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OCR for page 36
36
THE LIFE SCIENCES
out the history of biology, far-reaching general principles have been deduced
by comparative study of related structures or functions across a variety of
species, genera, or phyla.
THE LANGUAGE OF LIFE
The last two decades have witnessed a prodigious gain in understanding
of life at all levels. Undoubtedly, however, the crowning achievement of
this era has been the spectacular growth of understanding of that process
central to life itself-the chemical encoding of genetic information, the
mechanism whereby it is read out to give direction to the life of the cell,
and the mechanism whereby it is reproduced in the course of cell division.
This area of understanding variously termed molecular biology, bio-
chemical genetics, the chemistry of reproduction, or the biochemistry of
nucleic acids and proteins-flowered when the stage had been set. It could
not have happened earlier and probably was inevitable when it did occur
because of the centrality of the questions at issue.
Until 1940, genetics had been studied, in the main, with conventional
higher species of plants and animals, the hereditary traits studied being
those most readily observed eye color, distribution of hair, flower pig-
ments, etc. Through such studies, much of the language of formal genetics
was generated, although the molecular mechanisms responsible were un-
known. Biochemists had been concerned principally with identification
and characterization of the chemical compounds characteristic of living
forms and the pathways by which they are synthesized or degraded in cells.
Microbiologists, long concerned with techniques for the identification and
taxonomic classification of micro-organisms, then studied their susceptibility
to sulfonamides and antibiotics as an adjunct to medical practice. Viruses
had been a subject of study largely because of the diseases they engender
in plant and animal species. By the mid-1940's it was possible to combine
the understanding generated by these seemingly disparate disciplines into
a concerted effort to understand the nature of the genetic apparatus.
It had long been apparent that the genetic complement of any individual
must be encoded in some chemical form; and, although morphological
traits had served the geneticist well, in fact, there can be no gene for height,
or eye color, or number of teeth, or age of onset of baldness. Clearly, the
genes that govern such parameters must actually govern specific chemical
events, the consequences of which are evident in these more readily dis-
cerned characteristics. Slowly, the concept grew that each individual is a
reflection of his complement of proteins; the latter serve both as structural
OCR for page 37
FRONTIERS OF BIOLOGY
materials and as the enzymes that synthesize all other types of biological
chemicals. Each cell is whatever its proteins make possible, and the num-
bers of cells of each type and the manner in which they are distributed are,
in some way, a consequence of genetic instructions with respect to which
proteins to make and the relative amounts of each.
Belief in this concept began with the observations of Garrod in 1908,
who assembled then-existing information concerning six hereditary diseases
of man, indicating that each was the consequence of loss of some enzymic
ability. This concept was solidified with studies of a bread mold, Neuro-
spora crassa, which ordinarily can be grown on extremely simple nutritional
media and synthesizes for itself all the usual amino acids, carbohydrates,
purines, pyrimidines, vitamins, etc. Irradiated cultures of this organism
were found to contain mutants that had lost the ability to make one or
another of these vital components. By appropriate procedures it was
ascertained which step in the sequence of chemical reactions by which such
synthesis normally occurs (a "metabolic pathway" such as those shown
in Figure 3) was actually blocked. In each instance, the mutant had lost
the ability to catalyze one specific step in such a sequence. A large body
of information accumulated from studies of a variety of bacterial forms
confirmed this concept, encapsulated in the axiom, "A single gene deter-
mines the synthesis of a single enzyme."
The general relationship between the structure of genetic material and
that of proteins, however, could not be established until the structural plan
of proteins themselves had been revealed. The first protein to be studied
appropriately was the pancreatic hormone, insulin, the structure of which
is shown in Figure 4. Insulin proved to be constructed by the head-to-tail
combination of 20 different kinds of amino acids; in all molecules of insulin,
at each position along the chain, one and only one of the 20 possible amino
acids does in fact exist. When the techniques developed for determination
of this linear sequence became generally available, they were quickly
applied to other proteins with similar results. Although a few differences
were found among the insulins obtained from various species, in each
species all the molecules of insulin are identical, as are all the molecules
of cytochrome c, of myoglobin, and so on. These findings made explicit
the nature of the information that must be encoded within the genetic
material, i.e., instructions with respect to the linear sequence of amino
acids in each of the proteins to be synthesized. If this concept. is correct,
the amino acid sequence of a given protein must, in some manner, be
colinear with the instructions within the gene responsible for its synthesis.
The special advantage of utilizing micro-organisms is that it is possible
to screen for mutants in populations of billions of individuals at one time
and, by applying to them appropriate modifications of the classical tech
OCR for page 38
38 THE LIFE SCIENCES
OCR for page 39
FRONTIERS OF BIOLOGY 39
U. ~ ~
o ~ o Ct
c ~ fi ~
~ ~ Ct
v o ~
. ~ ~ ~
o
o ~
C ~ 3~ C; D
C) ~ _]
~ ~ ~ _
. ~ ~ ~ Ct
~ ~ 04
· _ ~ ~ ,
so O ^-S
C) ~ ~ O
O ~ ~
~ ~ _
3 'A ~ ~
° V'
=: ~ . _
~ C~ >
o Ct ~ ~
· _ 0,, _ o
C) ~ ~ ~
Ct _ ~ 9
L' _
- 5 5_
Cal ._ ._
~ ~0
._ ._ ~
~ 3 o ~
~ ~ o
o ~ -
9
~ O P:
V,
ha ~
~ (O Ct
~ ~ a
- C., a:
Cal ~ ~ Cal
~ ~ O
3 .= "C
Ct ~ Z
~ Ct C,=
.o
Ct ~ ~
~V ~ O '
~ O O
s~ ·- O
~ ~ . ~ 9
`,L1 ~ ~ ~
p~ ~ ~ O
C~) .C) ~ ^ O
~ .= 3 ~1 0
-
Ct
1 P~
o
C)
~n
~o
o
o
C~
~ _
. _ ~
3 z
~i
C~
_
cn r~
~ _
~ V)
.C4
OCR for page 40
40 THE LIFE SCIENCES
HE ~ S S ~ ~H2 ~H2 ~H2
Gly Ileu Val Glu Glu Cy Cy Ala Ser Val Cy Ser I`eu Tyr Glu I`eu Glu Asp Tyr.Cy Asp
1 23 4 5 6 1 7 8 9 10 11 12 13 14 15 16 17 18 19 120 21
S S
1 1
NH NH S S
1 21 2 1 1
Phe Val Asp Glu His Leu. Cy Gly Ser. lIis Leu Val Glu Ala. Leu Tyr Leu Val Cy Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
FIGURE 4 The amino acid sequence of bovine insulin. (From Principles of Bio-
chemistry, 4th ea., A. White, P. Handler, and E. L. Smith. Copyright (I) 1968
McGraw-Hill, Inc. Used with permission of McGraw-lIill Book Company.)
niques worked out by geneticists for larger organisms, construct "maps" of
the genes. The earliest such maps, of the genes of fruit flies, simply related
the position of each gene to that of other genes along a chromosome. The
refinements possible with bacterial genetics enabled construction of de-
tailed maps indicating the relative positions of individual mutations along
the length of a single gene. Such techniques have been applied to a con-
siderable variety of bacterial and viral genomes (the totality of genetic
material in a cell). A rather thoroughly studied gene in this regard is that
which directs the synthesis of an enzyme in E. cold called "tryptophan
synthetase." The positions along the map of hundreds of mutants of this
gene have been scrutinized and related to the accompanying change in the
amino acid sequence of the protein itself. A partial summary of these
studies is shown in Figure 5, which illustrates the colinearity of the gene
that provides the instructions for making tryptophan synthetase and the
amino acid sequence of that protein itself.
B51 A38 A3 A33 A487 A23 A46
A7 8 A5 8 A96
A446 A223 A187 A169
Genetic map l l l l
(not to scale) - =r I I I I =Jja
Genetic map
distances W.4~.7+0~1.6~.04~.3+.4~.0014.06~.55.001~.02~.3 -
Amino acid in
wild-type protein
1
Amino acid in
m uta nt prot2 in I
Position of change H2N-1-48 48 174 - 176-182 - 210 - 210 - 212 - 233-233 - 234 267-COOH
In the protein , I
:48+041 26~2+6~28+0+2~21~0+1-33 -
Glu Glu Tyr Leu Thr Gly Gly Gly Gly Gly Ser
Val Met Cys Arg He Arg Glu Val Cys Asp
Leu
Residue distance in
polypeptide chain
FIGURE 5 Colinearity of the amino acid sequence of the enzyme tryptophan
synthetase and the substructure of the gene that governs its synthesis. ( From C.
Yanofsky, G. R. Drapeau, I. R. Guest, and B. C. Carlton, '`The Complete Amino
Acid Sequence of the Tryptophan Synthetase A Protein (car subunit) and Its Colinear
Relationship with the Genetic Map of the A Gene," Proc. Natl. A cad. Sci. U.S. 57:296,
1967.)
OCR for page 41
FRONTIERS OF BIOLOGY
Figure 5 also reveals a concept first made clear by an understanding of
the defect in sickle cell anemia. This disorder is the consequence of an
alteration in the structure of hemoglobin, in which, at only one specific
position (,86) along a chain of 146 amino acid residues, there occurs a
substitution of the amino acid valine for glutamic acid. This observation,
with its profound implications for the understanding of genetic disease,
reveals the nature of the simplest possible kind of mutation: A change in
the structure of the genetic material at one point along the strand of genetic
instruction results in substitution of one amino acid for another in the strand
of amino acids.
The Genetic Material
Meanwhile, an ever-growing body of evidence indicated that the genetic
material itself must be the polymer called deoxyribonucleic acid (DNA),
which is peculiar to the cell nucleus and is the stuff of which chromosomes
are made. The ultraviolet-absorption maximum of this material occurs at
the wavelength most effective in creating mutants. As techniques for the
purification of viruses accumulated, each in turn was found to contain a
nucleic acid as a major component. The capstone in this argument, which
was not truly recognized as such at the time it occurred, was a study under-
taken for rather practical clinical purposes. Two strains of Pneumococci
were known, one of which was characterized by an outer coat of a carbo-
hydrate polymer that the other strain lacked. When, however, cell-free
preparations of cultures of the former were added to cultures of the latter,
they were "transformed," acquiring the ability to make the carbohydrate
polymer and retaining that ability through an indefinite number of subse-
quent cell divisions. In effect, these cells had acquired a gene they formerly
lacked.
The material in the cell-free culture that made this possible was found
to be DNA-in retrospect, categorical evidence that DNA is indeed the
material of which genes are made. The lack of immediate appreciation of
the profound implications of that finding was a consequence of earlier
studies of the structure of DNA, which were misleading in that they sug-
gested it was a dull repetition of a fundamental repeating polymer unit
without variation. In light of such studies it had seemed unlikely that the
structure of DNA could be the basis for genetic instruction, which was
known to require immense variation. Only as the structure of DNA was
re-examined was this incorrect impression rectified (Figure 6~.
More careful analyses, using newer techniques, demonstrated great
variability even in the gross structure, the relative composition varying
from species to species, yet with one pair of cardinal rules. All DNA's
OCR for page 42
42 THE LIFE SCIENCES
o
O=P-OH
o
/ O
\ H
H ~
13, 12'
o
l
O=P-OH
41'
H
H
Adenine
H~C ~ O Guanine
it,,: 41'
H i, H
13,
o
l
O=P-OH
O=/-OH
l
H
O Thymine
-11'
:/H
FIGURE 6 A segment of the backbone structure of
a single strand of DNA. Representation of a portion
of a DNA chain, showing the position of the inter-
nucleotide linkage between C-3' and C-5'. (From
Principles of Biochemistry, 4th ea., A. White. P.
Handler, and E. L. Smith. Copyright (if) 1968 Mc-
Graw-Hill, Inc. Used with permission of McGraw-
Hill Book Company.)
are constructed of only four fundamental units called nucleotides: adenylic
acid (A), guanylic acid (G), thymidylic acid (T), and cytidylic acid
(C). This small number of units, nevertheless, permits encoding of a
vast amount of information; indeed, in Morse code, with only two symbols,
it is possible to transmit all the works of Shakespeare. In all specimens of
DNA, A - T and G - C, whereas there is no consistent relationship
between A and G. The meaning of this constancy was not apparent until
combination of this information with studies of the x-ray-diffraction pattern
of nucleic acids led to the now well-known depiction of DNA as two very
long strands wrapped about each other in the familiar double helix, and so
aligned that, on the strands, every A is opposed by a T. and each G is
opposed by a C (Figure 7~. In each case the pair of bases is linked by
the relatively weak forces of hydrogen bonds, as illustrated in Figure 8.
The great stability of the double helix is the consequence of the sum of
thousands of such unions. Importantly, there is no rule with respect to the
actual consecutive sequence along one strand. If one knows the sequence
OCR for page 43
FRONTIERS OF BIOLOGY 43
along one strand, one automatically knows the complementary sequence
along the opposing strand, as shown in Figure 7.
This structure immediately solved the two basic questions concerning
the chemical structure of genetic material. This material must, as an in-
trinsic property, both achieve its own self-duplication as cells divide and
provide for the great variability that would permit the encoding of instruc-
tions to make the enormous diversity of proteins found in nature. Self-
duplication is achieved in a most ingenious way. Decades of bafflement
concerning how any chemical could achieve its own self-duplication were
resolved by the recognution that as cells divide, the double strand is, in
some manner, disengaged and each strand then governs the synthesis not
of itself but of its complement. Where one double strand existed, two
double strands are brought into being, each of which contains one of the
original strands and a complementary new partner (Figure 91. A great
body of evidence now supports that concept, although some of the details
remain obscure. The base pairing described is the feature of the structure
that determines how the new strands shall be formed. Occasionally mis-
takes are made, and when this occurs there is poor fitting of the strands.
Under those circumstances a set of additional enzymes comes into play.
One snips out the ill-fitting sections, thereby permitting another to reinsert
/ ~
. . · .
`~ .~ i
,~
1
To
4~.\
f 1-
At 13.4 A
~ A
FIGURE 7 Schematic representation of the
double helix of DNA. The ribbons represent the
deoxyribosephosphate backbone chains. The op-
posing arrows indicate that one strand is running
from the 5' position of one sugar to the 3' posi-
tion of the next, while the other strand is running
in the opposite sense. The horizontal lines repre-
sent hydrogen bonds between opposing pairs, two
for each AT couple, three for each GC couple.
(From I. Herskowitz, Genetics, 2nd ea., 1962.
Copyright (I) 1962 Little, Brown and Company.)
OCR for page 44
44 THE LIFE SCIENCES
cytosi ne
~3
Th ~ ~ ~ ~
--in ~
-POSSE
\
\
.\
51.5°]
hi\
iTo chain
To chain = ~_ A
\
-
-
/~'. ~° 1 1 ~,
~ . ~.
- 10 add (id) ~ To chain
\ ~-
-
\
-
-
-
-
-
(b)
-
/
FIGURE 8 Molecular dimensions and hydrogen bonding of base pairs of DNA.
(Adapted from S. Arnott, M. H. F. Wilkins, L. D. Hamilton, and R. Langridge,
"Fourier Synthesis Studies of Lithium DNA, Part III, Hoogsteen Models," J. Mol.
Biol., 11:391~02, 1965, p. 392.)
OCR for page 45
FRONTIERS OF BIOLOGY 45
I_ ~L
I_ _
(~ ~ ---I \
it- _ ~
\ _ _ fit
1: >~-----iLK 1
~, , _
~An_
~ As_
1 > - E-----5K
I___ _
FIGURE 9 Scheme of replication of a DNA model. Boldface chains represent the
newly synthesized strands of the two daughter molecules. (From J. posse, A. D.
Kaiser, and A. Kornberg, "Enzymatic Synthesis of Deoxyribonucleic Acid," J. Biol.
Chem., 236:864, 1961. Copyright (A 1961 The American Society of Biological
Chemists, Inc.)
the proper bases, which then make the normal tight fit of the double helix.
Interestingly, the double helix of a bacterial chromosome, like that of most
viruses, is a circular molecule, the head, as it were, being joined to the tail.
This was originally recognized by the mapping procedures noted above, and
then it was visualized by electron microscopy. Duplication of the DNA,
therefore, must commence by opening this circle.
In this concept, instructions for protein synthesis must be provided by
the linear sequence of bases along the DNA strand; given the fact that the
chromosome of E. cold consists of a single helix of about 10 million con-
secutive base pairs, and that any one of the four bases may lie to left or to
right of any other base, there is essentially an unlimited number of statistical
possibilities, only one of which is the actual structure of a specific DNA
chromosome. And the possibilities become astronomical in a human cell,
the DNA complement of which is 1,000 times as great as that of E. coli.
Subsequent research efforts concentrated on the mechanisms by which the
~ :,:`,
(~1 _____
OCR for page 46
46
THE LIFE SCIENCES
instructions encoded in the DNA are utilized to give direction to protein
synthesis. This is now understood in remarkable detail, although there are
a few serious gaps in this knowledge. The summary that follows encom-
passes the work of hundreds of investigators over a period of two decades.
PROTEIN SYNTHESIS
At an early stage it was recognized that, although the DNA holds the
primary instructions for protein synthesis, it does not itself directly par-
ticipate in that process. In the cells of higher organisms, DNA is locked
in the nucleus, whereas protein synthesis occurs in small bodies called
`'ribosomes" stippled throughout the cytoplasm. It followed, therefore,
that the instructions for protein synthesis in the DNA must be dispatched
from the cell nucleus to the ribosomes. Ribosomes were found to be com-
posed of a mixture of about 20 different proteins plus several forms of
ribonucleic acid (rRNA); RNA differs from DNA in that the sugar com-
ponent is ribose rather than deoxyribose, and most RNA is single-stranded
rather than double-stranded. Ribosomes are leaflets constructed of a small
component and a larger component, disposed much as a partially open
clam. The messages from nucleus to ribosome were shown to consist of
yet another form of single-stranded ribonucleic acid, messenger RNA
(mRNA), which is fabricated in the nucleus by a specific enzyme called
the DNA-dependent RNA synthetase. This form of nucleic acid is tran-
scribed from one strand of the DNA helix by the same base-pairing rules,
except that the pyrimidine nucleotide uridylic acid (U) is utilized, rather
than the thymidylic acid (T) of DNA, so the four letters of the RNA
alphabet are A, G. U. and C. Just as in DNA itself, the growing mRNA is
formed on one of the DNA strands in an antiparallel manner (see Figure
91. In a living cell the long fibers of mRNA can be seen threaded through
as many as 10 ribosomes at once (a polysome), so different areas of the
message are being "read" by each of the ribosomes consecutively.
Assuming some kind of colinearity of the mRNA and the protein to be
synthesized, it must be the base sequence of the mRNA that specifies the
amino acid sequence of the protein. Since there are 20 different amino
acids in proteins and only four letters in the RNA alphabet (A, G. U. C),
obviously these cannot bear a one-to-one correspondence; moreover, one
can form only 12 two-letter words with four letters. Hence, the minimal
number of letters that would suffice is three per "word," i.e., the "codon"
that specifies the exact amino acid next to be incorporated in a growing
protein chain. Indeed, one can form 64 three-letter words with a four-
letter alphabet. The problem then, was to establish the relationship be-
tween the four-letter alphabet of the RNA and the 20 words in the amino
acid dictionary.
OCR for page 47
FRONTIERS OF BIOLOGY
If three-letter words are utilized and a large body of evidence now
indicates that the code words for amino acids are built of three letters
each there seemed no way physically to relate the structure of the amino
acids to a sequence of three nucleotides in RNA. Accordingly, it was
postulated that some form of "adapter" would be required. That adapter
proved to be yet a third general type of RNA, termed "transfer RNA"
(tRNA), the smallest kind of RNA known. Several pure tRNA's have
been isolated, each of which serves as the adapter for one specific amino
acid; Figure 10 shows a complete structure of one tRNA. All tRNA's
appear to be built along the same general plan.
Within the cell there is a family of "amino acid-activating enzymes," and
the fact that the entire apparatus actually works successfully rests on the
remarkable properties of these enzymes. In absolutely specific fashion,
each such enzyme esterifies one and only one of the 20 amino acids to the
hydroxyl group at the 2-position of the ribose at one end of one specific
form of tRNA; it is imperative that the enzyme make no error since any
such error would necessarily become an error in protein synthesis. What
is shown in Figure 10 as the projecting round knob of the tRNA molecule
is the "anticodon," a sequence of three bases which fit, by the usual base-
pairing rules, against three consecutive bases in the messenger RNA, the
codon for an amino acid. The amino acid is attached to the tRNA at a
position quite remote from the anticodon. In a cell engaging in protein
synthesis, there is a pool of all 20 amino acids, each affixed to its specific
tRNA by virtue of the activity of the appropriate activating enzymes. As
the long mRNA (500 to 10,000 nucleotide units) threads through the
ribosome leaflet, it is attached to the smaller ribosomal component, while
an amino acylated tRNA, which can achieve the necessary complementary
base pairing to the message, is fixed in position on the larger member.
The first amino acid to be laid down is that at the amino terminus of
the chain. The protein chain grows by the reaction
O R1 O
Ret O
11 1 11 1 ~1
------C- N CH C tRNA1 + HINT CH C tRNA.,
H
O R1 O R
11 ~ 1 11 1
------C N CH C N CH tRNA~ + tRNAl.
H H
As each such reaction is completed, the freed tRNA departs, and the
mRNA must move through the ribosome so that the next three "letters"
47
OCR for page 48
48 THE LIFE SCIENCES
o
1
G)
1 1
G) ~
1 1
G)
1 1
1 1
G)
~1
c
1 1
G)
_ ~ _ ~ c
_ :~ 9_ :~ _ c~
1 1 1 1
, G- C- G- C - <;,
am. \
c' /
c
\
'G- O'
o;~
v_9_9_~> _ >_ (~ - n ~
1 1 1 1 1
u-c-C-G- G-r ~ c
A)\
o-~
1 1
C AS
1 1
~V
1 1
1 1
l
c
1 1
C _ ~
-
I /- G _C I
| ~ | Anticodon
FIGURE 10 Base sequence and gen-
eral structure of a tRNA for alanine.
The anticodon, the three bases that
pair with the three-base codon of
mRNA, are shown at the bottom. As
in DNA, and DNA-RNA hybrids the
strand of tRNA is running in the op-
posite sense to that of the mRNA,
to which it must attach on the ribo-
some surface. (From J. T. Madison
and H. K. Kung, "Large Oligonucleo-
tides Isolated from Yeast Tyrosine
Transfer Ribonucleic Acid after Par-
tial Digestion with Ribonuclease T1,"
J. Biol. Chem., 242: 1324-13 3 O. March,
1967, p. 1329. Copyright (it) 1967 by
The American Society of Biological
Chemists, Inc. )
are aligned at the working site. The mechanism by which this ratchet-like
process is accomplished is totally unknown. These events are schematically
shown in Figure 11.
There remained the task of establishing the dictionary; as the decade
of the 1960's began this appeared to be a herculean task. A fortunate
combination of accident and experimental virtuosity rapidly broke through
this problem, the solution to which is shown in Figure 12. All 64 pos-
sible three-letter words are utilized and, hence, show considerable redun-
dancy. Where more than one code word is utilized to signify the insertion
of a given amino acid, an equal number of tRNA's must also be available
OCR for page 49
FRONTIERS OF BIOLOGY
to the cell; in several instances this has been shown to be the case. As will
be seen, three of the three-letter words do not relate to any amino acid;
where these occur in the mRNA sequence, no amino acid can be inserted,
and synthesis of the protein chain terminates and the tRNA at the end of
the cliain is removed by hydrolysis. This, therefore, is the "punctuation" in
the message, the "period that ends the sentence."
Understanding of exactly how chain initiation is accomplished is less
satisfactory. It is all-important that reading of the message begin at a pre-
cise point; if the reading frame were to shift by one letter, the entire message
would be garbled and a completely different set of amino acids would be
assembled. In bacterial forms, it would appear that the first amino acid
in the chain is always the same-methionine, the amino group of which
bears a formyl group. Message reading, therefore, begins by utilizing formyl
methionine tRNA as the first "word" and is terminated when any one of
the three nonsense words shows in the message. Acetyl serine tRNA may
serve the same role in animal systems.
One of the most powerful tools in the multitude of studies that gave rise
to the remarkable picture just described was the experimental demonstration
of the effectiveness of base pairing. The force that holds the two strands of
DNA together is the sum of a multitude of tiny forces hydrogen bonds
between nitrogen and oxygen atoms. These are the forces that also hold
Formyl-Met-Phe-Lys~
/Pro
5/\
A U G U
1 1 1 1
into of\ 1
\~\ ~G
U U A A A C C \C U
1 1 1 1 1 1 1 ~1
I yr~ Ala -~
~1 ~ '
Tyr
tRNA
Ala
tRNA
1 1 1
C G I
1 1 1
1 1 1
/~ /
1 1 1 1 1 1
1 1 1 1 1
A C/ G C C / U C C
1 J<% 1 1 ~ 1 1 1 1 1
- _' ~- ; ~mRNA1
(50 S) Ribosome (30 S)
Movement of
ribosomes
FIGURE 11 Schematic representation of protein synthesis on a ribosome. The pro-
tein tRNA is departing, having been used in the previous step; the bond between
tyrosine and its tRNA is being displaced by the amino group of alanine; and a seryl-
tRNA is just coming into position. (From Principles in Biochemistry, 4th ea., A.
White, P. Handler, and E. L. Smith. Copyright (I) 1968 McGraw-lIill, Inc. Used
with permission of McGraw-IIill Book Company.)
OCR for page 50
50 THE LIFE SCIENCES
z
o
-
E~
V)
o
Pi
-
2nd POSITION
U C A G
PHE SER TYR CYS
PHE SER TYR CYS
U LEU SER CT- 1 CT-3
LEU SER CT-2 TRY
LEU PRO HIS ARG
LEU PRO HIS ARG
C LEU PRO G LN ARG
LEU PRO GLN ARG
ILU THR ASN SER
A ILU THR ASN SER
ILU THR LYS ARG
MET THR LYS ARG
VAL ALA ASP GLY
VAL ALA ASP GLY
G VAL ALA GLU GLY
I VAL ALA GLU GLY
U
C
A
-
U
C
A
-
U
C
A
G
U
C
A
G
z
o
-
v,
o
FIGURED The genetic code. Each amino acid in a protein is specified by a nu-
cleotide triplet in RNA, e.g., aspartic acid (asp) is specified by the triplets GAU and
GAC. UAA,UAG, and UGA are utilized for punctuation, viz., to indicate when
to terminate the chain.
crystals of water, i.e., ice, together and, like ice crystals, they can be
melted by warming. If double-stranded DNA is brought to an elevated
temperature, the double helix comes apart and the DNA becomes indi-
vidual random flopping coils. If the temperature is then lowered very slowly,
the coils find each other and the double helix is restored. By the same
technique one can prepare in the laboratory hybrid DNA-RNA complexes,
but only-when the latter can be aligned and can join by multitudinous base
pairing. In this way it was shown that a rather substantial fraction of DNA
of E. cold and a much larger fraction of mammalian DNA are used to code
for the preparation of rRNA, but only a tiny fraction, less than 0.1 percent,
specifies the formation of all the tRNA's. And by the same procedure one
can artificially achieve attachment of tRNA onto RNA. Indeed, this tech-
nique was the basis for the most successful procedure for determination of
the genetic code. Thus? a trinucleotide of RNA of known base sequence
can be added to a ribosome suspension. To a sample thereof is added a
tRNA charged with its amino acid, the latter labeled with ~4C. If the
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FRONTIERS OF BIOLOGY
radioactivity adheres to the ribosome-RNA complex, the tRNA has paired
its anticodon with the trinucleotide and the code word is established.
Much of these concepts had been elaborated by deduction from a great
variety of experimental observation. A capstone on this intellectual struc-
ture was provided by a series of maneuvers, conducted with great technical
skill, which achieved the isolation, in pure form, of a single gene from among
the many of the genome of E. colt, that which directs the synthesis of any
enzyme called 3-galactosidase. The final proof of this overall picture has
been provided by a remarkable tour de force. From knowledge of the struc-
ture of the tRNA for alanine, an antiparallel, complementary length of DNA
was synthesized chemically. The usual DNA-synthesizing enzyme was
utilized to form its complementary DNA strand. This relatively short
double-stranded DNA was then used with the DNA-dependent RNA-
synthesizing enzyme to form the tRNA for the amino acid alanine and
yielded the predicted structure. This constitutes true chemical synthesis of
a gene! Similarly, synthetic lengths of RNA have been used with ribosomes
as mRNA and have yielded small polypeptides of the structure predicted
by the code.
Self-Assembly Such studies demonstrated that base pairing is the primary
mechanism involved in DNA duplication, in the synthesis of RNA on DNA,
and in message reading in the ribosome. But they also demonstrated a
cardinal principle of the biological world, the principle of self-assembly.
Organisms are assemblages of cells, and within the cells there are myriad
organized sub-cellular bodies, within which, in turn, are macromolecules
that are aggregates of smaller molecules. Yet, as we have seen, all that
genetic instructions can provide is information descriptive of the synthesis
of protein chains. Are other types of instructions required, or does all
else follow from the fact of protein-strand synthesis? The answer, unques-
tionably, is that all else is derivative, that all other structures combine,
because they do indeed fit together and are held together by a collection
of small forces, much as are the two strands of DNA that make such a
remarkably tight fit.
For example, the hemoglobin molecule consists of four subunits, two
a-chains, and two ¢-chains. The two chain types can be separated by
appropriate means but, when remixed, the normal tetrameric units recon-
stitute themselves without assistance. The enzyme ribonuclease is a single
protein chain within which are three internal disulfide bridges ~ S S-).
These can be opened by appropriate chemical means (reduction). In this
form the enzyme is a random flopping coil and lacks catalytic activity. When
allowed to reoxidize slowly, virtually every molecule regains its enzymic
activity, despite the fact that there are eight different ways in which the
Representative terms from entire chapter:
amino acids
