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Chapter 21. The Genetic Code and Protein Synthesis
that genes are DNA molecules themselves. Thus, it becomes necessary to discuss here the
organization of chromosomes in order to understand the chemical nature of genes.
21.1 THE ORGANIZATION OF THE CHROMOSOME
The chromosomes are regarded to control all kinds of biochemical and physiological activities of the
cell through tiny chemical units called genes. They replicate with precision during cell division and
gametogenesis and help in the transmission of characters from one generation to another. Although
chromosomes vary in number in different organisms, they maintain unique similarity in terms of their
physical and chemical organization.
The genetic material of all cells is contained in the chromosomes which have a complex
composition. The human chromosomes contain approximately 15 per cent DNA. 10 per cent RNA
and 75 per cent protein, but the genetic material is almost always double stranded DNA. The viruses
are the only exceptions which contain single stranded DNA and in certain cases the single stranded
RNA functions as the genetic material. The chromosomes of viruses and bacteria are circular whereas
in all other organisms the chromosomes are linear structures. A typical haploid chromosome of a
complex organism is a cylindrical structure composed of two identical units, the chromatids. The
chromatids are intimately twined around each other and each one is supposed to be containing about
8 fibrillar threads. Each fibril is composed of two double helices of DNA. Both the chromatids are
attached with each other through a common point, the centromere, which represents a constricted
region on the chromosome.
The chromosomes of complex organisms contain long stretches of non-informative DNA and
some segments of DNA exist in multiple copies along a single chromosome. Although each species
has a characteristic amount of DNA, the eukaryotes vary greatly in DNA content which is always
more than the prokaryotes. One picogram of DNA, when properly stretched, would be equivalent to
31 cm of DNA. The chromatids are comprised of chromatin material which is heavily stained with
The chromatin is a viscous, gelatinous material which contains DNA, RNA, basic proteinshistones and non-histone proteins. Histone proteins and DNA are present in a fixed ratio of 1:1, but
the non-histone proteins always vary in different tissues. Histones are small basic proteins rich in
arginine and lysine, and bind intimately with DNA. There are four types of histones: H2A, H2B, H3,
H4, each of which are present in equimolar amounts. There are certain regions in the chromosomes
which remain condensed during interphase and early prophase. These are defined as heterochromatin
regions which are genetically inactive. Besides these regions, the remaining chromosome remains in a
non-condensed state and is called euchromatin.
What are Genes?
Cytogenetic observations suggested that the chromosomes contain DNA and that it is the hereditary
material. Mendel gave the idea of discrete factors as hereditary determinants which are inherited.
These factors were later termed as genes and defined as particulate units arranged on a thread-like
The Genetic Code and Protein Synthesis
Morgans work on Drosophila melanogaster, and later work with bacterial genetics on frequency
of recombination led to the formulation of a correct definition of gene. Thus the gene is defined as a
unit of recombination which cannot be subdivided by chromosomal breakage or crossing over and can
be separated from its neighbouring segments by a crossing over event. As our knowledge of the gene
advanced, it was discovered that the chromosomal unit functioning as the gene can undergo mutation
and thus affect the physiological function or expression. It was soon realized that the effectiveness of
a gene depends upon its relation to other neighbouring genes and as such there may be overlapping
regions of gene function. From the studies of mutation and mutagenic agents it has been suggested
that the unit of mutation could be much smaller than the functional unit, that is much smaller than the
unit of recombination.
From the mutation studies in Drosophila, it has been found that the so-called white-eye gene
exists in a number of alleles, hence a variety of mutations of this gene may give rise to various eye
colours. From this, it has been surmised that a gene can exist in many forms with a difference in
functions which account for distinct observable phenotypes.
21.2 REPLICATION OF DNA
From biochemical studies, it has been proved beyond doubt that DNA is the universal genetic
material of all forms of life except certain viruses. The building blocks of DNA have been described
in Chapter 1. In 1947, Chargaff demonstrated that DNA contains equal proportions of purine and
pyrimidine bases, so that adenine and thymine, and cytosine and guanine are present in equimolecular
proportions. These observations were quite significant which became clear from the elucidation of the
double helix model of DNA by Watson and Crick in 1953.
The double helix model consists of two twisted polynucleotide chains in which the deoxyribose
sugar units on adjacent nucleotides are linked with phosphodiester bonds to form a sugar-phosphate
backbone. The purine and pyrimidine bases project inwards perpendicular to the sugar-phosphate
backbone and are linked by hydrogen bonds. There is, however, a specific base pairing, occurring
between adenine and thymine and between cytosine and guanine (Fig. 21.1). The polynucleotide
chains are complementary to each other and the hydrogen bonds between the polynucleotide bases
stabilize the double helix, The hydrogen bonds, however, are sufficiently weak and capable of
breaking and reforming at room temperature. The genetic information in the DNA molecule depends
on the sequence in which the four bases are arranged along the polynucleotide chains. In the
complementary chains, the phosphate-sugar linkages run in the opposite directions. If in one chain,
the sugar-phosphate links go from a 3'-carbon to a 5'-carbon, then in the complementary chain the
sugar-phosphate linkages would run from a 5'- carbon to 3'-carbon.
The double helical structure of Watson and Crick suggests the manner in which the DNA
molecule can undergo replication. By a variety of ingenious experiments it has been shown that DNA
replication is semi-conservative, that is, each strand in the double helix serves as a template for the
synthesis of a new strand simultaneously, while the original strand remains intact in daughter cells for
Watson and Crick model of the double helix of DNA. The two chains are held together by hydrogen bonding
between the bases.
Semi-conservative Replication of DNA
In 1958, Meselson and Stahl experimentally demonstrated in E. coli bacteria that DNA is replicated
through semi-conservative mechanism. The chromosome of E. coli is a continuous DNA molecule,
whereas in plants and animals the chromosomes are more complex in organization. Hence, the
process of replication can be precisely elucidated in E. coli. Meselson and Stahl grew the bacteria
initially in a medium containing labelled nitrogen (l5N). After growing them for several generations, it
was found that all the DNA in bacteria was labelled. This was called heavy DNA. Then the bacteria
with heavy DNA were grown in an ordinary medium for one generation only. Analysis showed that
the next generation consisted of an intermediate form comprising of one heavy and one normal strand
of DNA. When the bacteria were grown in the ordinary medium, in the next generation, half of the
DNA was normal and another half of the intermediate form synthesized on the heavy strand
The Genetic Code and Protein Synthesis
15 N 14 N
14 N 14 N
14 N 14 N
15 N 14 N
The mechanism of semi-conservative replication of DNA.
The in vitro synthesis of DNA was demonstrated by Kornberg in 1956 and he showed that DNA
polymerase catalyzes the replication of DNA molecule by addition of deoxyribonucleotides to the
free OH group at the 3' end of a chain. The enzyme has two outstanding properties. Firstly, it
requires a mixture of the four types of DNA nucleotides (ATP, CTP, GTP and TTP) to function, and
secondly, a DNA primer which acts as template. In the absence of DNA primer no DNA synthesis
+ DNA primer
(dTP dGP dAP dCP)n
+ 4 (n) PP
It was found that DNA polymerase acts on the strand which is copied in the direction from 3' to
5' end. Since there are two strands in each DNA molecule, it is envisaged that the two strands unwind
which run in opposite directions so that the replication begins on each of the unwound strand.
Experimental evidence suggests that both strands are copied at the same time. In that case, the strand
which runs from 3' end to 5' end can be copied continuously. The other strand which runs in the
opposite direction from 5' to 3' end is copied in the opposite direction, in the 3' to 5' direction. The
synthesis cannot be continuous, hence newly synthesized DNA is formed in short segments which are
later joined by DNA ligase as discovered by Khorana and his associates.
We have seen that the parent DNA molecule unwinds before replication takes place but the
molecule does not unwind completely. Crick has pointed out that replication and unwinding take
place simultaneously. As soon as unwinding takes place, the formation of two new chains starts. The
mechanism is outlined in Figure 21.3.
3 ¢ 5¢
Mechanism of discontinuous replication of DNA. (a) partial unwinding of the two chains showing synthesis at
the 3¢ end by the action of DNA polymerase. (b) newly synthesized DNA is in short length chains. (c) the new
chains are joined by DNA ligase and reform new helixes.
The Genetic Code and Protein Synthesis
The rate of DNA replication in E. coli is a fast process, but in higher organisms it is rather slow.
In mammals, since the chromosome replication includes histone synthesis as well, the process is much
slower. At the same time, autoradiographic studies show that in eukaryotic chromosome, there are
several initiation points.
21.3 THE GENETIC CODE
While proposing the double helix model of DNA. Watson and Crick postulated that DNA is the
information system in which the bases were incorporated in a manner so as to determine the sequence
of amino acids in a protein molecule. This hypothesis visualizes that DNA utilizes a language of four
letters, adenine, guanine, cytosine and thymille, in different combinations. Through these four bases
DNA utilizes information to be transmitted to the cytoplasm for cellular control and its functions. But
then, this code has three variants in the form of DNA, tRNA and mRNA. In the information theory,
it was suggested that the code is operationally a system of codes and anticodes and if one was
deciphered the other one could be automatically determined.
In the DNA molecule, there is a mutual attraction for adenine and thymine, and for cytosine and
guanine which make the base pairs. DNA acts as the template for RNA synthesis. In the transcription
of RNA the attractive forces would be between adenine and uracil since RNA does not contain
thymine. Only one strand of double-helical DNA can act as a template for RNA synthesis. All types
of RNA molecules (rRNA, tRNA and mRNA) are single stranded and complementary to the DNA
The messenger RNA (mRNA) contains a linear sequence of bases which dictates the sequence of
amino acids of all polypeptide chains. The sequence of bases on mRNA is known as the genetic code.
There are twenty different kinds of amino acids and therefore there must be a specific code for each
amino acid. It was proposed by George Gamov that a combination of three bases seems to be most
probable which would yield 64 different combinations (43). In 1964, Khorana synthesized a
messenger RNA of known sequences though which he determined the triplet-base sequences that
could code different amino acids. The codes on the mRNA are called codons specific for each amino
There are a few generalizations about the genetic code (see Table 21.1). Several of the amino
acids have more than one codon, hence redundancy is present in the codes. For example, leucine is
coded by at least six codons, CUA, CUU, CUC, CUG, UUA, and UUA. The code is commaless and
universal for all protein synthesizing organisms. There are three codons, UAA, UGA and UAG which
do not code for any amino acid hence, specify termination of a peptide chain. The code is nonoverlapping.
21.4 SYNTHESIS OF POLYRIBONUCLEOTIDES
RNA molecules differ from DNA in two respects. RNA is a long-chain molecule in which there is
ribose sugar instead of deoxyribose and the fourth base thymine is replaced by uracil. Experimental
evidence indicates that RNA is synthesized iron a DNA template by a process analogous to the
replication of DNA and this process is known as transcription.
The Genetic Dictionary: Scheme for the Genetic Code
A specific enzyme called DNA-directed RNA polymerase is required to catalyze the synthesis of
RNA on DNA templates. Like DNA polymerase, this enzyme also requires nucleoside triphosphates
which align themselves on one of the DNA strands. When RNA synthesis starts, the DNA strands
unwind. In vitro synthesis of RNA has revealed that the enzyme RNA polymerase is Mg2+ dependent
and requires appropriate nucleoside triphosphates and a DNA template. Synthesis of RNA starts from
the 5' end of the RNA chain, hence in this respect it resembles DNA polymerase. Chemical analysis
has shown that RNA is copied as single strand from only one strand of the DNA molecule. Hence, the
newly synthesized RNA strand should be complementary to the DNA strand which is copied. This
has been amply proved with the help of hybridization experiments.
While a strand of RNA is synthesized on DNA template, the RNA molecule rapidly detaches
itself and the two unwound DNA strands come together after the reformation of hydrogen bonds. The
rate of formation of the phosphodiester bonds is rather slow (9000 per minute at 37°C) which is
suggestive of a number of starting points along the DNA chain. It has been experimentally proved
that a number of RNA molecules can be transcribed simultaneously on a DNA template. This shows
that the enzyme RNA polymerase can attach to DNA at a number of places. The growth of RNA
chains takes place from 5' to 3' direction as in case of DNA.
It is now known that RNA polymerase has a complex structure and functions on signals when to
start synthesis of RNA and when to stop. A factor sigma (s) gives the signals to start RNA synthesis
and it is an integral part of the enzyme molecules as such. The termination message to stop synthesis
of RNA is given by a protein factor rho (r) which is not considered as part of the polymerase.
As a general rule, RNA is formed on a DNA template. However, there are some exceptions to
this general rule, for example, viruses whose genetic information is contained in a single RNA strand,
The Genetic Code and Protein Synthesis
must first replicate a complementary RNA strand which may be copied to produce more. This
synthesis is carried out by RNA-directed RNA polymerase.
According to the central dogma in molecular genetics the genetic information is transferred from
DNA to RNA only. But in 1970, Temin reported that in some cancer producing RNA viruses copies
of DNA are produced on RNA templates by an RNA-dependent DNA polymerase, called reverse
transcriptase. The newly synthesized DNA may be incorporated in the host cell chromosome or it
may be used to synthesize more of viral RNA. This discovery generated tremendous interest among
the workers and the possible role of viruses in inducing cancer in humans was postulated.
Nevertheless, it is not necessary that the presence of the reverse transcriptase activity may be the
cause of malignancy of the virus to the host cells. Normal cells have also been shown to contain
reverse transcriptase activity, which according to some, may play some role in gene amplification
during differentiation of amphibian oocytes, and accidently the enzyme may produce new DNA
sequences which may lead to malignant cancerous growth.
Function of Ribonucleic Acids
RNA is always found as a single stranded structure but the molecule may occur as secondary and
three dimensional structures which are no doubt related to their functions. RNA is found to occur in
several species as described below.
Transfer RNA (tRNA) is also referred as soluble RNA or adaptor RNA. The molecules are relatively
small, about 75-85 nucleotides long. In a prokaryotic cell (E. coli), there are at least 60 different
species of tRNA, while in eukaryotic cells, there may be as many as 100 tRNA molecules. Hence
there may be two or three or more species of tRNA which can bind a single amino acid. Its molecular
weight is about 25,000 to 30,000 The RNA chain is bent upon itself and this brings the compatible
bases close to each other through hydrogen bonding. As a result, the tRNA molecule assumes a
cloverleaf pattern (Fig. 21.4) which relates to its secondary structure. Another special feature of tRNA
molecule is that it possesses a number of unusual bases.
A sequence of 3 bases is found on the middle lobe of the tRNA which is complementary to the
triplet code on messenger RNA. All tRNA have a terminal base sequence cytidylic - cytidylic adenylic acids (CCA) at the 3' terminal. The terminal nucleotide adenylic acid residue serves as an
attachment site to the energy-rich enzyme-bound amino acid. Such an amino acid is attached to the
2nd or 3rd carbon of the ribose sugar of the terminal nucleotide. At the 5' end of the strand, the tRNA
has an unpaired base guanine. The middle lobe with the sequence of three bases is known as the
anticodon and the base sequence of this varies with the type of tRNA. The amino acyl-tRNA complex
enters the ribosome and with the help of anticodon triplet recognizes its corresponding codon on the
mRNA molecule. Thus the function of tRNA is to carry various amino acids from the pool and
arrange them in the ribosomes as required by the messenger RNA.
Ribosomes and Ribosomal RNA
The ribosomes are submicroscopic particles occurring in every living cell (70 S ribosomes in
prokaryotes and 80 S ribosomes in eukaryotes). These are the sites for protein synthesis which are
The cloverleaf pattern of a typical tRNA molecule.
present throughout the cytoplasm in bacteria, whereas in higher organisms they are found attached to
the endoplasmic reticulum. About 60 per cent of the ribosome is RNA and the remaining 40 per cent
is protein. The ribosome is formed by the union of two subunitsa smaller 30 S* subunit, and a
larger 50 S subunit. The smaller subunit has one large ribosomal RNA (rRNA) which is 16 S
consisting of 1,600 nucleotides and 21 different proteins. The larger subunit has one large RNA (23
S) consisting of 3,200 nucleotides and 34 different proteins, and another smaller RNA (5 S) having
120 nucleotides. The task of ribosomal RNA is to join up the sequence of amino acids brought on the
assembly line of the messenger RNA molecule to form a peptide chain.
Both the 30 S and 50 S subunits are heterogenous mixture of three groups of proteins viz., unit,
marginal and fractional proteins. The ribosomes, owing to the existence of heterogeneous proteins in
them, serve different functions and fall under different classes. Some of these types of proteins are
known for their participation at various steps during protein synthesis. The 50 S subunit, in addition
to the above three groups, has another type of protein known as functional repeat (FR) protein.
rRNA is mainly synthesized in the nucleolus on extrachromosomal DNA as template. rRNA acts
as the framework on which the ribosomal particles are assembled during biosynthesis of ribosomes.
They maintain ribosomal particles in a configuration permitting them to fulfill their role in protein
*S is a Svedberg unit at which a particle sediments in a high speed ultracentrifuge.
The Genetic Code and Protein Synthesis
In 1961, Jacob and Monod demonstrated that the species of RNA which carries genetic information
from DNA to the ribosomes is the messenger RNA (mRNA). It has a thread like configuration which
may be coded for protein synthesis in small segments. It is synthesized enzymatically on DNA
template whose base sequence is complementary to DNA. In bacteria, messenger RNA has a short
life span, probably about 2 minutes, which is evident from the rapid turn over of viral proteins when
a phage infects the bacterium. The eukaryotic mRNA appears to be more stable, its half-life ranging
from a few hours to perhaps weeks.
Isolation of mRNA is a difficult process because of its short life and small quantity available in
the cell. However, techniques have been devised to isolate globin mRNA which synthesizes largely
haemoglobin from rabbit reticulocytes. The globin mRNA sediments at a small peak 9 S whose
estimated length is about 700 nucleotides. Messenger RNA has a base sequence complementary to
DNA of the same cell. This has been demonstrated by DNA-RNA hybridization experiments. Out of
the total-700 nucleotides of the globin mRNA, only 589 are coded by the DNA and the rest are
attached to the mRNA after transcription to its tail end. The globin molecule (protein) consists of 146
aminoacids, hence it would require only 436 nucleotides on the mRNA chain. Thus it is evident that
the mRNA has some extra nucleotides.
The prokaryotic mRNA is synthesized on one of the two strands of DNA acting as the template
and functions as a template for protein synthesis. However, the eukaryotic mRNA has two noncoding
regions which do not synthesize proteins. These regions are located at the 5' end and the 3' end. The
5' end has about 50 nucleotides whereas the 3' end has about 100 nucleotides. In some mRNAs the 3'
end is extraordinarily large.
21.5 PROTEIN SYNTHESIS
We have briefly described the structure and role of different types of nucleic acids. DNA is the
informational molecule which contains the genetic information in a coded form and this information
is transcribed on the mRNA molecules before it is translated by ribosomes for the synthesis of
specific proteins. Protein synthesis is thus a gene directed process which is carried out in the
cytoplasm utilizing an elaborate protein-synthesizing machinery. For convenience, the polypeptide
(protein) synthesis is discussed here under four stages: activation of amino acids; chain initiation;
chain elongation; and chain termination. As far as possible, a brief comparison will also be made
between protein synthesizing processes occurring in prokaryotes and eukaryotes to illustrate
Amino Acid Activation
There are 20 different types of amino acids in the cytoplasm of the cell forming an amino acid pool
and these are picked up one by one for assembly in a polypeptide chain. Activation of amino acid is
a prerequisite for its attachment to the specific tRNA molecule. Each one of them is selected and
bound by a specific enzyme, amino-acyl tRNA synthetase each of which is specific for each amino
acid. Each of the enzyme-bound amino acid reacts with ATP from the cytoplasm and forms amino
Steps in the activation of an amino acid.
acid adenylate-enzyme complex (Fig. 21.5). The enzyme-bound activated amino acid would then
readily react to bind with its specific tRNA. There is an amino acid specific for each tRNA, therefore
there will be 20 different amino acid tRNA complexes.
The activating enzyme has two separate catalytic sites. The activation reaction occurs in two
steps. The first reaction involves a reaction between ATP and the amino acid resulting in the
formation of aminoacyl adenylic acid and pyrophosphate along with AMP. The carboxyl terminal of
the amino acid binds with the 5'-phosphate group of the AMP through anhydride linkage (Fig. 21.5).
The second step involves the transfer of aminoacyl group to the tRNA molecule releasing adenylic
Initiation of the Chain
The process of translation has to ensure that translation does not commence in the middle of mRNA
chain and also correct alignment of the first anticodon with the first triplet codon of the messenger
RNA. In case of wrong alignment the reading frame will be shifted to cause frame-shift mutations.
Hence, chain initiation follows a series of events and includes a process of complex formation in the
The following components are involved:
1. mRNA chain.
2. A special tRNA known as methionyl tRNA (fmet-tRNA).
3. Three protein factors that initiate protein synthesis: IF1, IF2 and IF3.
4. Guanosine trophosphate (GTP).
The process of protein synthesis is perhaps best understood in E. coli, therefore the description
presented here would pertain to this organism unless otherwise stated. It has been well established in