Early Speculations About What Genes Are And How They Act

• It was natural to be curious about what is the molecular/chemical nature of these genes?
• And how they function?
• The realization that chromosomes have both nucleic acids and protein wasn’t helpful since the
structure of neither was understood.
• The most fruitful speculations was concerning the fact that genes need to duplicate.
• How a complicated molecule which carry such diverse types of information could be precisely
copied to yield exact replicas?
• How could such information be coded?
• What could be the nature of such molecule which is mostly stable, but can also succumb to
spontaneous mutation that is permanent in nature?
• Many Physicist/Mathematicians became intrigued but enough details was not known to say
anything.
 Preliminary Attempts To Find A Gene–Protein Relationship

• As it was observed that many of the functions in the cells were performed by proteins, it was
suspected that genes effect proteins.
• The most fruitful early endeavors to find a relationship between genes and proteins examined the
ways in which gene changes affect which proteins are present in the cell.
• It soon became clear that genes with simple metabolic functions would be easier to study than
genes affecting gross structures like eyes and wings.
• Based on a study of a hereditary disease affecting amino acid metabolism, English physician
Archibald E. Garrod suggested in 1909, that genes work by controlling the synthesis of specific
enzymes (the one gene–one enzyme hypothesis).

• However, no assurance could be given either that most genes control the synthesis of proteins, or
that all proteins are under gene control.
 Nature of Genetic Information
• Until the mid-1940s, there appeared to be no direct way to attack the chemical essence of the
gene.
• It was known that chromosomes possessed a unique molecular constituent, deoxyribonucleic
acid (DNA).
• Despite this, there was no way to show that DNA carried genetic information, or was serving as a
scaffold.
• It was generally assumed that genes would be composed of amino acids because they appeared
to be the only biomolecules with sufficient complexity to convey genetic information.
• By early 1940s, research was generating increasingly strong evidence supporting the 30-year-old
hypothesis of Archibald E. Garrod that genes work by controlling the synthesis of specific
enzymes.
• Thus, given that all known enzymes had been shown to be proteins, the key problem was how
genes participate in the synthesis of proteins.
• The simplest hypothesis was that genetic information within genes determines the order of the
20 different amino acids within the polypeptide chains of proteins. How is it coded?
 Nature of Genetic Information
• Initial guess was that there are enzymes that determine the order of each amino acid added to a
polypeptide chain.
• However, such scheme would require, for the synthesis of a single type of protein, as many
ordering enzymes as there are amino acids in the respective protein.
• But because all enzymes known at that time were themselves proteins (we now know that RNA
can also act as an enzyme), still additional ordering enzymes would be necessary to synthesize
the ordering enzymes.
• Chicken and egg problem?
• Fortunately, limitations in chemistry knowledge did not deter researchers from doing genetic
experiments with chemically simple molds, bacteria, and viruses.
 Nature of Genetic Information
• The idea that DNA might be the key genetic molecule emerged from studies on pneumonia-
causing bacteria.
• In 1928 English microbiologist Frederick Griffith made the startling observation that non-virulent
strains of the bacteria became virulent when mixed with their heat-killed pathogenic
counterparts.
• That such transformations form non-virulence to virulence represented hereditary changes was
shown by using descendants of the newly pathogenic strains to transform still other non-
pathogenic bacteria.
• ⇒ when pathogenic cells are killed by heat, their genetic components remain undamaged.
• Moreover, once liberated from the heat-killed cells, these components can pass through the cell
wall of the living recipient cells and undergo subsequent genetic recombination with the
recipient’s genetic apparatus.
 lele of the capsule gene is present, a capsule is formed around the cell
that is necessary for pathogenesis (the formation of a capsule also gives a
smooth appearance to the colonies formed from these cells). When the R

capS
(capsule gene) chromosome
capsule

heat to kill
capS fragment
released capS

pathogenic
S (smooth) cell capS
capR capR recombination capS
and cell division

nonpathogenic entry of chromosome

S cell
R (rough) cell fragment bearing
capS into capR cell

FIGURE 2-1 Transformation of a genetic characteristic of a bacterial cell (Streptococcus

pneumoniae) by addition of heat-killed cells of a genetically different strain. Here we show an
R cell receiving a chromosomal fragment containing the capsule gene from a heat-treated S cell.
Since most R cells receive other chromosomal fragments, the efficiency of transformation for a

• Pathogenicity reflects the action of the capsule gene, which codes for a key enzyme involved in
given gene is usually less than 1%.

the synthesis of the carbohydrate-containing capsule that surrounds most pneumonia-causing

bacteria.
• When the S allele of the capsule gene is present, a capsule is formed around the cell that is
necessary for pathogenesis.
• When R allele of this gene is present, no capsule is formed, the cells are not pathogenic.
 Nature of Genetic Information
• In 1944, after some 10 years of research, U.S. microbiologist Oswald T. Avery and his colleagues
demonstrated that the active genetic principle was DNA.
• Supporting their conclusion were key experiments showing that the transforming activity was
destroyed by deoxyribonuclease, a recently purified enzyme that specifically degrades DNA
molecules to their nucleotide building blocks but has no effect on the integrity of protein
molecules or RNA.
• In contrast, the addition of either ribonuclease (which degrades RNA) or various proteolytic
enzymes (which degrade proteins) had no influence on the transforming activity.
 Viral Genes Are Also Nucleic Acids
• Equally important confirmatory evidence came from chemical studies with viruses and virus-
infected cells.
• By 1950 it was possible to obtain a number of pure viruses and to determine which types of
molecules were present in them.
• This work led to the very important generalization that all viruses contain nucleic acid.
• Crucial insight came from study of the multiplication of T2, a bacterial virus (typically called a
bacteriophage, or phage) composed of a DNA core and a protective shell built up by the
aggregation of a number of different protein molecules.
• In these experiments, performed in 1952 by Alfred D. Hershey and Martha Chase in New York, the
protein coat was labeled with the radioactive isotope 35S and the DNA core with the radioactive
isotope 32P.
• The labeled virus was then used to follow the fates of the phage protein and nucleic acid : which
labeled atoms from the parental phage entered the host cell and later appeared in the progeny
phage.
 Viral Genes Are Also Nucleic Acids 24 Chapter 2

35
S-labeled THE DOUB
coat protein

• Clear-cut results emerged : much of the parental nucleic acid and 32

P-labeled
DNA
While work
none of the parental protein was detected in the progeny phage. mixing of virus
smaller num
pattern of D
with host cells
liam Astbur
• Moreover, it was possible to show that little of the parental protein Caspersson.
even enters the bacteria; instead, it stays attached to the out-side of fraction pho
lin (Fig. 2-4
the bacterial cell, performing no function after the DNA component 35S
DNA structu
lynucleotide
has passed inside. 32
P
violent bonds of DN
agitation
of organic c
• This was demonstrated by violently agitating infected bacteria after protein “ghost”
that 30 –50 ph
DNA (Fig. 2
the entrance of the DNA; the protein coats were shaken off without labeled with 35S
In 1951,
affecting the ability of the bacteria to form new phage particles. 32P-labeled
(which we
of helical
DNA H. Crick, an
DNA structu
mentary do
• The primary function of viral protein is thus to protect and transport multiplication of
viral chromosome
James D. W
John Kendre
its genetic/nucleic acid component in its movement from one cell to and production
of new phage
answer dep
configuratio
another. Franklin.
In the dou
release of bonds (a we
new progeny
particles
of bases on
specific: the
whereas the
In double-h
FIGURE 2-3 Demonstration that only
the DNA component of the bacterio-
 protein “ghost” DNA (Fig. 2-5).
labeled with 35S
In 1951, because of interest in Linus Pauling’s a helix pro
(which we shall consider in Chapter 6), an elegant theory of

The Double Helix 32P-labeled

DNA
of helical molecules was developed by William Cochran
H. Crick, and Vladimir Vand. This theory made it easy to te
DNA structures on a trial-and-error basis. The correct solution,
mentary double helix (see Chapter 4), was found in 1953 by
multiplication of James D. Watson, then working in the laboratory of Max P
• While work was proceeding on the X-ray analysis of protein structure, a smaller number of viral chromosome
and production
John Kendrew in Cambridge, United Kingdom. Their arrival at
answer depended largely on finding the stereochemically mos
scientists were trying to solve the X-ray diffraction pattern of DNA (makes sense as protein got all
of new phage
configuration compatible with the X-ray diffraction data of W
Franklin.
the limelight initially). In the double helix, the two DNA chains are held together by
release of bonds (a weak noncovalent chemical bond; see Chapter 3) betw
• The first diffraction patterns were taken in 1938.
new progeny
particles
of bases on the opposing strands (Fig. 2-6). This base pairi
specific: the purine adenine only base-pairs to the pyrimidine
whereas the purine guanine only base-pairs to the pyrimidine
• It was in the early 1950s that high-quality X-ray diffraction photographs were taken by Maurice
2-3 Demonstration that only
FIGURE
In double-helical DNA, the number of A residues must be eq

Wilkins and Rosalind Franklin, in the UK. the DNA component of the bacterio-
phage T2 carries the genetic information
and that the protein coat serves only as a

• These photographs suggested not only that the underlying DNA structure was helical
protective shell.

but that it was composed of more than one polynucleotide chain—either two or
three.
• At the same time, the covalent bonds of DNA were being unambiguously
established.
• In 1951, thanks to Linus Pauling’s work on 𝛼 helix protein motif, an elegant theory of
diffraction of helical molecules was developed by Francis Crick, among others. 2-4 The key X-ray photograph involved in the elucidation of the D
FIGURE

• The correct solution, a complementary double helix was found in 1953 by 1953,Crick and
This photograph, taken by Rosalind Franklin at King’s College, London, in the win
confirmed the guess that DNA was helical. The helical form is indicated by

James Watson, working in Cambridge, UK.

pattern of X-ray reflections ( photographically measured by darkening of the X-ra
center of the photograph. The very heavy black regions at the top and bottom r
3.4-Å-thick purine and pyrimidine bases are regularly stacked next to each other,
to the helical axis. (Printed, with permission, from Franklin R.E. and Gosling R.G.
171: 740–741. # Macmillan.)
 26 Chapter 2 Chargaff’s Rule
} K E Y E X P E R I M E N T S
• Biochemist Erwin Chargaff used “paper chromatography” to analyze the nucleotide composition
B O X 2-1 Chargaff’s Rules
of DNA.
Biochemist Erwin Chargaff used a technique called “paper chro- residues in all DNA samples was equal to the number of

• By 1949 his data showed not only that the four different nucleotides are not present in equal
matography” to analyze the nucleotide composition of DNA. By
1949 his data showed not only that the four different nucleo-
thymine (T) residues, and the number of guanine (G) residues
equaled the number of cytosine (C) residues. In addition, re-
amounts, but also that the exact ratios of the four nucleotides vary from one species to another.
tides are not present in equal amounts, but also that the exact gardless of the DNA source, the ratio of purines to pyrimidines
ratios of the four nucleotides vary from one species to another was always approximately 1 ( purines ¼ pyrimidines). The fun-
• Chargaff’s experiments also showed that the relative ratios of the four bases were not random.
(Box 2-1 Table 1). These findings opened up the possibility
that it is the precise arrangement of nucleotides within a DNA
damental significance of the A ¼ T and G ¼ C relationships
(Chargaff’s rules) could not emerge, however, until serious at-
molecule that confers its genetic specificity. tention was given to the three-dimensional structure of DNA.
• The fundamental significance of the A = T and G = C relationships (Chargaff’s rules) could not
Chargaff’s experiments also showed that the relative ratios of
emerge, however, until serious attention was given to the three-dimensional structure of DNA.
the four bases were not random. The number of adenine (A)

BOX 2-1 T A B L E 1 Data Leading to the Formulation of Chargaff’s Rules

Adenine to Thymine to Adenine to Guanine to Purines to
Source Guanine Cytosine Thymine Cytosine Pyrimidines
Ox 1.29 1.43 1.04 1.00 1.1
Human 1.56 1.75 1.00 1.00 1.0
Hen 1.45 1.29 1.06 0.91 0.99
Salmon 1.43 1.43 1.02 1.02 1.02
Wheat 1.22 1.18 1.00 0.97 0.99
Yeast 1.67 1.92 1.03 1.20 1.0
Hemophilus influenzae 1.74 1.54 1.07 0.91 1.0
Escherichia coli K2 1.05 0.95 1.09 0.99 1.0
Avian tubercle bacillus 0.4 0.4 1.09 1.08 1.1
Serratia marcescens 0.7 0.7 0.95 0.86 0.9
Bacillus schatz 0.7 0.6 1.12 0.89 1.0
After Chargaff E. et al. 1949. J. Biol. Chem. 177: 405.
 Replication of DNA
• Most of the excitement came not merely from the fact that the structure was solved, but also
from the nature of the structure.
• There had always been the worry that it would turn out to be dull, revealing nothing about how
genes replicate and function.
• Rather, the two intertwined strands of complementary structures suggested that one strand
serves as the specific surface (template) upon which the other strand is made.
• Rigorous proof had to await the development of in vitro systems for DNA synthesis.
 How DNA Replicate: Finding the Polymerases that make DNA

• These came fast due to expertise of biochemists in enzyme isolation.

• Like U.S. biochemist Arthur Kornberg, who by 1956 had demonstrated DNA synthesis in cell-free
extracts of bacteria.
• Kornberg’s studies revealed that the nucleotide building blocks for DNA are energy-rich
precursors (dATP, dGTP, dCTP, and dTTP)
• Further studies identified a single polypeptide, DNA polymerase I (DNA Pol I), that was needed
for catalyzing the synthesis of new DNA strands.
• Was shown that it works only in the presence of DNA.
• Also, that DNA Pol I depends on a DNA template to determine the sequence of the DNA it is
synthesizing.
• This was demonstrated by allowing the enzyme to work in the presence of DNA molecules that
contained varying amounts of A:T and G:C base pairs.
• In every case, the enzymatically synthesized product had the base ratios of the template DNA.
 How DNA Replicate: Strand Separation during DNA Replication
• Simultaneously with Kornberg’s research, in 1958 Matthew
Meselson and Franklin Stahl, then at CalTech, US, carried out an
elegant experiment which showed that the two strands
permanently separate during DNA Replication.

cha-
ruc-
dels
epli-
peri-
tahl
dels,
ated
dispersive semiconservative conservative

“semi” in semiconservative). These experiments ruled out two other models

at the time: the conservative and the dispersive replication schemes
(Fig. 2-10). In the conservative model, both of the parental strands were pro-
posed to remain together and the two new strands of DNA would form an
entirely new DNA molecule. In this model, fully light DNA would be formed
after one cell generation. In the dispersive model, which was favored by
many at the time, the DNA strands were proposed to be broken as frequently
as every ten base pairs and used to prime the synthesis of similarly short
regions of DNA. These short DNA fragments would subsequently be joined
to form complete DNA strands. In this complex model, all DNA strands
would be composed of both old and new DNA (thus nonconservative) and
fully light DNA would only be observed after many generations of growth.
How DNA Replicate : Strand Separation during DNA Replication Nucleic Acids Co

• Showed that the two strands of the double helix permanently bacteria growing in 15N;
all DNA is heavy
transfer
to 14N medium
continued growth
in 14N medium

separate from each other during DNA replication.

• They used heavy isotope 15N as a tag to differentially label the
parental and daughter DNA strands.
• Bacteria grown in a medium containing the heavy isotope 15N DNA isolated from the cells is mixed with CsCl solution
(6M, ρ (density) ~1.7g/ml) and placed in ultracentrifuge

have denser DNA than bacteria grown under normal

conditions with 14N. ρ = 1.65 ρ = 1.80

• One can then separate heavy DNA from lighter DNA by light 14N-15N heavy
creating density gradients in cesium chloride in a centrifuge. DNA hybrid DNA DNA
solution centrifuged at

• DNA molecules, in which both strands are heavy (H-H DNA) 140,000 x g for ~48 hr

will form band closer to the bottom of the tube.

• If an H-H DNA transferred to 14N medium and allowed to grow,
14
N-14N
light DNA ρ = 1.65

the new DNA will be distinguishable from original. 15

N-14N
hybrid DNA
• With this it was clear that semiconservative model is the right 15N-15N

model. heavy DNA

ρ = 1.80

before transfer one cell two generations

to 14N generation after after transfer FIGU
transfer to 14N to 14N (CsCI)
the location of DNA molecules within the centrifuge cell the se
can be determined by ultraviolet optics during
 How DNA codes information?
• It was a genuine mystery how the genetic information of DNA functions to order amino acids
during protein synthesis.
• All DNA chains capable of forming double helices.
• Therefore, the essence of their genetic specificity has to reside in the linear sequences of their
four nucleotide building blocks.
• Even from a four-letter alphabet (A, G, C, and T), the number of potential DNA sequence is?
DNA Cannot Be the Template That Directly Orders Amino Acids
during Protein Synthesis
• Experiments showed that protein synthesis occurs at places where DNA is absent ⇒ ruled out a
direct role for DNA in protein synthesis.
• Especially in eukaryotic cells it occurs in the cytoplasm, which is separated by the nuclear
membrane from the chromosomal DNA.
• ⇒ At least for eukaryotic cells, a second information-containing molecule had to exist that
transmits the information to cytoplasm.
• Prime suspect was RNA : found to reside largely in the cytoplasm ⇒ was easy to imagine that
single DNA strands, when not serving as templates for DNA synthesis, act as templates for
complementary RNA chains.
• RNA Is chemically very similar to DNA:
• a long, unbranched molecule containing four types of nucleotides.
• Has the ability to form complementary helices of the DNA type.
• Unlike DNA, however, RNA is typically found in the cell as a single-stranded molecule.
 ther the additional hydroxyl group nor the absence of the methyl group
found in thymine but not in uridine affects RNA’s ability to form double-
helical structures held together by base pairing. Unlike DNA, however,
The Central Dogma
RNA is typically found in the cell as a single-stranded molecule. If double-
stranded RNA helices are formed, they most often are composed of two parts
of the same single-stranded RNA molecule.

• By end of 1953, the working

THE CENTRAL hypothesis
DOGMAwas adopted that chromosomal DNA functions as the
template for RNA molecules, which subsequently move to the cytoplasm, where they determine
the arrangement of amino acids.
By the fall of 1953, the working hypothesis was adopted that chromosomal
DNA functions as the template for RNA molecules, which subsequently
• In 1956 Francis Crick move
referred
to the to this
cytoplasm,pathway
where theyfor the
determineflow of genetic
the arrangement of information
amino
acids within proteins. In 1956 Francis Crick referred to this pathway for
as the central
dogma: the flow of genetic information as the central dogma:

Transcription Translation
Duplication DNA RNA Protein.

Here the arrows indicate the directions proposed for the transfer of genetic
• The Adaptor Hypothesis: It was
information. Therealized by Crick
arrow encircling DNA that it that
signifies willDNA
be isdifficult for Nucleotides to
the template
for its self-replication. The arrow between DNA and RNA indicates that
distinguish between the
RNA 20 amino
synthesis acids.
(called transcription) is directed by a DNA template. Corre-
spondingly, the synthesis of proteins (called translation) is directed by an
• Crick thus proposed that prior toMost
RNA template. incorporation into
importantly, the last twoproteins, amino acids
arrows were presented as uni- are first attached to
specific adaptor molecules,
directional;which in turn
that is, RNA possess
sequences unique
are never surfaces
determined thattem-
by protein can bind specifically to
plates nor was DNA then imagined ever to be made on RNA templates.
bases on the RNA templates.
The idea that proteins never serve as templates for RNA has stood the test
of time. However, as we will see in Chapter 12, RNA chains sometimes do
 Discovery of Transfer RNA

• Key to the discovery:

1. Ability to use cell free extracts for protein synthesis
2. Recent availability of radioactively tagged amino acids, which was used to mark the trace
amounts of newly made proteins.
3. High-quality, easy-to-use, preparative ultracentrifuges for fractionation of their cellular extracts.

• This led to the seminal discovery that prior to their incorporation into proteins, amino acids are
first attached to transfer RNA (tRNA) molecules (accounts for some 10% of all cellular RNA).

• Crick had indeed previously speculated that his proposed “adaptors” might be short RNA chains,
because their bases would be able to base-pair and “read” the appropriate groups on the RNA
molecules that served as the templates for protein synthesis
 The Paradox of the Nonspecific-Appearing Ribosomes

• Early on, the cellular site of protein synthesis was pin-pointed to be the ribosomes.
• Ribosomes contain both RNA and proteins (About 85% of cellular RNA is found in ribosomes).
• Amount of ribosomes in the cell is greatly increased in cells engaged in large-scale protein
synthesis (e.g. rapidly growing bacteria).
⇒ Ribosomes are responsible for protein synthesis.

• Initially ribosomal RNA (rRNA) was thought to be the template for ordering amino acids.
• But it can’t be: the RNAs and proteins are all the same in all ribosomes.
• Moreover, base composition of rRNA was seen to be high in G and C in all known bacteria, plants,
and animals, despite wide variations in the AT/GC ratios of their respective DNA.
 Discovery of Messenger RNA (mRNA)

• In 1960, work on bacteriophage and also in E.coli evidence for a separate messenger class of RNA,

• Only a few percent of total cellular RNA is mRNA; easy to understand why it was first overlooked.
 Enzymatic Synthesis of RNA upon DNA Templates
• As mRNA was being discovered, the first of the enzymes that transcribe RNA using DNA templates
was being independently isolated: RNA polymerases
• These enzymes function only in the presence of DNA.
• Direct evidence that DNA lines up the correct ribonucleotide precursors came from seeing how
the RNA base composition varied with different DNA molecules: In every case, the RNA AU/GC
ratio was roughly similar to the DNA AT/GC ratio.
• By this time, there was firm evidence for the postulated movement of RNA from the DNA-
containing nucleus to the ribosome-containing cytoplasm of eukaryotic cells.
• By briefly exposing cells to radioactively labeled ribonucleotides, then adding a large excess of
unlabeled ribonucleotides (a “pulse chase” experiment), mRNA synthesized during a short time
window was labeled.
• They were observed to be created in the nucleus, and then transported to cytoplasm.
 Establishing the Genetic Code
• Given the existence of 20 amino acids but only four bases, groups of several nucleotides must
somehow encode a given amino acid.
• Group of 2 gives?
• Group of 3 gives?
• The assumption of colinearity was important: It held that successive groups of nucleotides along a
DNA chain code for successive amino acids along a given polypeptide chain.
• A mutational analysis on bacterial proteins, carried out in the early 1960s by Charles Yanofsky and
Sydney Brenner showed that colinearity does exist.
• Also, genetic analyses by Brenner and Crick, in 1961, first established that groups of three
nucleotides are used to specify individual amino acids.
 Establishing the Genetic Code
38 Chapter 2

TA B L E 2-3 The Genetic Code

• But which specific groups of three bases (codons) determine second position
U C A
which specific amino acids could only be learned by biochemical
G

UUU UCU UAU UGU U

analysis. Phe Tyr Cys
UUC UCC UAC UGC C
U Ser
UUA UCA UAA stop UGA stop A
Leu
• Completion of the code in 1966 revealed that 61 out of the 64 UUG UCG UAG stop UGG Trp G

possible permuted groups corresponded to amino acids, with CUU

CUC
CCU
CCC
CAU
CAC
His
CGU
CGC
U
C

most amino acids being encoded by more than one nucleotide C

CUA
Leu
CCA
Pro
CAA CGA
Arg
A

third position
first position
Gln
CUG CCG CAG CGG G
triplet. AUU ACU AAU AGU U
Asn Ser
AUC Ile ACC AAC AGC C
A Thr
AUA ACA AAA AGA A
Lys Arg
AUG Met ACG AAG AGG G

GUU GCU GAU GGU U

Asp
GUC GCC GAC GGC C
G Val Ala Gly
GUA GCA GAA GGA A
Glu
GUG GCG GAG GGG G

FIGURE 2-18 Demonstration that

RNA is synthesized in the nucleus and
moves to the cytoplasm. (Top) Autoradio-
graph of a cell (Tetrahymena) exposed to ra-
showed that colinearity does in fact exist. Equally important were the genetic
dioactive cytidine for 15 min. Superim-
posed on a photograph of a thin section of
analyses by Brenner and Crick, which in 1961 first established that groups of
the cell is a photograph of an exposed three nucleotides are used to specify individual amino acids.
silver emulsion. Each dark spot represents But which specific groups of three bases (codons) determine which spe-
the origin of an electron emitted from a 3H cific amino acids could only be learned by biochemical analysis. The major
(tritium) atom that has been incorporated breakthrough came when Marshall Nirenberg and Heinrich Matthaei, then
into RNA. Almost all the newly made RNA working together, observed in 1961 that the addition of the synthetic poly-
is found within the nucleus. (Bottom) Auto-
nucleotide poly U (UUUUU . . .) to a cell-free system capable of making
radiograph of a similar cell exposed to radio-
active cytidine for 12 min and then allowed
proteins leads to the synthesis of polypeptide chains containing only the