper 47.
http://digitalcommons.iwu.edu/bio_honproj/47
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NUCLEIC ACIDS AND PROTEIN SYNTHESIS
b�
Sheldon Nicol
Senior Honors
growth of
among
biochemists
a result of, a:ad has contributed to,. our
adva:acing
o f the chemistry a:ad
Over the past two
intimate
J.�"'�iJ..!.\LJ.J
of the
, evidence has accumulated
Q�.V;��
an
between cellular nucleic acid and protein
A large number of experiments in
shown
systems
that ribonuclease disrupts the cell's protein synthetic
that ribonucleic acid (RNA) ca:a frequently restore it.
Studies on bac­
terial tra:asformation ( Hotchkiss, 1957) and the discovery of the autonomous infectivity
1956)
tobacco mosaic virus RNA ( Gierer
the neces-
that nucleic acids
to direct
on
body of
demonstrates conclusively
of
acids
particles ( ribosomes) .
was
received much
to
synthesis was
another, and
a
-2-
he
He
a
and
aware o f
, and he was
But he
of
of its function, because the
no
at the time, even to Mendel.
the nucleus in heredity was
It was
well over half a century later, long after the death of both men, before
their work could be fused.
The fusion required much more nucleic acid
and protein chemda�ry than existed
Miescherts time and a vas�
of new biologyt including the later unearthing of Mendel's work"
During the last thirV years of the nineteenth century, work
in
biology, led by Weism�, suggested the continuity of the germ plasm
heredity"
and implied that the nucleus of the cell plays a central
Attention soon
equal
chromosomes..
on
,",UWU.L",lJlJ.=A
Since sperm and egg
uY.'v.l.<:,.I.
chromosomes and
of
and
are concerned with
and the
redisc overy
£h",�'I!!rI�h
more
..
from a .......,""-,,,......
far more
that
became
onY.U.VIlJ;LV
The
than
the genes as
"' ...1--,....,.,''',
were shown to contain the
was
of
The now
that
DNA came
in the
a given organ-
cells was measured a.nd found,
of chromosomes"
be constant per
DNA as the essential material of the genes.
This
pointed to
Numerous experiments
could be transmitted from one
pneumococci showed
strain of bacteria to another by transferring, to cells of the latter,
DNA extracted from the former.
These experiments proved conclusively
that DNA is the carrier of genetic information.
These developments made a great impression on geneticists, for they
went a long way toward answering questions concerning the nature of the
genee
The impact on biochemists was far greater; biochemists couad now
study the molecular baSis of gene action, tracing the effect of DNA in
discrete observable events in the synthesis of molecules composing the
cell.
In the
years, it has become possible to
molecular level, some of the fundamental questions of genetics and
came in 1953, when
The most
scientists, J.
D.
Watson and F. H. Go Crick, after
for
lography, devised a new molecular
on a double helix model
with x-ray crystalDNA molecule,
and Crick, 1953) @
hypothesis of the tetranucleotide
For
According to this
n�7·"",-!cn
These
results from
are
ester
aJZ,Q'r�"'f:l'I'I."t.e,$l
of four
each of which
was
of
seemed
to
""'Y.I"''''''''y
from the combination of
the
amounts of the
in the
(2)
to
of
early determinations of the molecular weight of the acid
gave figures which seemed to indicate a molecule of about tetranucleotide
(3)
size, and
titration of the acid indicated four or five free acid
hydrogens in the phosphate radicals.
ture
Th�s was best explained by a struc�
in which one mononucleotide is linked to another through a phosphate
ester linkage from the phosphate of one nucleotide to a hydroxyl group
It had even seemed possible on the basis of
of the sugar of another.
their relative rates of hydrolysis to decide Which nucleotides were the
outer ones and which were the oentral ones.
In accordanoe with the avail­
able etidence, various structures were proposed for the tetranucleotide,
including open chain compounds and giant rings.
The Watson-Crick model for the moleoular structure of
DNA
is based
on the construction of moleoular models arranged to conform to the
cal and chemical characteristics of the
DNA
molecule.
.Inthe�.�prop6sed
structure, two molecular strands are coiled around a common axis to form
a fairly rigid helix..
Each chain is a long polynucleotide, resulting
from the linkage of many individual nucleotide units.
The
are formed by the combination of molecules of desoxyribose sugar with
phosphoric acid molecules and nitrogenous bases.
There are two
bases
..
each
"
DNA:.
on the six-membered
a
and oan thus be
are
atoms..
and
in
compounds with two nitrogen atoms
bases are
ring.
or
one of two types:
The nitrogenous base
Two
, because it possesses an�
DNA:
group,
.....I.<,.u.<:'u.
.
can be
from
e
In the T2,
and
the place of
wbeat germ
and
of tbe cytosine
replaced by methyl-
Glucose bas been reported as a constituent of the DNA of cer­
cytosine.
tain bacteriopbages (Sinsbeimer,
1954) .
Tbe purine or pyrimidine base
is attacbed to tbe sugar at tbe one-carbon and the phospbate to the three­
carbon.
The nucleotides are linked togetber by phosphate ester groups
in sucb a way that the three-carbon of one sugar is linked to the five­
carbon of the next.
The unique feature of tbe Watson-Crick structure is the arrangement
of the bases.
The two chains are joined together by hydrogen bonds w�ich
join the opposing nucleotide bases.
The phosphates and sugar groups are
on the outside of tbe helix; the bases are on the inside and directed
perpendicularly to the fiber axis.
Bonding occurs only between adenine
and t�mine and between cytosine (or a substituted cytosine) and 51A.::'U.I.U'='"
Adenine-tbymine bonds contain two �drogen bonds, while cytosine-guanine
bonds contain three hydrogen bonds.
Hydrolysis of DNA preparations yields
the desoxynucleotides of adenine, guanine, cytoB�ne, and thymine.
There
are differences in the base ratios of DNA preparations from,different
species; that
the molar amounts of adenine, guanine, cytosine, and
thymine are not
1:1: 1: 1
of DNA
, but there are definite analytical regularities"
as required by tbe old tetranucleotide bypothesis
to cytosine seems
to thymine and
one.
to equal
vary
of nucleotide
The
and are
Tbe
for each
ratio, which
•
from the so-
This
ratio of
and
to
1
The
the sea
o
and l
m an
no known restriction on the sequence of bases in one chain
There
but it is obYcious that the chains are complementary; that is, once the
sequence of bases in one chain is known, the necessity for specific pair­
ing determines the sequence of bases in the second chain.
tide
rotated
sive base pairs are
molecule
20
36
degrees with respect to its neighbor and succes­
3.3
Angstroms apart (DeRobertis,
very long (about
��gstroms)in diameter ) ,
(Strauss,
Each nucleo­
30,000
nucleotides),
1958) .
The DNA
quite narrOw (about
and has a molecular weight of several million
1960) .
The experimental
the double helical
..."'"
.u.,::;.u.
other than
lllV'�O""
evi-
two
evidence.
dence and the
The chemical evidence shows
that the molar ratio of purine and pyrimidine bases is very c:1ose
for
sources of DNA.
There is now much physical evidence to
Titration curves
support a
form
that the bases
bonds and that these are bonds within the
measurements show
viscosity, and
DNA in
at
DNA, so that the
does not come
one
there are
oppo-
and
combines
The phosphate-sugar backbone repeats regularly, both
chemically and structurally.
This repitition necessarity implies that
the phosphate-sugar groups are related by symmetry, in this case by a
screw axis, and i t
this which makes the backbone a simple helix.
Again, the two separate phosphate-sugar backbones are related to each
other by symmetry,
in this case by two-fold rotation axes perpendicular
to the fiber axis..
The arrangement of the bases, however, does not repeat,
a.nd only shows pseudosymmetry; that is, the region occupied by a pair of
bases is fixed, and successive regions are related to each other by sym­
metry, but there is no restriction on which pair of bases occurs at any
point t as long as one of the allowed pairs is used
e
It remains to be seen
whether there are structural reasons for the particular base pairings.
It sho�ld be noted that while x-ray diffraction shows that a sub­
stantial portion of the DNA must be in the double helix form it is an
extremely poor method for deciding how much of the DNA is in this con­
figuration.
The titration curves
and
the analytical data suggest that
the great majority of bases are paired..
However, it seems certain that
the molecule is folded in its biological condition, and that
•
be occassional regions where';.the configuration is somewhat
of DNA with the crystalline
desoxyribonuclease results
dialyzable fragments, but a non-dialyzable frac-
in a
, whose
'"'VJ... "''-''''' ....
ginal DNA and the dialyzate.
from the ori­
This indicates a complex and asymmetrical
of the DNA
of DNA form fibers with intense
and
viscosity"
1'1""''''''':
microscopy shows that DNA
and
fibrous
indicating the
asymmetry.
This molecular
and the tendency of DNA particles to become oriented under
)
stretching is also revealed by the dichroism shown in ultra- violet light.
result from the combination of nucleic acids and
proteins and, in certain cells, constitute the majorJart of the solid
material.
Nucleic acids combine with the proteins by ionic bonds which
It is suspectedihat a basic protein
are dissociated with relative ease.
lies
in
the narrow groove that spirals around the
DNA
molecule and that
the component basic amino acids of the protein neutralize the phosphoric
acid residues of the DNA.
Two
main types of basic proteins have been
one of low molecular weight
found associated with chromosomes:
mine)
molecular weight (histone ) .
and one of
the DNA structure
In addition, there
..
occurs an acidic protein of high molecular
Evidence suggests that the protamine
( prota-
...
•••'"
....AJ.."" ....,.,)"
wound helically around
the smaller of the two grooves between the backbones.
Models show that an extended polyarginine chain can be fitted in this
basic groups
, with the
groove without
the
side-chains going alternately up and d�wn to the negatively charged phos-
In
phate groups of the DNA backbones..
nucleoprotamine there appears
for every phosphate, yet only two-thirds of the protamine
be one
are basic.
whenever the
V'l,",'"_'''''
to
tively easy with two
the
the
This suggests that the polypeptide
amino
U..I. ..L\"J.J.J.�
occur ..
"',u,.........
shows that
, but rela-
a fold with one
succession..
on the amino acid sequence
residues do occur in
between the
is folded
is
and the
clear
of
or
folds go
or outwards.
are that (
nor
The main features of the preliminary x-rays
part of it - maintains its character-
the DNA - or
istic structure; and that (2) some larger repeating structure is also
present.
These results are obtained wi th nuclei, swollen in water and
into fibers,
with artificial combinations of DNA with
lysine-rich histone.
One equatorial spacing (of about 60 Angstroms)
little on drying; another, around 40 Angstroms, alters with hum�­
dity (Crick, 1951).
These results show that nucleohistone has a structure
some sort.
In the chromosome, each of the ultimate fibrils consists of two
cemplementary DN:l\;malecules intertwined about the basic protein.
fibrils are 3-4
mu
in diameter and several mici:en�.lon:g:�.c (,J..)�UJ.I.L..L";l:>,�JI;:::tnEase
.5-1 micron
form the microscopically
meter.
These
dia-
Whether or not a single DNA molecule extends the entire length
the chromosome
matter of debate.
The chromonema
UHli,l"",,_',,",,,
characteristic coiling cycle which may be the result of cyclical
the chemical links between DNA and�its basic protein partner.
VLL'_'�>Q�
The
behavior of chromosomes m� be experimentally modified by treatpotassium cyanide"
ment with metabolic
state, the chromonema has a
When at a
of
chromomeres.
chromosome
Chromomeres
form compact
path
of the chromonema"
The
of DNA
various means has
of
does occur.
been observed in observation
A
"
often
40
form
1'���,mN� and Butler,
1955).
or more,
of
i:ij'C'I.A..l..L!!l::'Ll.
observed
DNA
a
specimen
Therefore, there must be considerable varia­
It has not yet been
tion of particle configuration Qf DNA specimens.
possible to prepare
constants,
specimens by fractional centrifugation in suf­
DNA
It appears that
ficient quantity to determine their composition.
does not exist as fairly straight rods of constant diameter and differ�t
length, but rather that some kind of aggregation occurs.
RNA
,
general,
structurally similar to
nucleotide but is generally single-stranded.
the sugar ribose
of desoxyribose"
DNA.
It
a long poly-
In its backbone it contains
The bases linked to the ribose
units c an be any of four diff�rent types, all similar to those found in
DNA
except
, which
of
found
DNAts
"
RNA
abun-
dant in the cytoplasm, particularly in the ribosomes.
DNA
can betbopographically located within the cell by means of the
Feulgen nucleal reaction.
This method consi sts of treating the tissue
with Schiffts reagent (basic fuchsin bleached with sulfurous anhydride,
The
after acid
a positive reaction (they combine with
occur
acid
to
but it
s
two
recolor the basic
negative with RNA"
This reaction
of
DNA
of the cell
In
free
bases are
groups in the
DNA
the
sometimes
second
reaction between the
mole­
by means of
groups and the decolored fuchsin.
When
the
is
, the
in the cytopl asm.
and
the
the nucleus, the
is nega-
tive and chromonemata (especially the chromocenters) are intensely pos­
itive.
In ultra- violet light, nucleic acids show a characteristic absorp­
2600
tion peak at
bases.
Angstroms due to the presence of purine and pyrimidine
Ultra-violet micro spectrophotometry permits the localization of
the two types of nucleic acid without distinguili1 ing liJetween them,
the nucleal reaction of Feulgen shows DNA presence.
Some
time ago it was thought that the double-stranded DNA structure
occurred in all organisms.
Recently, however, there have been reports of
a virus which has a single-stranded DNA structurel' judged from its suscep­
tibility to inactivation py�'phosphorus-32 decay
(an
efficiency of one
inactivation per disintegration which is about ten times
the efficiency of inactivation of the double-stranded phage DNA) and by
the unusual physical properties of the nucleic acid (Sinsheimer, 1959).
The finding of a virus with a single-stranded DNA implies that the gene­
tic information can be obtained in a single strand.
DNA
located exclusively in cell nuclei.
cell nucleus is relatively constant.
Although the content of
drastically with
of the cell, the DNA
DNA content
The amount of DNA per
.
and
the physiological condition
remains constant as would be expected if
the gene number.
and does not
It
DNA
metabolism.
ina
of
shows that among
""'<Nt""''';;....
in size of the
are
of
content
lowest DNA
and
values are
found
The values
coelenterates.
tJles, .and birds suggest
DNA content
the most
that
in lung
of DNA
such as sponges
primitive
fishes, amphibians,
during evolution there has
been a decline
(Mirsky and Ris, 1951) .
The;.zceal experimental)ev:irdence that ;f6rces'ex:clusi1le;;c�msideratii.:olb..,
genetic mljl,teI!ial; come.s d}Ilom '.studies:;with .:1fliCIlOO rganisms;,
fromLs:'bud:j.es on:.the ..tr:anstkll:rmi;p.gfprinciplesEQf1bacteflia... ;
Pne umoc ocoi : o rdinarily . possess:;a smooth polysaccharide
after oulture
the
�
vitro,
is
. polysacoharide
This
oells
not
and whioh have a
transformation has
been shown
the capsular
to
be DNAe
quite
polysaccharides; transformations of
good that
the
eliminate the
transforming
that protein
Although many
are transformed
an
..
The DNA
the
of
is
one at a time..
therefore
Only a
donor DNA
the
of one
set
of
genes
does
for
of
but
into the
..
of
is
Transformation
donor, when introduoed
the
It
really
characteristics
at one time.
a complete SUbstitution
drug
principle is
plays a spacifio
contained in
the
not
have been achieved.
1957) .
DNA unoontaminated by protein
transformation process.
responsible
Transformation
, biochemioal oharaoteristios, etc.
in
which
rough appearance.
The substance
develop;t
� he ability
The evidenoe
oolonies in
are abtained which yield
produ ced
Occassionally
irreversible; rough cells never spontaneously
transformation
restricted to
c61at.
still
to speculation, some basic facts have been demonstrated.
Most interpre­
tations are influenced by the Watson�Crick model, wbdbhgives use to the
theory of replication by a template (model ) process by which each strand
acts as a mold
a
synthesized one.
reproduction,
this a positive print.
in
This
.. can be compared
... ,u.."'"....
"""'
which a negative is made first and
Since one of the polynucleotide chains determines
the synthesis of the other during interphase, all that is needed to set
the template mechanism into action is the s�paration of each strande
This hypothesis presupposes that the simple nucleotides fall into phase
and that the parent helix of DNA unwinds while the daughter helices are
formed <and unwound"
Calculations indicate that energy requirements for
such an uncoiling �e not too great.
Nonetheless, a detailed model for
the duplication of the coiled DNA as it occurs
��
difficult to
make even though there have been several attempts.
In nuclei of living, proliferating cells, a
violet absorbing component has been found which
cells.
ultraabsent in non-dividing
It is postulated that this component comprises relatively unpoly­
merized DNA precursors that are lost by fixation.and hydrolysis.
This
; it
in the metabolism of folic
the
of
Some
which is
a precursor of coen;"
act on the
may produce a chromosome
A deviation�from normal
rfl�r��nc:e with the
from.
has been
few
DNA (Kornberg, 1951)3
The synthesis
deteoted by the inoorporation of labelled desoxyribo-
nuoleotides into DNAuus1ialg a polymerase enzyme preparation obtained
from Esoheriohia .2.2.ll- oells.
reported.
A net syntheslis of 50% or better has been
all
The enzyme system has the following characteristios:
four desoxyribonucleatide triphosphates are required for appreciable
synthesis (the diphosphates will not substitute), magnesium
required,
the system is essentially irreversible (as would be required to explain
the metabolic stability of DNA), and about 1% of the maximum reaotion
rate is obtained when a single nucleotide is eliminated from the reaction
mixture.
Polymerized DNA
required to serve as a primer for the reaotion,
upon which complementary chains are
to serve as the
constructed.
Overall proportions of the bases in the product DNA
the base composition of the primer and the sequence of the
the
DNA.
the same as
synthesized DNA
_�':;w..&l"'''''''''
Incorporation ratios
of their concentrations in the reaction
""'"""7'."
is a
In cells of
the
the
by
as is the case for the
an
The
in
LL�!U���-
RNA (Oohoa and
is increased by
strand of DNA
serves as a model.
to the
llJ.'M;!.l.�jJ.·
the chromosomes are still in their extended
the
form and are not
that
the
lll .l.\... U..... v
method
of the
There
not a continuous activity of the
going since this
cell
set
sort of
be
higher organisms.
the
of
DNA
requires the
activation of the Kornberg enzyme but there is no knowleq;ge of what sort
of signal
DNA
required.
itself cannot be the template material for protein synthesis.
The major site of protein synthesis in most organisms
of the cell, a region devoid of
DNA
protein synthesis from
DNA.
It
the small
possible to dissociate
synthesis; that is, inhibition of the synthesis
of DNA by ultra-violet light, x-ray irradiation, mitomycin, or thymine
starvation does not inhibit protein synthesis (Spiegelman,",jlf)9��,;,\;;The
high concentration of
the cytoplasm suggests that this substance
RNA
has a major role in protein synthesise
RNA
in
in which enzymes are being pro-
rapidly
duced.
cells and in
in
Cells involved
activities that do not include protein synthe­
sis contain very small quantities of
RNA,
inactivates
RNA.
The
DNA
RNA,
out to the cytoplasm (to the
w hich
The template RNA mole-
to direct
are produced from
e
and protein synthesis
the genes controls the production of a specific
RNA
template or
in
Ribonuclease, an
inhibits protein synthesiSe
The relationship between the genes,
now
concentration is always high
DNA
molecules by specific base
The amino
are
to the
template RNA molecules by low molecular
appears to occur
the
in the
There have been
just as
molecular
transfer
and
a
)
seems to occur in a system other than the
ribosomes
.... .1,.....
",..,.... and Ochoa, 1958) but in higher organisms the synthe-
of protein seems to take place m ainly in the ribosomes.
Ribosomes
appear as uniform, round, electron-dense particles having an average
diameter of 100 to
200
the total cellular
RNA (Hoagland,
Angstroms. (iThe.l.rio<fS0meS}Contain 80 to 90% of
1969).
They exist apparently free
in the cytoplasm in some tissues (notably bacteria and rapidly growing
mammalian cells) , perhaps sometimes attached to the limiting membrane of
the cell (bacteria),
usually arenfound
in
association with lipopro-
tein-rich membranous material of the cytoplasm.
The ribosomes, in natu-
association w ith this membrane-like material comprise what has been
variously named the cytoplasmic
ground
substance, ergastroplasm, or
reticulum by electron microscopists.
of
individual
The
on the ribosome may be bound to each other
particle protein by magnesium ions and base interaction
for
that RNA is the
synthesis is the ability of tobacco mosaic virus
RNA to
direct the forma­
tion of a specific protein and of the RNA from certain animal viruses to
infect
and
lead to the production of complete virus"containing protein
,
RNA
1959).
There are many other experiments which show that
formation.
must
For
"'A':;w.u�)...""
thymine-requiring bacteria can still form induced enzymes in the absence
of
factor
and
1955) ,
with the
cannot.
formation of
or
RNA
sis on RNA can occur
the absence of
The direct
between DNA and RNA has been demonstrated
The enzyme RNA polymerase, when
by experiments with cell-free systems.
placed
a cell-free medium containing the four nucleotide units of RNA,
Addi­
fails to induce RNA synthesis even when RNA is added as a primer.
tion of a small quantity of)DNA, however, results in rapid synthesis of
RNA.
Thus it is actually DNA that stimulates RNA synthesis.
is appar-
ent that the four nitrogenous bases appear in the synthetic RNA in the
same proportions as they oocur in the DNA molecule used as a primer, with
uracil substituted for thymine.
Further evidence of the specificity of
the reaction is obtained by using as primers two synthetic DNA polymers,
one containing only thi)'1Iiline (poly-T) and one containing alternate units
of
and thymine (AT oopolymer).
the
With poly-T as the DNA
,
RNA contains only thymine's regular partner, adenine.
With AT copolymer as the primer, the. RNA consists of a copolymer of
uracil
and
adenine in perfectly alternating sequence.
even
all four
the reaction (Hurwitz and Furth, 1962).
by DNA is also seen in living cells.
bacteria,
RNA
an
host
This specific
are
The control over RNA synthesis
When bacterial viruses infect
formed that resembles the virus DNA and not that of
base composition (Volkin, Astrachan, and Countryman,
1958).
One
can
conjecture
the
helix of
molecule
in the C!DUrSe of the reaction so that ribonucleotides (in triand
off
the
the aid of the enzyme RNA
one strand of the DNA.
of the
the ribonucleotides to
then
from the DNA
groups
up into an RNA polymer.
The RNA
and thelDNA reverts to its previous
double-stranded form.
The
of a messenger RNA sent out from the genes to the ribosomes
been confirmed by experiments with T4 virus.
Colon bacilli were grown
on SUbstances containing heavy isotopes of nitrogen and carbon.
The cells
were then infected with T4 virus and simultaneously transferred to a
medium containing ordinary nitrogen and carbon.
:Imroediately after infec­
tionthe(cells were exposed briefly to radioactive phosphorus to label
the messenger RNA produced by the virus DNA.
two ribosome fractions
After centrifugation, the
examined for radioactive messenger RNA.
It
was found that only the heavy ribosomes (those present before infection)
were radioactive (Brenner, Jacob,
.and
Meselson, 1961).
new ribosomes were synthesized after infection.
Therefore, no:
Ribosomes, then, are
responsible for synthesizing protein.
Not until the machine
has been supplied with the proper instructions, supplied by messenger
RNA, can a
be
•
Developments in understanding the cell-free system illuminate another,
and more direct, participation of RNA in protein synthesis..
the soluble, nonparticulate
fraction (pH 5 fraction), found
for the overall incorporation of amino acids
acids by formation of enzyme-bound amino
of reaction
mnner.
The
compounds from
that
and amino
same
ribosome
the carboxyl activation of amino
that
(adenosine
It is known
amino acids
activating enzymes not
a
the
amino
thus
but
to
acid
of
RNA molecules.
, low mole-
These molecules, once charged with amino
acid, are able to transfer the amino acid to the protein of the ribo­
nucleoprotein parti�les.
Ultracentrifugal and electr�etic analyses
of transfer RNA (sRNA) preparations indicated that they are heterogeneous.
It is apparent that sRNA must consist of a number of distinct
acids ii&;re attached to sRNA additively and terminally"
A characteristic of transfer RNA's is their content of a relatively
high proportion of unusual bases, particulary pseudo-uridine, of which
one or two molecules per chain are present
1960).
dilic,
(Dunn,
Smith, and Spahr,
The amino acid-carrying terminal is uniformly cytidilic, cyti­
adenylic acid in all 20 amino acid"specific sRNA's, while the
phosphate terminal of the chain appears to be uniformly guanylic acid"
The
of
30,000, corresponding to
molecular
6
10
20,000-
little
65-85
nucleotides
comparison, the high
template RNA molecules have a molecular
U960).
Protein synthesis is often measured by the incorporation of radioactive amino
a
into the
(ATP and guanosine
of
5
lLUL/U«J.J.
seems to be
There is
in
and medium of the
many other
""T,,,+,,,m,,,
have been
the best results.
that the
The
the
of amino
are
(
of
into the
are incorporated
have
��'JQ"m�'�
protein
the
of reticulocytes.
a ratio characteristic of the com-
of hemoglobin, the protein produced by these cells
are not incorporated
protein.
system.
The amino
�
vivo, and
the ratio of the total amino acids in the ribosome
This indicates that a specific protein is being formed by the
It is important to show that amino acid incorporation really
represents protein synthesis sinc� in some systems, it was thought that
the process of amino acid incorporation could be dissociated from true
protein synthesis although the processes were related.
The first process in:pprotein synthesis consists of the activation
of amino acids.
The process of peptide formation from free amino acids is
not spontaneous and the amino acids must therefore be converted to compounds with a
chains
chemical potential.
The lengthening of
does not necessarily require large amounts of energy and
can occur by transpeptidation.
This process depends on a supply of
peptides and can be catalyzed by the proteolytic enzymes.
known
It is not
amino acids
has
contain both amino and carbolKiyl groups and since both types of groups
can be activate�it was not obvious how the activation would
The evidence now is that activation occurs by reaction of the cargroups of the amino
of
as
of
the
group which
•
This was suspected because of the beha-
that form simple peptides and could
for protein synthesiSe
Such systemslare the
the acetylation of sulfonilamide, and t he
In
be
of
all these
A or by
of
At
synthesis
the
ATP to form an adenylic acid deriva-
amino acids
tive of the
acids
the combination of
which the c arboxyl group of the amino acid
of the\'A:TP G
is combined with the
In the presence of enzyme and amino acid, phosphorus-32 labelled
ATP will e�change with non-labelled pyrophosphate�
this reaction
that all
20
involved
in
protein synthesis
One evidence that
the demonstration
of the common amdno acids can be activated
( Lipman, 1958);
another is the behavior ofcertain analogues of trypophano
Certain ana­
logues of the amino acids are actually incorporated into protein.
(1951)
and Lipman
Sharon
demonstrated that those tryptophan analogues which
were incorporated into protein were activated by the tryptophan-activatenzyme.
Those
vated but
which were
This correlation indicates
inhibited
that the activation reaction
acti-
incorporated were
a part of the protein synthesis mechanism.
Activation occurs in the soluble or supernatant fraction of the cell.
is, each amino
enzymes are
vated by its own single specific enzyme.
vating enzymes
Several relatively pure acti­
been isolated and the work continues.
amino acids do not compete for
enzymes
that activation is entirely
The fact
all
sites indicates
be located.
There is
of RNA.
This
affect the
based on the fact that
amino acids by crude or
enzymes
the more
RNA
are fully
not
the absence of RNA.
The
bou..ll d to
a very small
acf�81)"[;O:I'" for the amino
amino
of an
the
Thus the activating enzyme and
bound
can be thought of as acting effectively as a single
The
of amino acid to sRNA is mediated directly by the
activating enzyme without the involvement of other enzymes.
protein synthesis, the amino acid becomes
In the second phase
attached
ester bond to the terminal adenosine of the
an
corresponding sRNA
1960).
Glassman, and Schweet,
This RNA
have built into its nucleotide sequence a specificity for the particular
amino acid to which it becomes attached on the particular activating
enzyme.
It is important that the reactivity of an sRNA with its eor­
responding amino acid varies with enzymes from different species.
transfer RNA for a particular amino
affinity to the
"
in ester
by
for all
therefore mot
enzyme,
now well known that
The
amino acid is joined
on the sRNA to one of the adjacent free hydroxyl groups
of a
of
The third step in the synthesis of proteins is the transfer of the
amino acid f�om the sRNA to the template RNA of the ribosome"
that
been demonstrated
The
RNA
reacts
fact that sRNA
A
into
seen for
sRNA
and
to
deserves
a cyclic, coenzymatic
the
in the
complementary.sequence of
combines
��'�\JQ�.ll�
RNA
for
is
It has
for
very broad
in
code
a
CWoJ....
the
"''''....''' world"
The amino
RNA,
therefore, seems to carry two apparently separate specificities:
the activating
, and
(2)
for position on
ribosome..
(
for
Work with
the electron microscope has provided a direct and remarkable view of
appears
rib0somes in the act of synthesizing the protein hemQglobin"
that several ribosome particles are linked together at the time of synthesis, probably by messenger RNA»
1963).
Knopf, and Rich,
It
has been proposed that the ribosome attaches to the messenger RNA at
one end and travels across to the other end.
In the process, the ribo­
some synthesizes hemoglobin according to the code it reads off the RNA
molecule ..
The coding problem can be explicitly stated as
problem of how
the sequence of the four bases in RNA determines the
the protein.
acids
and one
"
The problem has two aspects, one general
Specifically one would like to know just what sequence
of bases codes each amino acid.
has
20
of
DNA
units,
of the
do w ith the
they are arranged in the
The more general aspect of the coding
molecule, and the way in Which
m essage
The simplest sort o f code would be one in which a small group of
stands
one particular
16
this would
More
the
One
and at
code group
"
a codon"
This gro�p can scarcely be a
A group
o f such a code
.I.J.,=,,'::ou<::'u.
which would
a
bases that codes one amino
schemes
20 are
called
that
only certain
can
consequence
leads to a change in
that
in
amino acids.
cG,de seem unlikely
G
Recent evidence makes
In the first place, there seems to
be no restriction of amino acid sequence in any of the proteins so far
examined.
has also been shown that typical mutations change only a
Although it 1'3
amino acid in the polypeptide chain of a protein"
theoretically possible that the genetic code may be partly overlapping,
� more likely that adjacent codons do not overlap
all.
Since the backbone of the DNA molecule is comp�y regular, there
nothing to mark off the code into groups.
To solve this difficulty,
various ingenious solutions have been proposed.
It was thought, for
example, that the code might be designed in such a way that if the wrong
non-
of triplets were chosen, the message would always
sense and no protein would be produced"
message
and
But it now looks as though the
at a fixed starting pmint, probably one end of the gene,
read
bases at a time.
If the
started, at the
wrong point, the message would fall into wrong sets of three and would
If this idea were correct, it would immediately explain why the
or deletion of a base
most
of the gene would make the
message
the reading of the
from
point onward would be
incorrect.
In
with
were always without
them were
of a base and one causing
Il)ocur
on the
If
addition and
a
, the gene would work"
a, base
molecule, the gene
are too far apart, the
be
To understand
64
t
the code must again be
20
triplets but only
to
amino acids to be coded.
two or more triplets may stand for each amino acid.
e
There are
Conceivably
On the other hand,
reasonable to expect that at least one or two triplets may
to start or to stop protein
other meaning, such as a
formation.
�o'n��'_
Although such hypothetical triplets may have a meaning of
some sort, they have been named nonsense triplets.
It is possible that
the misreading produced in a region lying between an?i::eLddition and a dele­
tion might give rise to a nonsense triplet, in which(case the gene might
work.
Investigations w ith a number of addition-with-deletion com­
binations in which the intervening distance was relatively short have
that these combinations were indeed inactive when they might be
to function.
Presumably an intervening nomrense triplet
:to
In confirmation of this hypothesis� it has been possib�e to modi�y
blame.
that turn one base into another, thereby
such nonsense triplets by
restoring the gene's activity.
At the same time it has been possible
of nonsense
mes-
It
is read from a fixed point, most likely one end ,iof the
So far, all the evidence has fitted very well into the
the
read
in
groups
idea
one end.
starting
The
same results should have been._,�u�'�J'QU' however, if the message. had been
read
in groups of
or indeed in groups of any size.
addition mutations were
this,
either
of the gene.
"
But when all three are
The Same
fairly close together in one gene.
function
will
or in
have been
To test
the s� gene,
by
three base dele-
tio ns ( Cr ick , 1 9 62).
o nd
of
wr o ng r e ading.
w il l br
ion will
t co d e ,
B u t if the c o d e is a tr
,
the me s s age b a ck in
w il l b e r e ad cor r e ctly .
ion w ill
ion is obvious.
The
a third a d d itio n
and fr o m the n o n to the e n d it
A l tho ugh the m o s t l ik e l y e x p l anation is that
the me s s a ge is r e ad thr e e b as e s at a time , this is n o t comp l e te l y
cer tain .
T he r e a d in g could b e in multip l e s o f thr e e .
T he f ie l d o f ce ll ul ar pr o te in
s is and the ins e p ar ab l e
f ie l d o f nucle ic a cid chem is tr y w il l continue
0
and n e w inf or ma tion will cer tain l y b e d is co v e r
r ap id p ace .
A t the pr e s e nt tim e ,
the m o s t inte r e s t.
b e w id e ly s tu d ie d
�t a n incr e a singly
the co d in g p ro b l e m is pr o b ab l y
Dis co v e r y o f the c o d e s f or s p e cific
amin o acid s w il l a l l ow e x p e r im e ntatio n with a l te r nation, by chemical
means ,
o f g e n e tica l ly inhe r ite d char a cte r is tics .
A pr o b l e m in
am.:L"LO a cid co d e s is the d if f icul ty in nucl e ic a cid de
mapp
d a tio n .
a-
T his d if ficu lty could p o s s ib l y b e a l l e v ia te d b y s e par ation
of d if f e r e nt p ur e DNA s .
C hr omat
me tho d s o ccupy a r ather
ion
u niqu e p o s ition among r e ce nt d e v e l opme nts in the f ie ld o f s e
T he ter m chr omatogr ap hy now cov e r s se v e r a l
m e thod s .
s:
cie n t lab or atory
m o l e cu lar s ie v e ,
gas ,
ads or p tion,
tio n .
in
e
ions with a s o lu-
ctr
I
a mate r ial must b
es.
I on
ionic
tic ion
time
at
ther e f or e ,
p
is b a s e d on the
of a s ol id mater ia l
To be us e fu l a s an ion
co l umns .
I u s e d ion
chr omat
o f ioncexc
comm o n
io n
and e l e ctr o chr omat
F or my e xp er im e nts with DNA ,
T he pr in c
par tition ,
e f f i-
cr o s s -link e d or
and inor
r s ins ar e e la s tic
Is that ab s or b
er
other
s o l v ents and in
in s
erior o f a res in
c on c entrated elec tr
11 c
o
s
r
f
The p ro perties o f the p o lymer
e s ol u tio n .
n etw o rk , inc l u d in g its l ow d ielec tric c ons tant, a f f ec t the ion
b ehavior�
and ,
in a d d itio n , the po l ymer a c ts to a c er­
tain extent as a s ol v en t o r a d s o rb en t f o r certain c om p o u nd s .
I on exc hange c ol umns a re u s ually o p erated by elution d ev el opmente
I n elu tion c hroma to grap hy , the s u b s tanc e to b e s ep arated
is f irs t s o rb ed on a narrow band o f res in at the top o f the c olumn .
I ons o f this s u b s tanc e a re then elu ted ,
o r c a rried d own the c o l umn ,
b y a s ec o nd s p ec ies of ions whic h c ompetes f o r b onds o n the res in .
ep aratio n is a c c omp l is hed b y v a ry ing the conc entratio n o f the
c ompeting ions .
F ra c tio n s are c o l l ec ted at the b ottom o f the
c o l umn .
T he a p p l ic ation o f ion
to
elu tion c hromat
iv e p ro b l ems in the nuc l eic a c id f ield
ic a 1 a nd pr
d irec tly f o l l o w ed the inv ention o f the t
tio n to in o r
!
c at
Khym ,
and its
and C o hn ,
1 9 4 7 ) and w a s
a c tually the w a y i n w hic h true el ution c hr
was introd u c ed into b io c hemis try .
exc ha nge
T he c omp l exes b etween the
p o l yv a l en t ino rganic c ations a nd the p
or
a c id s
that p erf o rmed s o s p ec taCUl a rl y o n the f irs t tes t o f ion exc
c hromato gra p hy may be c on s id ered a s
d egree and k ind o f
range by
teric ,
inc
o f c ha r
contro l
to them w ithout the nec es s
d irec
ion s .
o v er a w ide
;;:lin c e the nuc l eic a c id s a re a l so
it was a p p a rent that the
c
inasmuch as the
may be v aried c an t
a d j us tmen t .
c o ntro l could
eric,
It
als
apparent tha t s ep arations
of
ith
anion o r c
i on
w ere f eas i bl e.
o
n uc leic ac id f ie
o
( Cohn , 1 9 4 9 )
lie
t
t ouc h ed of f a s eries of d i s c overies of n ew n uc leo­
t id es , b ot h t hos e d eri ved f rom nuc l ei c a c i d s t ruc t ure, a s wel l a s
I t als o mad e a va ilab l e a c h em i c a l
t h os e t ha t exi s t free in na t ure.
t ec hn i que of b road us e f ul n es s in a w i d e varie
of analy t i c a l and
i on p rob l ems .
I n my experiment s ,
I s
by ammon i um hyd roxid e elut i on,
t o ef f ec t a s ep a ra t i on of DNA,
on an i on exc h ange c ol umn .
Th e
c ol umn was prep a red b y p ouring a s lurry of Dowex- 5 0 , a c ommerc ial
c a t i on - t y pe exc ha n ge res in, i n t o a b uret p l ug ged a t t h e b ot t om b y
a wad of glas s wool .
T h e c ol umn was w a s h ed w i t h d i s t i l l ed w a t er
A small q uant i t y of DNA,
t o as s ure a neut ra l p H .
admini s t ered t o the t op of the c ol umn .
inc reas
in s ol ut ion , was
T o a c c omp l i s h elut i on ,
c on c ent ra t i on s of ammon i um hyd rox i d e were ad d ed t o t h e
t op o f t h e c ol umn .
F rac t i ons w ere c ol lec t ed a t t h e b ot tom of t h e
c ol umn ( el ut i on rat e
=
2 ml . /m in . )
in w e
s ampl e b ot t l es .
A f t er t h e c ont a ined s ol ut i on was evaporat ed i n an oven,
b ot t l e was
eac h s amp l e
A f t er
and the mas s o f t he DNA was c
eac h s amp l e was el ut ed ,
t he c ol umn was regenera t ed b y p a s s i n g
t h rough d il ut e hyd roc h l oric a c id and t h en ri ns ing w i t h d i s t i l l ed
wat er unt i l a n eut ra l
was ob tai ned .
F or t h e f i rs t c ol umn, ap p roxima t ely 0 . 3 g. of c ommerc
p rep ared DNA was d i s s olved b y h ea t
and s orb ed on t h e c ol umn (
of el ut i on c on c ent ra t i ons
f ra c t ion 5, 0 . 10
f ra c t ions
in a 0 . 05 M .
. ; f ra cti on 6, 0 . 20
1 . 00
.; f ra c t ion s
Th e
mm . ) .
used:
s ol ut i on of
s ch edule
f rac t i ons 1 -4 , o.
M.;
!
f ra c t ions
o.
2.00 M . ;
f rac i on
.
;
t he f o I l
t
re s id u e s w e re o b t a i ne d :
f ra c t i o n 1
2
3
4
5
6
7
8
\9
10
11
12
13
14
15
16
F o r t he ne xt c o l umn, 0 .1 g.
0 .0 2
Ivl.
- o.
g.
- o.
-
01
0 . 00 3 8
0 .0037
0 .0 0 3 2
0 . 0 0 27
0 . 0037
0 .0 06 3
0 .0 0 3 9
0 . 0017
0 .0 0 3 1
0 .0 24 5
0 . 00 4 7
0 . 0037
0 .0 0 1 2
0 .0 0 23
o f DNA w a s d i s so lv e d b y he a t
NH4 0H a nd s o rb e d o n t he c o lumn ( 3 5 mm.).
re s o lve t he l a rge i ni t ia l DNA re s id u e
c o l umn i nt o pos s ib l e c omp o ne nt
was u s e d :
f ra c t io ns
, 0 .0 2
,
1'1.
f ra c t i o n 7 , 0 . 10 H . ; f ra c t i o n 8 ,
f ra c t i o ns 1 2-1 4 , 1 .0 0
1 4 .8 0 M .
S e v ent e e n m I .
.
;
I n an att
in
to
o b t a ine d in t he f i rs t
t he f o l l o wi ng e lu t i o n s c he d u l e
NH 4 0H; f ra c t i o ns 4-6 , 0 . 0 5 H . ;
0 .20 H. ;
f ra c t i o ns 1 5 -1 6 ,
f ra c t i o ns
, 0 . 50 Iv[. ;
2.0 0 M . ; f ra c t i o ns 17 -18 ,
f ra c t i o ns w e re c ol l e c t e d a nd t he f o I l
re s idue s w e re o b t a i ne d :
f ra c t i o n 1 - 0 .0 6 4 1 g .
2 - 0 . 0035
3 - 0 . 0015
4 - o.
5 - 0 . 0010
6 - 0 .0010
7 - 0 . 00 06
8
9
10
11
12
.
- 0 . 00 8 5
- o.
- o.
a c ti o n
- 0 . 00 1 4
F or t h e t h ir d c o l umn , 0 . 1 g.
o f DNA was d i s o l v e d in h o t
wat e r and s or b e d o n t h e c o l umn ( 4 0 mm . ) .
t o r e s o l v e t h e ini t ia l p
.;
,
int o p o s s ib l e c omp o ne nt
frac t i o n s 1 - 6 , 0 . 0 2
f o l l ow ing e l u t i o n s c he du l e was u s e d :
f r a c t i ons 7 - 9 , 0 . 05
I n s t il l a n o t h e r a t t e mp t
t he
1'1 .
NH40H ;
fr a c t i ons 1 0 - 1 2 , 0 . 50 M . ; fr a c t i o ns 1 3 - 16 ,
1 . 00 M . ; fr a c t i ons 1 7 - 18 ,
2 . 0 0 M . ; fr a c t i o ns 1 9 - 22 , 7 . 4 0 M .
Fr a c t i o ns o f 6 mI . wer e c o l l e c t e d and t h e f o l l ow ing r e s id u e s
w e r e o b t a ine d :
fr a c t i o n 1
2
3
4
5 6 7 8 9 10 11 12 13 14 16 17 19 20 21 22 -
I n a l l t hr
is
is
nt
1 . 00
c
0 . 0 0 1 9 g.
0 . 00 28
0 . 0 4 24
0 . 0 0 22
0 . 0009
o.
o.
0 . 000 6
0 . 0010
0 . 0007
0 . 0005
0 .0 0 0 9
0 . 0 0 10
0 . 00 16
0 . 0015
0 . 00 16
0 . 0 0 23
0 . 0019
o.
0 . 0012
o.
0 . 0000
t w o app ar e n t
c one
ar e n o t e d .
i o n s and
l ar ge
smaller p e ak
no o ther
ic ab l e
or c
e s e nt in
r s id u e
small frac -
t i ons .
o
D NA s o f d i f fe r e n t c h emic al
sc
dule ,
c o uld
ass
d own int o s ma l l e r
e par a t i o n o f D NAs by c hr oma t o gr ap hy h a s b e e n pr e v i ou s ly
a c c o mp l i s h e d b y v ar i o u s me a ns b u t , b e c au s e b u f fe r s o l u t i o ns ar e
u su a l l y u s e d ,
t h e D NA fr a c t i o ns ar e mix e d w i t h s al t r e s id u e s wh i c h
make b as e s e qu e nc e d e t e r m i na t i o n b e d e gr ad a t i o n d i f f i c u l t .
D NA
s e p ar a t i o n by ammo nium hydr o x i d e e l u t i o n h a s d e f init e app l i c a t i o ns
b e c aus e , a f t e r e vap or a t i o n, G: o l l e c t e d fr a c t i o ns c o nt a in o nly D NA ,
o n whi c h d e gr ad a t i o n e xp e r ime nt s C a n b e p e r f or me d .
B
E. ,
1 , "H OVl C e I l s I'4ake 11 0 1 e 205 ( 3 ) , p p . 7 4 - 8 2 .
1.
A l l fr e y , V . G . ,
c u le s , t I S c i e nt i f ic Ame r ic an ,
2.
, A . E . , a n d Os awa , S . , 1 9 5 7 , The Nuc l e u s
is i n S ymp o s ium o n the Ch e m i c a l
�---��������
M c E lr oy and B . G l as s , B a l t imor e ,
Pr e s s , p p . 200 - 23 1 .
•
•
3.
B e l j ansk i , !vI . , and O c h o a , S . , 1 9 5 8 , ! l Pr o t e in B i o s yn t h e s i s b y a
C e l l -Fr e e B a c t er ial
t e m , !! P r o c . Nat . A c ad . S c i . ,
, p p . 4 94 501 .
4.
B e nz e r ,
t ific
5.
Br e nner , S . , J a c o b , F . , and �ll e s e l s o n ,
. ,
1 , "An Uns t ab l e
I n t er me d ia t e
I n f or ma t i on fr om G e ne s t o R i b o s o m e s f or
Pr o t e in S yn th e s is , II Na t ur e , 1 9 0 , p p . 5 7 6- 58 1 .
6.
B u t l er , J . A . V . , a nd S h o o t er , K . V . , 1 9 57 , The Phys i c al H e t e r o ­
ge n e i t y o f D NA i n S ymp o s ium o n t h e Ch emic a l B a s is o f H e r e d i t y ,
e d . b y W . D . M c E lr oy and B . Glas s , B a l t i m or e , The J ohns H op k i ns
Pr e s s , p p . 5 4 0 - 5 5 1 .
8.
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