Basic

Chapter 7 Processes

7.1 DNA as the Genetic 7.1 DNA As The Genetic Material

Material
You have studied in previous chapter that characters or
7.2 Prokaryotic and traits are inherited from parents to offspring through
Eukaryotic Gene genes. You are also aware that these genes are present
Organisation on chromosomes which are made up of nucleic acids and
7.3 DNA Replication proteins. However, understanding the nature of gene which
7.4 Gene Expression is responsible for expression of trait was one of the biggest
challenges before the scientific community. Answer to this
7.5 Genetic Code
question came after a few experimental evidences that
7.6 Translation deoxyribonucleic acid (DNA) determines the trait or feature
7.7 Gene Mutation of any organism except a few viruses.
Credit of discovery of DNA goes to Johann Friedrich
7.8 DNA Repair
Miescher, who for the first time isolated an acidic substance
7.9 Recombination from nuclei of pus cells and named nuclein having DNA
7.10 Regulation of Gene and protein. Due to presence in chromosome and nucleus
Expression these two chemical components; nucleic acid (mainly DNA)
and protein became possible candidates to be the genetic
material. Still, the nature of genetic material remained
unknown for a long time. Gradually, experiments with
microorganisms by different investigators yielded results
that provided evidences in favour of DNA as genetic material.

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7.1.1 Discovery of the transforming principle

In 1928, a British medical officer, Frederick Griffith made
an observation in the course of developing a vaccine
against pneumonia caused by bacterium Streptococcus
pneumoniae (also called Diplococcus pneumoniae) in
mammals, which causes pneumonia in humans and is
normally lethal in mice. He identified two different strains
(varieties) of the bacterium i.e. virulent (disease causing)
having a polysaccharide capsule around cell and non-
virulent (harmless). In virulent strain, each bacterium
is surrounded by a polysaccharide capsule because of
which the bacterial colony when grown on an agar plate
appear smooth and are referred to as smooth strain (S).
The non-virulent strain lack polysaccharide coat and
produce rough looking colony and are referred to as
rough strain (R). The S type bacteria kill mice by causing
pneumonia.
Griffith made a series of experiments with S and R type
bacteria (Fig. 7.1). When he injected live S bacteria into
mice, the mice developed pneumonia and died. However,
when he infected mice with R type bacteria mice showed
no ill effects. The results of these two experiments
confirmed that the polysaccharide coat present in S type
bacteria was apparently necessary for virulence.

+
Rough strain Smooth strain Heat-killed Rough strain Heat-killed
smooth strain smooth strain

Mouse survives Mouse dies Mouse survives Mouse dies

Fig. 7.1: Griffith’s transformation experiment

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In order to understand further, Griffith killed some

virulent S bacteria by boiling them and injected the said
heat-killed bacteria into mice. As per his expectations,
mice survived. However, quite unexpectedly, mice died
due to pneumonia when it was injected with a mixture of
heat-killed S bacteria and live R bacteria. Examination
of blood and tissue fluid of the dead mice revealed the
presence of live S type bacteria. Based on the above
observation, Griffith concluded that the R-strain bacteria
must have taken up what he called a ‘transforming
principle’ from the heat-killed S bacteria, which
allowed them to ‘transform’ into smooth-coated bacteria
and become virulent. He called the phenomenon as
transformation, which means transfer of genetic material
from one cell to another that alter the genetic makeup
of the recipient cell. But the nature of transforming
substance still needed to be determined.

7.1.2 Biochemical characterisation of

transforming principle
Three scientists, Oswald T. Avery, Colin Macleod and
Maclyn McCarty conducted a series of experiments
to identify the Griffith’s transforming principle, and
it was confirmed in 1944 that the transforming agent
is DNA (Fig. 7.2). In the design of experiment, they
focused on three main components of smooth strain
of bacteria, i.e., DNA, RNA and protein. They prepared
an extract of heat-killed smooth strain of the bacteria
from which lipids and carbohydrates were removed.
Remaining components of the extract having proteins,
RNA and DNA were retained for further experiment by
dividing the extract into three parts. These extracts
were separately treated with hydrolytic enzymes like
ribonuclease (RNase), deoxyribonuclease (DNase) and
protease to degrade RNA, DNA and protein, respectively,
for their transforming ability by transferring each of the
enzyme treated extracts into three different cultures of
rough strain of bacteria. Transformation of rough strain
into the smooth strain was observed in those colonies
in which RNase and protease treated extract were added
and not in the colony to which DNase treated extract
was added. These results established beyond doubt that
it is DNA which acts as a likely transforming principle.

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Fig. 7.2: Confirmation of transforming principle

7.1.3 The Hershey – Chase experiment

Later on, yet another experiment conducted by Alfred
Hershey and Martha Chase (1952) with T2 bacteriophages
provided evidence in favour of DNA as genetic material.
The virus T2 bacteriophage that infects Escherichia coli
bacteria contains DNA surrounded by a protein coat.
When it infects a bacterial cell, it attaches onto the outer
surface followed by injecting its DNA into the cell. In a
series of their experiments with T2 bacteriophage and
E. coli, the purpose was to establish as to which component
is responsible for multiplication of phage particles, DNA or
protein. To identify easily, T2 bacteriophages were initially
grown with the colonies of E. coli in medium containing
radioactive phosphorous (32P) and radioactive sulfur (35S)
separately (Fig. 7.3). This led to labelling of one set of
bacteriophages with radioactive phosphorous (32P) and the
other set with radioactive sulphur (35S).
35
S and 32P labelled T2 phages were now inoculated
into two separate cultures of unlabelled E. coli bacterial
colony. After infection, the bacterial colonies were agitated

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in a blender for removing any remaining phage and phage

parts from the outside of the bacterial cells. The mixture
of the blender was then centrifuged to separate the
bacteria (present in pellet) from the phage debris (present
in supernatant). Pellets of bacterial culture which showed
radioactivity were infected with phages having radioactive
DNA, whereas, radioactivity was observed in the
supernatant which was infected with 35S bacteriophage.
This indicates that proteins did not enter the bacteria from
the phage. It was therefore, concluded that the material
which enters into bacterial cell, i.e., the DNA can be the
genetic material.
Though the above experiments provided strong evidence
in favour of DNA as the genetic material, it was not clear
DNA molecule is the repository of genetic information.
Subsequent studies made by Erwin Chargaff, Maurice
Wilkins, Rosalind Franklin, James Watson and Francis
Crick led to the discovery of DNA structure, clarifying how
DNA can encode large amounts of information (described
in Chapter 3).

BACTERIOPHAGES

sulfur labeled phosphorus labeled

protein capsule (red) DNA (green)

1. Infection
cell cell

2. Blending

3. Centrifugation

After centrifugation After centrifugation

no sulfur in cells phosphorus in cells

Fig. 7.3: Hershey-Chase experiment

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7.2 Prokaryotic and Eukaryotic Gene

Organisation
It is well understood that traits get inherited from parents
to offspring as ‘gene unit’ and DNA is the genetic material
in all organisms except some viruses (where the genetic
material is RNA). This led to the question about the
organisation of gene, whether this organisation is similar
in prokaryotes as well as eukaryotes and how does it
function at molecular level? Gene is the unit of inheritance
that controls a specific trait or character and may also
be expressed in alternative forms known as alleles. In
other words gene is a segment of DNA that expresses
itself through the synthesis of polypeptide chain via RNA
synthesis, which is known to be the ‘Central dogma’ of
molecular biology.
In the beginning it was established that the traits
or characters are regulated by gene on the basis of the
rediscovery of Mendel’s principle of inheritance in 1900,
and a series of subsequent researches both in plants and
animals established the fact that characters or features
are regulated and controlled by some inherent principle
which are passed on from one generation to the other.
Factor or the inherent unit that controls traits or character
was later given the name ‘gene’ by Wilhelm Johannsen
in 1909.
Understanding the nature and functioning of gene was
one of the main focuses of scientific community during the
early twentieth century. The work of George Beadle and
Edward Tatum during 1930s on the mould Neurospora
crassa helped to establish the relationship of a gene to
control synthesis of one enzyme.
Considering the property that the mould Neurospora
crassa can be very easily grown on medium containing
simple sugar, inorganic salts and vitamin biotin, Beadle
and Tatum experimented on the premise that the organism
can synthesise other essential amino acids and nitrogenous
bases on its own (Fig. 7.4). It was considered to be obvious
that the synthesis is mediated by enzymes, which are
synthesised under genetic control. Design of the experiment
was very simple in which the conidia, i.e., the asexual

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spores were irradiated with X-ray to induce mutation

(Fig. 7.4). The offspring produced by irradiated spores
were identified by growing them on some specific minimal
medium. For the purpose of identifying strains with
mutation, the offspring of irradiated spores were crossed
with wild type and identified mutant strains by growing
the subsequent offspring on minimal medium either for a
specific amino acid or vitamin (culture medium that
contains all amino acids, nitrogenous bases and vitamins
except a specific vitamin or amino acid). Many such
mutations were identified by them and it was genetically
established that each of the mutation, in fact, results in
the non-functioning of a specific enzyme.

Fig. 7.4: Experiment showing detection of mutation in Neurospora

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Subsequently, it was observed that not all proteins

are made up of single polypeptide but more than one
polypeptide chain. The fact that one gene encodes one
polypeptide; the central dogma also got modified from one
gene one protein, to one gene one polypeptide.
Almost simultaneously in early 1940s, the cytological
investigations of chromatin fibre revealed somewhat bead
on a string like structure (Fig. 7.5) through electron
microscopy and it was
readily concluded that
each bead perhaps
represents a gene.
Later investigations
revealed that each
Fig. 7.5: Beads on string structure of bead is a nucleosome
chromatin (containing a core of
histone octamer and a
double stranded DNA of 146 bp) and a string between the
two beads, the linker DNA. It was also established that
each nucleosome with its linker region involves
approximately 200 bp. This cannot be considered to be a
gene, as the size of the genes in many cases has to be
much bigger than 200 nucleotides. The simple reason is
that many proteins have more than 100 amino acid
residues and their corresponding regulatory genes cannot
be less than three times of the same (based on triplet
nature of the codon).
It has now become evident that a gene is the segment of
DNA with specific promoter region, where RNA polymerase
can bind and transcribe mRNA. The transcribed mRNA then
gets involved in the process of translation. The mechanism
is same for all organisms from virus to bacteria, plants and
animals. Are genes of a virus, bacterium or higher organism
similar in their structure and function? The total DNA
content in one complete set of chromosomes (including all
the genes and other parts of DNA) is called a genome. As
we see in the case of viruses or bacteria, the size of genome
is comparatively much smaller than that of eukaryotic
genome. Eukaryotic genomes are much more complex
than prokaryotic genomes. Plant genomes are even more
complex than any other eukaryotic genomes. Estimated
size of genome in various groups of living organisms is
shown in Fig. 7.6.

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Mammals
Birds
Insects
Amphibians
Fishes
Plants
Protists
Fungi
Bacteria
Viruses

Base pairs 103 104 10

5
106 10
7
108 109 1010 1011 1012
3 5 6 7 8
Kilo bases 1 10 100 10 10 4
10 10 10 10 109
3 4
Mega bases 1 10 100 10 10 105 10
6

Fig. 7.6: Genome size variations in various groups of living organisms. Genome sizes are
measured in thousands of nucleotide pairs, i.e., 1000 bp = 1 Kilobase (kb) and 1000,000
bp = 1000 Kb = 1 Megabase (Mb)

In most of the eukaryotes a major part of the genome is

not expressed and remains as non-coding sequence. It has
also been observed that in eukaryotic gene expression the
size of active mRNA involved in the process of polypeptide
chain synthesis is much smaller than the primary
transcript. In fact, many of the eukaryotic genes e.g., the
β-globin gene of haemoglobin after transcription undergoes
the process of splicing in which a few interspersed segment
of primary transcript is excised (introns) and remaining
portions (exons) of the RNA transcript are joined together
to form mRNA.
A eukaryotic cell has two types of genome: (i) nuclear
genome and (ii) organellar genome.

Nuclear Genome
Majority of the DNA is found in the nucleus and is known
as nuclear DNA. In prokaryotes, most of the genome is
composed of coding DNA sequences while in eukaryotic
genomes coding regions makes relatively very small part
of the total genome. For example, size of the human
genome is about 3,000 Mb or 3 billion base pairs of DNA
and estimated to have more than 20,000 genes, which

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constitutes approximately 2% of the total genome. In non-

coding region of the genome, there are sequences which are
repeated thousands to several million times in the form of
tandem arrays. Size and number of these repetitive DNA
sequences in the genome varies significantly.
Nuclear genome in a eukaryotic cell is organised into
smaller condensed unit, known as chromosome containing
linear DNA molecules. One complete set of chromosomes in
a haploid genome of an individual is represented by letter
n. Most of the organisms carry two sets of chromosomes
in each cell and are known as diploid organisms (2n).

Organellar Genomes
In addition to nuclear DNA, few membrane-bound cellular
organelles like chloroplast and mitochondria contain
organelle-specific DNA. Organelle genomes are mostly
circular double stranded DNAs and are present in multiple
copies in each organelle (Fig. 7.7). These replicate in
semi-conservative fashion and are inherited separately
from the nuclear genome. Organelle DNA contains genes
that are required for organelle-specific functions and are
usually uniparental and inherited to next generation
through female gametes.
Chloroplast Nucleus Mitochondria

Outer DNA
membrane Stroma
Ribosome
Inner
membrane Thylakoid Ribosome
Mitochondria
Chromosome DNA
granum
(Nuclear Genome)

Fig. 7.7: Organellar DNA

7.3 DNA Replication

When the three dimensional structure of DNA was
proposed by James Watson and Francis Crick in 1953,
the feature that most excited the biologists was the
complementary relationship of bases of two polynucleotide
chains. Watson and Crick immediately suggested a model
that the basis for copying the genetic information is

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complementarity (For details refer to Unit II, Chapter 2).

According to the model proposed by them, the two strands
of DNA separate during replication; each strand serving as
a template for the synthesis of a new complementary
strand because of the specificity of base pairing (i.e.,
Thymine with Adenine and Cytosine with Guanine). Thus
the parental duplex DNA to form two identical daughter
duplex, each of which consists of one parental strand and
one newly synthesised daughter strand. This form of DNA
replication is called semiconservative replication (Fig.
7.8). The evidence for this mode of replication was provided
by Mathew Messelson and Franklin Stahl in 1958.

A T A T

C G C G

G C G C

A T A T A T A T
C G C G T A T A

G C G C

A T A T Parental Daughter
T A T A
strand strand

Original DNA Original DNA strands T A T

A

separated apart
G C G
C

C G C
G

T A T
A

A T A
T

Fig. 7.8: Semiconservative mode of DNA replication

7.3.1 Messelson and Stahl experiment

To distinguish between old and new strand, Messelson and
Stahl used two isotopes of nitrogen, 14N (the common form)
and 15N (a rare, heavy form). They grew E. coli bacteria in a
medium containing the heavier isotope of nitrogen as the
sole nitrogen source. After several generations, all the E. coli
cells had 15N incorporated into the purine and pyrimidine
bases of DNA. By using density gradient centrifugation, they
observed that the DNA extracted from E. coli grown in 15N
produced a single band at the lower side of the centrifuge
tube, while DNA extracted from bacterial cells grown in 14N
medium formed a band closer to the top. This indicated that
the DNA of E. coli cells grown in 15N medium was denser

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than that of bacteria grown in a medium

containing the lighter isotope (14N) (Fig. 7.9).
Messelson and Stahl then transferred the
E. coli bacteria from the 15N medium to the 14N
medium and collected DNA at various time
intervals as the bacterial cells multiplied (E.
coli divides after every 20 minutes). The DNA
extracted from E. coli cells after first round
of division (generation I) when analysed by
using cesium chloride salt in density gradient
Mathew Messelson and Franklin Stahl confirmed
centrifugation) produced a single band, semi-conservative mode of DNA replication
but at a position intermediate between the
bands of heavy DNA (15N) and light DNA (14N) bands. When
DNA was extracted from E. coli cells, after a second round
of replication in 14N medium (generation II) two bands of
equal intensity appeared in the centrifuge tube, one in the
intermediate position and other at the position expected of
DNA that contained only 14N DNA. When samples of DNA
were collected after additional rounds of replication they
produced two bands. The bands representing light DNA
became progressively thicker but the band at the intermediate
position remained unchanged.

Box 1

Density Gradient Centrifugation

It is a centrifugation technique for separating the molecules from a mixture on the
basis of their density. The centrifugation tube is filled with a heavy salt solution
of cesium chloride (CsCl) and the sample whose density is to be measured. The
centrifuge tube is then allowed to spin in an ultracentrifuge at a very high speed
for several days. The enormous artificial force generated by the ultracentrifuge causes
the Cs ions to migrate towards the bottom of the tube, creating a gradient with high
density at the bottom and low density at the top. The DNA strands float or sink in the
gradient till the density matches the density of the salt.

It is then spun in a A centrifuge tube is lled A density gradient develops within

centrifuge at high speed with a heavy salt solution the tube. Heavy DNA (with 15N) will
for several days and DNA fragments move towards the bottom; light DNA
14
(with N) will remain closer to the top

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Method

Transfer to
14
15
N medium N medium Replication in Replication in
14 14
and replicate N medium N medium

Spin Spin Spin Spin

Result
14
Light N
15
Heavy N

New
Strands
Original DNA (N14)

15 14
Parental Strand (N ) + New Strand (N )

Fig. 7.9: Messelson - Stahl experiment to confirm semiconservative mode of DNA replication
7.3.2 Interpretation of results by Messelson
and Stahl
After the first round of replication, each daughter DNA
duplex was a hybrid having one heavy strand containing
15
N from the parent and one light strand containing 14N
from the medium. When this hybrid duplex replicated, the
heavy template strand formed another hybrid duplex DNA
while the lighter formed light DNA duplex. Thus, Messelson
and Stahl experiment clearly confirmed the prediction of
Watson and Crick model that DNA replicates in a semi-
conservative manner.
Taylor and colleagues in 1958 also conducted similar
experiments in Vicia faba by using radioactive thymidine
to detect the distribution of newly synthesised DNA in the
chromosomes. They also proved that DNA in chromosomes
replicate semiconservatively.

7.3.3 The machinery of replication

In order to carry out the replication of double stranded
DNA molecule it must unwind for separating the strands
to expose the bases. Both DNA strands act as template
for the assembly of complementary bases to form new
polynucleotide strands which are anti-parallel to the
template strand. Several proteins and enzymes are
required for unwinding the double helix and synthesis

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of new strands of DNA. The key enzymes and proteins

involved in the replication of DNA in both prokaryotes and
eukaryotes include —

(i) DNA Polymerases

These are the main enzymes of replication as they
are responsible for synthesising new DNA strands.
They synthesise new DNA strands in 5'→3' direction by
catalysing the formation of phosphodiester bond between
the 3′ hydroxyl group at the growing end of DNA chain and
the 5′ phosphate group of incoming deoxy-ribonucleoside
triphosphate (dNTP).
In prokaryotes, there are three kinds of DNA
polymerases — DNA polymerase I, II, III. DNA polymerase
III is the main enzyme of DNA replication. It synthesises
new strand in 5'→3' direction and by its 3'→5' exonuclease
activity that enables it to correct errors in the growing
chain by removing mis-incorporated nucleotides in 3'→5'
direction. The DNA polymerase I has 5'→3' polymerisation
activity in addition to both 5'→3' and 3'→5' exonuclease
activity. By its 5'→3' exonuclease activity, it removes RNA
primers laid down by primase. The DNA polymerase II is
involved in repair of DNA. In eukaryotes, multiple DNA
polymerases are involved in synthesis of new strands and
repair of damaged DNA.

(ii) Primase
The drawback of DNA polymerase is that it cannot initiate
synthesis of a new strand of DNA. It needs a RNA primer,
an existing segment of nucleotides that provides 3′ OH
group to which it can add a new nucleotide. The primase,
a DNA dependent RNA polymerase synthesises short
RNA oligonucleotide chain (about 10-12 nucleotides long)
called RNA primer in order to get DNA replication started.

(iii) Helicases
In order to generate single strand templates, the DNA
double helix must unwind. DNA helicase breaks hydrogen
bonds that exist between the bases of two strands of a
DNA molecule by using energy in the form of ATP.

(iv) Topoisomerase
As the two strands of DNA separate, torsional strain
causes the DNA helix to coil up forming a knot in front of

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the replication fork. Topoisomerases relieve the tension by

nicking and religating the DNA that holds the helix in its
coiled and supercoiled structure.

(v) Single strand binding (SSB) proteins

After unwinding of DNA by helicase, the single stranded
nucleotide chains have a tendency to form hydrogen
bonds again and reanneal. SSB proteins attach tightly to
the exposed single stranded DNA and stabilise them in
single stranded form for replication to take place.

(vi) DNA ligase

DNA ligase enzyme joins the newly synthesised DNA
fragments by forming phosphodiester bond between 3′OH
group and 5' phosphate group.

7.3.4 Mechanism of DNA replication in bacteria

Replication usually starts at a specific site on the DNA
sequence known as origin of replication (ori) (Fig. 7.10).
In bacteria, DNA is circular and
double stranded. It has single
Old DNA
replication origin (in E. coli called
oriC ) and ends at a specific site,
New DNA knows as terminus. Initiator
proteins bind to origin of replication
and unwind a short section of DNA.
Origin Replication proceed
Further, separation of two strands
of replication (ori) in the both direction on both sides of initial opening is
brought about by helicase, by
Fig. 7.10: Bacterial chromosomes have a single point breaking the hydrogen bonds
of origin present between the bases of two
nucleotide strands. Replication forks are formed on both
sides and they move away from the origin in opposite
direction. This mode of replication is called bidirectional
replication (Fig. 7.10). As DNA synthesis requires single
stranded template, single strand binding proteins stabilise
the single stranded DNA by binding to it. A topoisomerase,
reduces torsional strain generated in front of replication
fork as a result of unwinding of DNA strands (Fig. 7.11).
DNA polymerase III synthesises a new DNA strand in
5'→3' direction, antiparallel to the template strand (Fig.
7.12). DNA polymerase cannot initiate DNA synthesis on a
bare template; rather can add nucleotide to 3'OH group of

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Fig. 7.11: DNA synthesis takes place simultaneously but in opposite directions
on the two DNA template strands

a primer strand. The primase synthesises a RNA primer, a

short stretch of about 10-12 nucleotides long in 5'→3'
direction on template strand in each replication fork at
the origin. The RNA primer provides 3'OH group to which
DNA polymerase add nucleotides. After the formation of
RNA primer on the template strand oriented in 5'→3'
direction in the replication fork, DNA polymerase III
elongates the polynucleotide strand by catalysing the
formation of phosphodiester bond between the 3'OH group
present at the growing end of DNA strand and 5' α
New strand Template strand
5' end 3' end 5' end 3' end

Sugar A T A T
Phosphate
C G C G

G C G C
OH
DNA polymerase III
3' end
A T A
T OH
P
P P P 3' end
P
C Pyrophosphate C
OH
Nucleoside 2Pi
triphosphate 5' end 5' end
Inorganic
phosphate

Fig. 7.12: DNA polymerase catalyses the addition of the 5′ phosphate group
to the 3′ OH group of the previous nucleotide

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phosphate group of incoming deoxyribonucleoside

triphosphate (dNTP). In each step, β and γ phosphates of
incoming dNTP are cleaved and the resulting nucleotide is
added to the 3'-OH group of the growing nucleotide strand.
On the template strand, oriented in 3'→5' direction, the
new strand is synthesised continuously in 5'→3' direction
and is called as leading strand (Fig. 7.13).

Fig. 7.13: Leading strand is synthesised continuously and

lagging strand synthesised discontinuously

Synthesis of new DNA strands on both template strands

in replication fork takes place simultaneously. On the
template strand, oriented in 5'→3' direction in replication
fork, replication proceeds in opposite direction to that of the
movement of replication fork. The primase synthesises a
RNA primer at the replication fork on this template strand.
The DNA polymerase III adds nucleotide to the 3′OH end of
primer and synthesises short stretch of DNA of about 1000
– 2000 nucleotides. As unwinding proceeds, another RNA
primer is formed which is elongated by DNA polymerase.
Synthesis on this template takes place discontinuously in
the form of short segments called Okazaki fragment named
after R. Okazaki, who along with colleagues first identified
them. Each Okazaki fragment begins with an RNA primer.
The DNA strands synthesised discontinuously is called
lagging strand.

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Since during DNA replication, one

strand is synthesised continuously
(leading strand) and the other strand is
synthesised discontinuously (lagging
strand), replication is said to be semi-
discontinuous. Thus, leading strand has
only one RNA primer at its 5' end while
lagging strand has multiple RNA primers
(equal to number of Okazaki fragments).
The question here arises is, how are Termination of
replication
these Okazaki fragments converted into a
continuous DNA strand? DNA polymerase Fig. 7.14: Termination of replication
I by its 5'→3' exonuclease activity
removes nucleotides of RNA primer and
replaces them with complementary DNA
nucleotides by its 5'→3' polymerisation
activity. The DNA ligase joins the Okazaki
fragments by catalysing formation of
phosphodiester bonds between them.
The exact details of the termination
process are not clear, but it is known
that a specific termination site is located
roughly opposite to origin of replication on
the circular DNA molecule. Replication is
terminated whenever two replication forks
meet (Fig. 7.14). A termination protein
binds to termination site and blocks the
Fig. 7.15: Eukaryotic chromosomes have multiple
movement of helicase thus stopping the points of origin
replication fork and preventing further
DNA replication.
The eukaryotic DNA replication resembles
bacterial replication in many respects. Eukaryotes
have multiple chromosomes containing linear,
double stranded, long DNA molecules. Instead
of single origin, each eukaryotic chromosome
has multiple origins and replication forks
are bidirectional. Replication can start
simultaneously from all origins. Several kinds
of DNA polymerases are found in eukaryotes
involved in replication and repair (Fig. 7.15).

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7.4 Gene Expression

As we have already studied that gene is a segment of DNA
which carries biological information for the expression of
a trait. One of the great challenges in understanding gene
is how the gene is expressed. In other words, how is this
information in the form of linear sequence of nucleotides in
a polynucleotide chain converted into the linear sequence
of amino acids in a polypeptide chain?
Every organism from bacteria to human beings has the
same basic mechanism of expression of genes (Fig. 7.16).
The information encoded in the sequence of the four bases
of DNA directs the assembly of amino acids in the correct
order, so as to produce the protein for which the given DNA
sequence is responsible. The DNA inherited by an organism
leads to specific traits by directing the synthesis of specific
proteins. In other words, proteins are the link between
genotype and phenotype. This unidirectional flow of
information from DNA to proteins involves two steps, i.e.,
transcription and translation and is called central dogma.

Fig. 7.16: Central dogma – unidirectional flow of information

During transcription the genetic information is

transferred from DNA to mRNA. In the second step of
central dogma, i.e., translation the information is
transferred from mRNA to polypeptide chain. Why would
the cell want to have mRNA as an intermediate between a
gene present in DNA and the peptide it encodes? First of
all, the information present in gene can be amplified by
having many copies of mRNA made from a single copy of
DNA. Second, in eukaryotes, DNA is present inside nucleus
and ribosomes, the protein factories are present in

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cytoplasm. Once the mRNA is synthesised as a

complementary copy of DNA strand (gene), it moves into
cytoplasm. In the cytoplasm, it serves as a template for
the synthesis of polypeptide chain in ribosome (Fig. 7.17).

Fig. 7.17: Gene expression in prokaryotes and eukaryotes

In some viruses like retroviruses, the genetic material

is RNA instead of DNA. The information present in genetic
RNA is transferred to a single stranded complementary
DNA strand which is then converted into double stranded
DNA. This process is called reverse transcription.
After the formation of DNA, the genetic information flows
to mRNA and then to polypeptide chain (Fig 7.18).

Fig. 7.18: Reversal of central dogma of molecular biology

7.4.1 Transcription
All kinds of cellular RNAs such as messenger RNA (mRNA),
transfer RNA (tRNA), ribosomal RNA (rRNA) are synthesised
on DNA template. Synthesis of complementary RNA

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polynucleotide chain on DNA is called transcription. The

transcription is in many ways similar to the process of
replication. One fundamental difference is that during
replication, the entire DNA template is copied to DNA
molecule but, during transcription, only small parts of the
DNA are copied into RNA. The DNA strand whose base
sequence is identical to that of the transcribed RNA (except
for T in DNA to U in RNA) that carries genetic information
is called sense or coding strand. The other DNA strand on
which RNA is transcribed and whose nucleotide sequence
is complementary to that of the transcribed RNA is called
the template or non-coding strand (Fig. 7.20). Thus,
within a gene, only one of the nucleotide strands is normally
transcribed into RNA.

7.4.2 RNA polymerase

The enzyme responsible for transcription in both prokaryotes
and eukaryotes is DNA-dependent RNA polymerase. In
prokaryotes there is a single type of RNA polymerase that
catalyses the transcription of all types of RNA (mRNA,
rRNA and tRNA). In eukaryotes, three distinct types of RNA
polymerases are present. RNA polymerase I transcribes
28S, 18S and 5.8S; RNA polymerase II transcribes hnRNA
(heterogenous nuclear RNA, the precursor of mRNA) and
RNA polymerase III transcribes tRNA and 5S rRNA. RNA
polymerase enzyme do not require a primer for initiating
synthesis of polynucleotide chain and to synthesise new
chain in 5'→3' direction using ribonucleoside triphosphates
(rNTPs) as substrates.

 
 

  

Sigma factor

Core enzyme Holoenzyme

Required for Required for correct

polymerisation activity initiation of transcription:
Binding to promoter

Fig. 7.19: Prokaryotic RNA polymerase

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The prokaryotic RNA polymerase is a large enzyme

comprising of two α, one β and one β' subunits (Fig. 7.19).
These subunits together constitute core enzyme (α2, β, β').
When core enzyme associates with a sigma factor it forms
holoenzyme (α2, β, β', σ).

7.4.3 Transcription unit

The stretch of DNA on which RNA is transcribed is called
a transcription unit. How does the RNA polymerase
recognise a transcription unit? How does it know which
DNA strand to transcribe and where to start and stop?
Each transcription unit has a start site from where
transcription start and a terminator site where transcription
is to end (Fig. 7.20). Upstream to the start site is a DNA
sequence called promoter, which is recognised by the RNA
polymerase and binds to it for accomplishing initiation of
transcription. In addition to providing the binding site for
the polymerase, the promoter also tells the polymerase
where to start synthesis and in which direction to proceed.
Within promoter is a most common consensus sequence
TATAAT called Pribnow box in bacteria where initial
melting of duplex DNA takes place.
Transcription Unit
Coding/sense
strand Promoter RNA-coding region

5' 3'
DNA
3' 5'

Template strand Transcription Terminator Transcription

(Non-coding strand) start site termination site

RNA transcript 5' 3'

Fig. 7.20: A transcription unit

7.4.4 Initiation
The binding of RNA polymerase to the promoter is the first
step in transcription. In bacteria, sigma subunit of RNA
polymerase recognises promoter and binds to it. Once
bound to promoter, the RNA polymerase holoenzyme
begins to unwind the DNA helix at the TATAAT Sequence.
It unwinds a DNA segment approximately 17 base pairs
long. To begin the synthesis of an RNA molecule, RNA
polymerase pairs the base of an incoming ribonucleoside

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triphosphate with complementary base at the start site on

the DNA template strand. No primer is required to initiate
the synthesis of the 5' end of the RNA molecule. The next
ribonucleoside triphosphate complementary to the second
nucleotide of template strand is added to the 3′ OH end of the
first RNA nucleotide by a phosphodiester bond catalysed by
RNA polymerase. During this, the two (β and γ) of the three
phosphate groups are cleaved as pyrophosphate from the
incoming ribonucleoside triphosphate. The sigma subunit
is usually released after initiation.

7.4.5 Elongation
The region containing the RNA polymerase, DNA and
growing RNA transcript is called transcription bubble
because it contains a locally unwound ‘bubble’ of DNA (Fig.
7.21). The RNA polymerase moves along the template strand

Coding strand Polymerase movement

Rewinding RNA
of DNA Polymerase Unwinding of DNA

3' 5'
5' 3'

Template strand Nucleotide being added

to the 3' end of the RNA
RNA
5'
NTPs
RNA-DNA
hybrid region

Fig. 7.21: Elongation of RNA chain by RNA polymerase

Fig. 7.22: Addition of rNTP to the 3'OH end of the growing

polynucleotide chain

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progressively and unwind the DNA at the leading edge of

the transcription bubble. It joins nucleotides to the 3'OH
end of RNA molecule according to the sequence of the
template (Fig. 7.22). As the elongation of RNA chain
continues, the new RNA molecule peels away from the DNA
template and the DNA double helix reforms behind
transcription bubble. The rate of transcription in bacterial
cells at 370C is about 40 nucleotides per second.

7.4.6 Termination
The elongation of RNA chain continues till the RNA
polymerase reaches the terminator sequence in DNA.
The RNA-DNA hybrid helix within transcription bubble
dissociates as RNA polymerase reaches terminator
sequence. The RNA polymerase separates from template
DNA, the two strands of DNA rewind and newly synthesised
RNA chain gets released. In prokaryotic termination of
transcription, RNA polymerase requires a rho protein (rho
dependent termination) in some cases, while in others it is
terminated with rho independent termination.
In prokaryote, a group of genes is often transcribed
into a single RNA molecule, called polycistronic RNA. It is
produced when a single termination sequence is present
at the end of a group of several genes that are transcribed
together. In prokaryote, since the mRNA does not
require any processing to become active, and also since
transcription and translation take place in the cytoplasm,
many times the translation can begin much before the
mRNA is fully transcribed.

7.4.7 Transcription in eukaryotes

The basic mechanism of transcription by RNA polymerase
is the same in eukaryotes as in prokaryotes. However,
a number of differences in the process of transcription
occur between prokaryotes and eukaryotes. A number of
accessory factors known as trascription factors are required
for proper initiation of transcription by RNA polymerase I,
II and III at promoters of rRNA, mRNA and tRNA genes
respectively. Most of the genes in eukaryotes are split genes
containing introns (non-coding regions) in between exons
(coding regions). The primary transcripts contain both
the exons and the introns. The primary transcripts called
heterogenous nuclear RNA (hnRNA) undergo processing

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RNA-coding sequence

DNA

Promoter
Transcription by RNA polymerase II.
Addition of 5' cap

Addition of 3' poly (A) tail.

Cap Exon Intron Exon Intron Exon Poly (A) tail

Pre-mRNA 5' AAAAAAA...3'

5' UTR RNA splicing: 3' UTR

introns removed

Protein-coding sequence

mRNA 5' AAAAAAA...3'

Translation

Polypeptide

Fig. 7.23: Post-transcriptional modifications of pre-mRNA

before getting transferred into cytoplasm from nucleus

called post-transcriptional modifications (Fig. 7.23).
1. Capping
In eukaryotic pre-mRNA, the first nucleotide is a purine (A
or G) at the 5' end. The 5' end of the pre-mRNA is modified
by the addition of GTP called a 5' cap. The guanine in the
GTP is also modified by the addition of a methyl group
often called 5' methyl G cap. The cap protects 5' end of
mRNA from degradation by exonuclease.
2. Splicing
The primary transcripts or pre-mRNA has both exons
and introns. During processing of pre-mRNA, the introns
are cleaved and exons are joined (spliced) together. This
process is called splicing.
3. Poly-A tail
After the transcription of pre-mRNA, a series of adenine

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nucleotides are added to the 3' end called poly-A tail or

poly-adenylated tail. The poly-A tail appears to play a role in
the stability of mRNA by protecting them from degradation.

7.5 Genetic Code

In case of replication and transcription, a polynucleotide
chain (template strand) is copied to form another,
polynucleotide strand, i.e., a DNA strand or an RNA strand
respectively. These processes are easy to conceptualise
on the basis of complementarity. But in the process of
translation, genetic information is transferred from a
polymer of nucleotides (mRNA) to a polymer of amino
acids. No complementarity exists between nucleotides
and amino acids. The question arises how the sequence of
four bases in mRNA specifies the amino acid sequence of
a polypeptide? Evidences suggest that a minute alteration
in the nucleotide sequence is accompanied by a change
in the amino acid sequences of the polypeptides. This led
to the proposition of a genetic code that could direct the
amino acid sequence during protein synthesis.
The proposition and deciphering of the genetic code
was indeed challenging as it necessitated the collective
involvement and efforts of physicists, organic chemists,
biochemists and geneticists. In 1961, Francis Crick
hypothesized the existence of a genetic code and suggested
base sequence as the carrier of genetic information. How
many nucleotides are necessary to specify a single amino
acid? Since there are twenty different amino acids and
only four different bases, it would be logically impossible
for each amino acid to be specified by only one nucleotide.
Similarly, combination of two nucleotides could also
specify sixteen amino acids as there will be 16 codons.
George Gamow, a physicist, argued that since there are
4 bases and if they have to code for 20 amino acids, the
code should constitute a combination of bases. Further
he added that in order to produce 20 amino acids there
should be 3 nucleotides constituting a genetic code. This
was indeed a bald proposition, because a permutation
combination of 43 gives 4×4×4 that would generate 64
codons, which is more than enough to specify 20 different
amino acids.
The next major step was to determine which groups of
three nucleotides specify which amino acids. In 1961, the

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Second letter
U C A G
UUU Phe UCU UAU Tyr UGU Cys U
UUC UCC UAC UGC C
U Ser
UUA UCA UAA Stop UGA Stop A
Leu
UUG UCG UAG Stop UGG Trp G
CUU CCU CAU His CGU U
CUC CCC CGC C
C Leu Pro CAC Arg
First letter

Third letter
CUA CCA CAA Gln CGA A
CUG CCG CAG CGG G
AUU ACU AAU Asn AGU Ser U
AUC ACC AGC C
A lle Thr AAC
AUA ACA AAA Lys AGA Arg A
AUG Met ACG AAG AGG G
GUU GCU GAU Asp GGU U
GUC GCC GAC GGC C
G Val Ala Gly
GUA GCA GAA Glu GGA A
GUG GCG GAG GGG G

Genetic Code
Fig. 7.24: Genetic code

experiment conducted by Marshall Nirenberg and Johann

H. Matthaei deciphered the first codon. They synthesised
an artificial mRNA by linking RNA nucleotides containing
uracil as their base. The poly (U) RNA was added to a test
tube containing all twenty types of amino acids, ribosomes
and the other components required for protein synthesis.
In each test tube, a particular amino acid was made
radioactive. A radioactive polypeptide chain was detected
in one of the test tubes containing phenylalanine amino
acids. Thus, Nirenberg and Matthaei determined that the
mRNA codon UUU specifies the amino acid phenylalanine.
The results of similar experiments using poly (C) and poly
(A) RNA demonstrated that CCC codes for proline and AAA
codes for lysine. The experiments of Nirenberg, Matthaei,
Leder, Ochoa and H. G. Khorana helped in deciphering all
64 codons of genetic code.
The salient features of genetic code are as follows:
1. The codons are triplet and there are 64 codons (Fig. 7.24).
2. Out of 64 codons, 61 codons code for 20 amino acids.
The remaining three codons UAA, UAG and UGA do
not code for any amino acids and are used to signal
the termination of protein synthesis.

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3. AUG has dual functions. It codes for methionine and

also acts as an initiator codon.
4. The genetic code is unambiguous, i.e., one codon
codes for only one amino acid.
5. The genetic code is degenerate, which means that
each amino acid may be specified by more than one
codon. Only methionine and tryptophan are encoded
by a single codon each.
6. The genetic code is non-overlapping. Each base along
the mRNA is a part of only one codon.
7. The genetic code is universal; for example, from bacteria
to human GUG would code for valine. Some exceptions
to this rule have been found in mitochondrial codons,
and in some protozoans.

7.6 Translation
So far you have learnt that genetic information present in
DNA is transcribed into mRNA. Thus, mRNA carries genetic
information for the sequence of amino acids of specific
polypeptide chains in the form of codons. In ribosomes,
information present in the language of codons 3'
is decoded into language of amino acids which A
join to form a polypeptide chain. The process C
C
of synthesis of polypeptide chain on an mRNA
bound to ribosome is called translation. 5'

The translation process can be divided into tRNA

four steps:
(i) the charging of tRNA
(ii) the initiation of translation
(iii) elongation
(iv) termination

Charging of tRNA Anticodon loop

The tRNA molecules deliver amino acids on to

ribosomes. Three bases present in anticodon Anticodon
region of a tRNA recognise specific base of a CUC
GA G
codon of mRNA and get paired with them (Fig. G UC CA G CCA UA G
7.25). For example, the mRNA codon GAG 5' 3'
mRNA
contains information for glutamic acid. The Codon
anticodon of tRNA that base pairs with codon
Fig. 7.25: Anticodon of tRNA
GAG by hydrogen bonding is CUC and carries recognises and base pairs with
glutamic acid at its other end (3' end). During codon of mRNA

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translation, when an mRNA molecule moves through

ribosome, glutamic acid is added to the polypeptide chain
whenever GAG is presented for translation.
Each tRNA molecule carries a specific type of amino
acid. The tRNA molecules are named according to the amino
acid they carry. For example, the tRNA carrying methionine
is called methionyl tRNA or tRNAmet. Similarly tRNA that
carries serine is called tRNAser. In genetic code, 61 codons
code for 20 amino acids. For 61 codons there should be 61
different tRNA molecules with different anticodon in the
cell. However, the number of tRNA
Leu Leu molecules is much less than 61.
3' 3' Hence, the anticodon of one tRNA
5' 5'
molecule can recognise more than
Identical one codon on mRNA and base pairs
leucine with it. But how does the anticodon of
tRNAs
one tRNA molecule recognise more
than one codon? The base pairing
between codon and anticodon follow
G A G G A G Watson and Crick base pairing, i.e., A
Normal
pairing
Wobble
pairing
pairs with U and G with C. The pairing
C U C C U U is precise in first two positions while
mRNA
5' 3 5' 3 it is flexible at third base of the codon.
Fig. 7.26: Wobble pairing at third position of codon This unusual pairing at third base
position is called wobble pairing
(Wobble hypothesis) (Fig. 7.26).
Amino acids are first activated in the presence of ATP
by aminoacyl-tRNA synthetase and transferred to its
specific tRNA molecules (Fig. 7.27). A cell has 20 different
Aminoacyl-tRNA
synthetase
Amino acid
binds
Amino acid ATP binds A
ATP loses two
A P phosphate groups
P P P P P and binds to the
ATP Pyrophosphate amino acid as AMP

P P
Phosphate
C A A

tRNA

A
A
C

A
P
aa-AMP-enzyme
C A A tRNA
tRNA released tRNA binds
Aminoacyl tRNA AMP released
(an activated
amino acid)
aa-AMP-enzyme

Fig. 7.27: Activation of amino acid and transfer to its specific tRNA to form aminoacyl-tRNA
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aminoacyl-tRNA synthetase enzymes, one for each of the

20 amino acids. Each synthetase recognises a specific
amino acid and transfers it to a specific tRNA molecule.
Before starting of protein synthesis in ribosomes the amino
acids are activated and transferred to their appropriate
tRNA molecules. This is called charging of tRNA and
involves two steps.
In the first step, amino acid reacts with ATP in the
presence of aminoacyl tRNA synthetase producing
aminoacyl-AMP and PPi. In the second step, the amino
acid is transferred to the tRNA. The activated amino acid
is attached to the hydroxyl group of terminal adenine
nucleotide present at the 3'OH end of the tRNA (CCA)
sequence. The charged aminoacyl tRNA enters the
ribosome.

Initiation of Translation
In the previous unit we have studied about prokaryotic
(70S) and eukaryotic (80S) ribosomes and their subunits.
There are three sites on a ribosome for binding of tRNA
molecules: the A site (aminoacyl tRNA binding site), P site
(peptidyl tRNA binding site) and E site (tRNA exit site).
Aminoacyl-tRNA molecules enter into A site one after
another during translation.
During initiation, the 30S subunit of ribosome in
prokaryotes binds to ribosome binding sequence present at
the 5' end of mRNA (Fig. 7.28). As a result, the initiation
codon AUG (codes for methionine) is positioned in the P site.
The initiating amino acid methionine in prokaryotes
is formylated (formylated methionine - fMet) but it is not
formylated in eukaryotes. The initiating charged tRNA or
aminoacyl-tRNA molecule in prokaryotes is fMet-tRNAfmet (in
eukaryotes—Met-tRNAmet) which attaches to the initiation
codon AUG present in the P site. The 3' UAC 5' anticodon of
fMet-tRNAfmet base pairs with 5' AUG 3' codon of mRNA. The
50S subunit then associates with 30S subunit to form 70S
initiation complex. The formation of this initiation complex
also requires the activity of certain protein factors known as
initiation factors and GTP. Hence, in the initiation complex,

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we have the initiator aminoacyl-tRNA (fMet-tRNAfmet) in the

P site, while the A site is empty, awaiting delivery of the
second aminoacyl tRNA.
The initiation in eukaryotes is almost similar with some
important differences. The small subunit of eukaryotic
ribosome (40S) recognises the cap with the help of initiation
factors, binds to it, and then moves along the mRNA until
it locates the initiating AUG codon.

AA1

tRNA carrying
rst amino acid

UAC
Anticodon Ribosomal
Large subunits
AA1

Small 5' 3'

AUG
INITIATION
5' 3'
AUG mRNA 1. During initiation, the
UAG components of the translational
Start codon Stop codon apparatus come together with an
mRNA, and a tRNA carrying the
rst amino acid (AA,) binds to the
start codon (AUG).
ELONGATION
2. During elongation,
amino acids are brought to
AA1
the mRNA by tRNAs and
AA1 are added, one by one, to a
AA1
growing polypeptide chain.
AA1 AA1

Release 5' 3'

Completed AUG
polypeptide factor

5' 3'
UAG
Stop codon

Fig. 7.28: Translation process in prokaryotes

Elongation
The next step in protein synthesis is elongation, in which
amino acids are joined to create a polypeptide chain. In the
initiation complex, P site is occupied by amino acyl-tRNA
with formylated methionine (fMet-tRNAfmet) in prokaryotes
and methionine (Met-tRNAmet) in eukaryotes (Fig. 7.29).
The A site is vacant. Now a second aminoacyl-tRNA enters
into A site with appropriate anticodon that base pairs

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with the mRNA codon. CH3

S
A peptide bond is now CH2

formed between the amino H CH O

2
O
acids that are attached to H
C H C C N
H C
2CH OH
H C O
tRNA at P and A sites. The
OH O
peptide bond is formed 3'
5' 5'
between the carboxyl Shine-Dalgarno
group of amino acid sequence

bound to initiator tRNA mRNA

UA C A G G
AU G UC C A A G G C C C U U
in the P site and the free 5' 3'
amino group of the amino
acid attached to tRNA in
the A site. This reaction Fig. 7.29: Formation of peptide bond between initiating amino
is catalysed by peptidyl acid (fMet) and second amino acid
transferase enzyme. The
formation of peptide bond releases the amino acid in the P
site from its tRNA. Thus A site has a dipeptidyl-tRNA and P
site contains an uncharged tRNA (without amino acid).
The ribosome now moves along mRNA in 5'→3'
direction three nucleotides at a time. This movement is
called translocation. The movement brings the uncharged
tRNA from P site to E site from where it is ejected from
ribosome. The peptidyl-tRNA with growing polypeptide
chain moves from A site to P site. A site of ribosome is now
again vacant with a new codon of mRNA. It is now ready
to receive the next aminoacyl-tRNA molecule specified by
the codon. The entire process is repeated and elongation
of polypeptide chain takes place. Several protein factors
called elongation factors and GTP are involved during the
elongation step.

Termination
Elongation of polypeptide chain continues until a stop
codon on the mRNA enters the A site of the ribosome.
The three stop codons—UAA, UAG and UGA do not code
for any amino acid. There are no tRNAs with anticodons
complementary to these stop codons. No tRNA with amino
acid enters into A site of ribosome when termination codon
occupies it. The protein factors called release factors
recognise stop codons and binds to A site (Fig. 7.30). The
release factors then release the polypeptide chain from
tRNA in the P site. Other protein factors bring about the

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Fig. 7.30: Release factor recognise stop codon and terminates translation process

release of tRNA from the P site, mRNA from ribosome and

finally the dissociation of ribosome.
Proteins synthesised in ribosomes then undergo post-
translational modifications (PTMs) to form the mature
protein product. Such modifications come in a wide
variety of types, and are mostly catalysed by enzymes that
recognise specific target sequences in specific proteins.

Polyribosomes
A single mRNA is translated simultaneously by several
ribosomes producing many copies of a polypeptide chain.
When the first ribosome attached to mRNA translocates far
enough past the start codon, a second ribosome attaches to
the same mRNA, eventually resulting in a number of
ribosomes attached to mRNA called polyribosomes
(Fig. 7.31). It is found in both prokaryotes and eukaryotes.
Completed
Polypeptide

Growing
Polypeptide
Incoming
ribosomal
subunits
Dissociated
ribosomal
Polyribosome subunits
Start of mRNA End of mRNA
(5' end) (3' end)

Fig. 7.31: Several ribosome bind to an mRNA during translation forming

polyribosome

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7.7 Gene Mutation

You have understood the fact that traits or characters
in an organism are regulated or controlled by genes,
which are a part of the DNA of the chromosomes. Traits
are faithfully inherited in the form of coded information
through these genes present on the DNA or chromosomes
from parents to the offspring. All the mechanisms and
processes such as DNA replication, transcription and the
distribution of chromosomes during the process of cell
division (mitosis or meiosis), etc. are extremely precise
and accurate under the control of specific enzyme. Yet
there are possibilities that some error may occur during
these molecular processes leading to changes in the
chromosomal organisation and molecular structure of the
DNA carrying genes to be transmitted. These changes are
broadly classified as mutation or sudden changes in the
genetic material. Thus, it is now clear that changes may
take place in the carrier of genetic information, i.e., DNA
or chromosome both in somatic as well as germinal cell.
However, such a change unless occurring in germinal cells
and getting inherited into offspring has no significance.
Let us now examine different categories of changes
or modifications that may happen. Among eukaryotes,
chromosomes present in a germinal cell carries the
hereditary information from parents to offspring. Hence,
any change, either in the structure of a chromosome,
(chromosomal aberration) or overall number of
chromosome (ploidy) is categorised to be chromosomal
mutation. Aberration may take place either due to loss
of a part or addition of some part in a chromosome. Even
rearrangement of chromosomal segment either within a
chromosome or between two chromosomes are categorised
as chromosomal aberration. Many extraneous factors like
ionising radiations or some chemicals may induce such
aberration in chromosomes called mutagen. You will
appreciate that all such rearrangements can be identified
by either specific chromosome staining technique called
banding or fluorescence in situ hybridisation (FISH),
which you will study later in Unit V. Similarly, there
may be some exceptional situations in which the overall
number of chromosomes (which remains constant from
one generation to other) may get changed either by

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increase or decrease by one or both the homologues. Such

a situation is described in the category of aneuploidy and
you will find later that this is responsible for different
types of hereditary syndromes observed in human beings.
Likewise, change in number may occur by multiplication
of the complete haploid set in such a way that the number
increases to that of 3n, 4n or even more which is called
polyploidy.
You may be thinking now that if such a change at
chromosomal level may take place or be induced, there may
be possibility that changes in the genetic material, i.e., DNA
or RNA may also occur at molecular level. Bacteria has only
a circular DNA or each of the chromosomes of a eukaryote
contains DNA and all are involved in the process of making
its copy by the process called replication before mitosis or
meiosis. Any error happening during replication or by other
means may alter the reading frame of the genetic code of
one or the other gene and may alter the code and thus
may affect the trait encoded by the gene. Such a change
in the genetic material at molecular level is categorised to
be gene mutation or point mutation. Sickle cell anaemia
is one such example of mutation in which substitution
of one nucleotide results in the formation of abnormal
sickle haemoglobin in human RBC and consequently the
disease. Extraneous agent (mutagen), physical (ionising
radiation, UV rays), chemical or biological (viruses) may
induce gene mutations. We will focus our attention mainly
on gene mutation.
It is now clear that alteration in the genetic material,
i.e., DNA (RNA in case of a few viruses) may occur during
the molecular processes happening. It has been observed
that there are some intrinsic properties of the molecule or
process that may result into changes in DNA or gene from
the point of view of structural organisation at molecular
level. These changes can be categorised in three different
groups, i.e., addition, deletion and substitution of one or
a few nucleotides. Among these, the two types of changes
namely addition and deletion change the entire reading
frame of nucleotide sequence on the DNA molecule. The
impact of such a change can be understood by the fact
that the changed coding of DNA may change its expression
during RNA transcription and ultimately during the
polypeptide chain synthesis. Obviously, the protein

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synthesized by such modified gene may not be a normal

one or even the specific protein may not be synthesised.
The change in the gene may also happen due to
substitution or replacement of single nucleotide. The
replacement of nucleotide may take place in which a
purine base is replaced by another purine base. Same may
happen for pyrimidine base too. Such a substitution type
of mutation is known as transition. Similarly, a purine
base may be replaced in a gene by a pyrimidine base or its
vice versa, which is called transversion (Fig. 7.32).

T
KEYS:

Purine

Pyrimidine
A G
Transition

Transversion

Fig.7.32: Different substitution mutations

7.7.1 Molecular mechanism of mutation

As discussed earlier, mutations may happen spontaneously
due to the intrinsic properties of the molecule or they may
even be induced by external agents called mutagens.

Spontaneous mutations — DNA carries the genetic

information in the form of nucleotide. Among these
nucleotides, there are functional groups present either in
C = O or C – NH2 form, normally called Keto or amino form
respectively. However, in these forms of nitrogenous bases
hydrogen atom may shift from one atom of the molecule to
the other atom within the base. Such a phenomenon is
known to be the tautomeric shift and results in to a
temporary phase of the nitrogen base called either enol (C
- OH) or imino (C = NH) form. These rare tautomeric forms
of nitrogenous base have changed property to pair with
other nucleotide in the DNA molecule. Therefore, at the
time of replication, when the imino form of guanine is

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present in the DNA, it makes a complementary pairing

with thymine nucleotide whereas the former would have
normally paired with cytosine. During the next replication
cycle, place where thymine is wrongly added may normally
pair cytosine leading to the substitution of G  C pair in
gene by A = T pair (Fig. 7.33). We have discussed the
consequence of single base substitution in case of sickle
cell anaemia.
5' 3'
A C G T C
3' T G C A G
5'
3' 5'
A C G T C
Wild type
T G T A G
3' 5'
5' 3'

Rare enol tautomeric A C A T C

form of guanine (G*) 3' T G T A G
3' 5'
C

Mutant
5'
T

G
G

3' 5'
A

5'
T

A C G T C A C
DNA
T G C A G T G replication 5' 3'
C

3' 5' 3' A C G T C

T
G

3'
C
A

DNA T G C A G
G

1 2
replication 3' 5'
5' Wild type

5' 3'
5' 3'
A C G T C
A C G T C
T G C A G
T G C A G
3' 5'
3' 5'
Wild type
3
4
First-generatiom
progeny Second-generation
progeny

Fig. 7.33: Showing spontaneous induction of substitution mutation

Induced Mutation — It is after the use of Hermann J.

Muller’s experiments of inducing mutation using X-ray, a
new area of induction of mutation using various external
agents were opened. These mutagens fall in the categories of:
• Physical— Radiations like X-ray, UV rays, etc.
• Chemical— Alkylating agents like Mustard gas,
ethyl methane sulfonate (EMS); base analogs like
5-bromouracil or 2-aminopurine; the deaminating
agents like nitrous acid, etc.

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Replication I Replication after

Incorporation

G C A T

Replication I Replication I

G BU G C A BU A T

Replication II Replication II

G C A BU A T G BU

Replication III Replication III

A T A BU G C A BU

G C A T A T G C

Or, total A T G C

Fig. 7.34: Effect of 5-bromouracil on DNA replication

In order to understand the mechanism of induction of

mutation by external agent let us try with a few examples.
Any cell, especially those responsible for the formation
of gamete when gets exposed to ionising radiation of
X-ray it may induce breakage of different bonds present
within the molecule. In case such a breakage occur in the
phosphodiester bond of DNA, it may result into the loss
of a few nucleotides in it. This may result into the frame
shift mutation due to deletion of one or a few nucleotides.
Consequence of such a change in the reading frame of the
genes is understandable as it may lead to a wrong RNA

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transcription and non-expression or altered expression of

the gene. Even non-ionising radiation like UV rays may
lead to the excitation of electron within the DNA molecule.
These excited molecules may become more reactive and
may lead to the induction of deletion or substitution type
of mutation.
Similarly, base analog like 5-bromouracil when present
in its keto form may get incorporated preferentially in the
DNA as a complementary base against adenine. However,
in the eventuality of its conversion into tautomeric enol
form 5-bromouracil may pair with guanine nucleotide
resulting into the substitution of A=T pair into G  C pair
(Fig. 7.34).
Alkylating agent like EMS ethylates DNA nucleotide
either at nitrogen at 7th position or oxygen at 6th position.
Such an alkylation alters the pairing property. For example,
the ethylated guanine nucleotide pairs with thymine,
which means a G  C pair in DNA will be mutated to A=T
pair. Many such chemical mutagens induce mutation by
the process of altered pairing property.

7.8 DNA Repair

From the above account on mutation it may perhaps be
understood that the rate of mutation either spontaneous or
induced must be very high. But such a high rate of mutation
is not observed. Also, a very high rate of mutation is not
expected considering the fact that the genetic material gets
transferred in a faithful stable way. Understanding of the
molecular processes in various organisms has revealed
that mechanisms exist so that most of the errors that
occur are corrected also. Many of such mechanisms have
been identified in the bacterial system E. coli. Out of these
we will see a few to understand the mechanism.
Excision repair — This is a mechanism in which
altered or modified bases in the DNA are removed or
excised sequentially by identifying the altered base and
subsequently removing them by enzymatic binding. Gap
thus created is eventually filled by a DNA polymerase
enzyme for which the unaltered strand is used as template.
One such mechanism involves recognition of altered bases
by enzyme DNA glycosylase, which specifically recognises
either deaminated or oxidised bases followed by cleaving of

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bond between the altered base and the deoxyribose sugar.

As a result, a site without any nucleotide is created in one
of the strands of DNA called AP site (which stands either
for apurinic or apyrimidinic site). A specific enzyme AP
endonuclease detects this AP site and removes the sugar-
phospate group creating a gap at the site. Lastly, a DNA
polymerase fills the gap by placing the correct nucleotide
as per the complimentary strand followed by joining the
nick by DNA ligase enzyme (Fig. 7.35). Such an excision
repair is also called base excision repair.
There exist other mechanisms of the excision repair
in which comparatively larger portions of altered portions
of DNA can be repaired. One such example is the repair
of damage formed by thymine dimer. Such a dimer may
be formed as a covalent linkage between carbons of two
adjacent thymine nucleotides due to photochemical
reaction caused by ultra violet rays. Consequences of such
a dimer formation can be easily understood by the fact
that, it may induce a deletion type of frameshift mutation
as the dimer would not be able to pair with any nucleotide
during the next replication cycle. The mechanism known
as nucleotide excision repair for this type of damage
is slightly complex, in which a specific trimeric protein
called Uvr (Uvr stands for ultra violet repair) protein
recognises and binds to the dimer site and bends it. Two
units of UvrA polypeptide of the trimeric protein leaves
the site and another protein UvrB forms a complex with
the DNA molecule at the damaged site and breaks the 5th
phosphodiester bond towards the 3' end. Another protein
called UvrC also acts on the damaged site and breaks the
8th phosphodiester linkage towards 5' end. Thus a portion
of 12 nucleotides are excised from the DNA strand having
damaged dimer. This 12 nucleotide long gap is filled by
DNA polymerase I followed by DNA ligase sealing of the
nick (Fig. 7.36).
Mismatch repair — Sometimes in the process of DNA
replication, wrong nucleotide is likely to be incorporated.
There exist a mechanism through which the same
is corrected in four different types of proteins MutH,
MutL, MutS and MutT. This repair process also helps in
proofreading of the replication. Mismatch is recognised by
MutS and subsequent to its binding MutH and MutL protein
also binds forming a complex. The specific endonuclease

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5' 3'
A G C T G C C C T A G C T T
T C G A C G G G A T C G A A
3' 5'

STEP 1 Deaminated Cytosine

5' 3'
A G C T G C U C T A G C T T
T C G A C G G G A T C G A A
3' 5'

Binding of uracil
STEP 2 DNA glycosylase

Glycosylase
5' 3'
A G C T G C U C T A G C T T

T C G A C G G G A T C G A A
3' 5'

STEP 3 Uracil Excision

AP site

5' 3'
A G C T G C C T A G C T T
T C G A C G G G A T C G A A
3' 5'

AP endonuclease and
Missing phosphodiesterase
STEP 4
nucleotide removes sugar-phosphate

5' 3'

3' 5'

Single-strand
STEP 5
break DNA polymerase

5' 3'

3' 5'

STEP 6 Site of repair

DNA ligase

5' 3'

3' 5'

Fig. 7.35: Base excision repair mechanism

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5' 3'

3' 5'

STEP 1 Trimer protein Uvr having 2 units

of A and 1 unit of B binds with A A
damaged DNA
B

5' A A 3'
B
3' 5'

DNA bends using energy from ATP

and conformational change in the
UvrB protein; UvrA dimer released
STEP 2 ATP

Excinuclease activity
ADP + Pi A A

A A
B
5' 3'

3' 5'
Binding of UvrC protein with
UvrB and cleaving of 3' and
5' phosphodiester bonds
C
STEP 3 respectively

5' nick
A A 3' nick
C B
5' 3'
3' 5'
UvrD helicase releases
excised oligomer C
ATP
STEP 4

ADP + Pi
12-mer

5' 3'
B
3' 5'

B UvrB is replaced by DNA

STEP 5 polymerase-I followed by gap lling

5' 3'

3' Pol I 5'

STEP 6 Gap lling by DNA polymerase-I and

nick sealing by DNA ligase

5' 3'

3' 5'

Fig. 7.36: Nucleotide excision repair

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activity of MutH breaks the strand either 5' or 3' side which
may be of 1000 nucleotide far or more and nicked portion
including mismatch is excised. Gap thus created is filled by
DNA polymerase followed by joining of nick by DNA ligase.
A few other DNA repair mechanism have also been
observed and studied both in prokaryotes and eukaryotes.
You will study them in your higher classes. However, based
on the account of mutation and repair mechanism the
stable nature of genetic information can be appreciated
vis-à-vis its ability to undergo changes.

7.9 Recombination
The concept of recombination was very quickly observed
by geneticists after the rediscovery of Mendel’s principles
of inheritance in the year 1900. Experiments performed
by W. Bateson and R. C. Punnet in sweet pea clearly
established that all genes do not assort independently.
This was evident in the dihybrid cross performed in
sweet pea. Crossing of pure line of red flower having long
pollen grains with white flower having short pollen grains
resulted into expected red flower and long pollen offspring.
But among the selfed F2 offspring more than 50% of the
offspring had parental combinations of red-long and white-
short for flower colour and pollen shape. Recombinants,
i.e., red flower having short pollen and white flower having
long pollen were less than 50%. This was a deviation from
the expected 9:3:3:1 ratio (Fig. 6.8).
Though, the initial explanation suggested by Bateson
and Punnet were different, but later, this was established
as the phenomenon of linkage in which genes situated
on one chromosome inherit together. The appearance
of recombinants was attributed to the phenomenon of
crossing over, in which exchange of parts of homologous
chromosome during meiosis may result into recombinants.
Obviously, percentage of such recombinants is expected
to be less in comparison to the independently assorting
traits as the same has been observed in the experiment
described earlier. Later, similar experiments performed
in Drosophila melanogaster by Thomas Hunt Morgan
provided many instances of linkage and recombination.
Not only this, based on the frequency or percentages of
recombinants, a physical map of chromosomes of a number

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of organisms were prepared. In preparing such physical

map of the chromosome 1% of recombinants between two
traits were designated to be apart by
1 map unit or 1 centimorgan (cM).
Further, the cytological studies of
meiosis and appearance of chiasmata
in the first meiotic division, when
the homologous chromosome pair
separate (reductional division) provided
insight about direct connection of
recombinants with the exchange of the
parts of homologous chromosome pair.
One of the initial evidence that crossing
over is responsible for recombination
came from the classical experiment
performed by Harriet Creighton
and Barbara McClintock (1931)
in maize. Based on their careful
observation they found that some of
the homologous chromosomes can
be morphologically distinguished.
In their investigation, two types
of chromosome 9 were identified.
One of the pair was a normal
one whereas the other had a
heterochromatic knob at one end of
the chromosome and a small piece
of another chromosome on the
other end of it. You have already
studied chromosomal aberration
and understood as to how these
may arise. In their experiment,
two traits were used as markers
to understand recombination. One
was the gene that is responsible for
colour of the kernel, i.e., coloured (C)
versus colourless (c). The other was
the gene responsible for texture of
the kernel, i.e., starchy (Wx) versus
waxy (wx). Performance of the cross Fig. 7.37: Experimental evidence of crossing over
as per Fig. 7.37 provides an evidence (a) showing cytological marker on one
that during crossing over exchange chromosome and other normal chromosome
of parts of homologous chromosome (b) Result of test cross showing non-cross
over and cross over progeny

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occur. When recombinant offspring were examined for

the exchanged part of chromosome, it was found that
recombinants, i.e., either coloured with starchy kernel or
colourless with waxy kernel had different morphological forms
of chromosome 9. Here only one of the aberrant markers
were found cytologically among recombinants, contrary to
the presence of both the markers on the same chromosome,
establishing the fact that during recombination exchange of
part of homologous chromosomes do take place.

7.10 Regulation of Gene Expression

Do you know in a multicellular organism there are
many types of cells differing in structure and function,
nevertheless their genes are identical? This is because
all cells are derived from zygote by mitotic divisions. All
activities of an organism are controlled by genes. Most of
the genes of an organism express themselves by producing
proteins. All genes are not expressed in all cells as their
products are not needed at one time. Only those genes
whose products are required in a cell are expressed
while other genes are not expressed as its products are
not required by the cell at that time. This ‘switching on
and switching off’ mechanism of gene action is known as
regulation of gene expression or regulation of gene action.
Lower organisms like bacteria encounter wide range of
environmental conditions. For example, E. coli live in our
large intestine. Our eating habits determine the nutrients
available to this bacterium. When glucose is available, it
expresses those genes whose products (enzymes) will break
it down for generation of energy. If lactose or any other sugar
is available then some other genes are expressed whose
products will break it to generate energy. This indicates that
specific genes are expressed at specific times according to
the need of the cell.
The expression of genes may be regulated at different
steps along the pathway of flow of information from
genotype to phenotype. Regulation may be at chromatin
level, transcription level, mRNA processing (eukaryotes),
transport of mRNA and at translational level (Fig. 7.38).
In both prokaryotic and eukaryotic systems, transcription
initiation is an important point of gene regulation as the cell
not only decides which gene has to be expressed but also their

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degree of expression. In most Gene on DNA

cells of a species or organism, Transcriptional
some genes are expressed at a Primary control
more or less constant level and transcript
are called ‘housekeeping genes’ RNA processing
mRNA control
or ‘constitutive genes’. The
product of these genes is required Nucleus
RNA transport
all the time. Genes encoding the Cytosol control
enzymes that catalyse the steps
in central metabolic pathways, Translation
control
such as the citric acid cycle Protein
fall into this category. Such
unregulated expression of genes Fig. 7.38 Levels of regulation of gene expression
is called constitutive gene
expression. But rate of expression of most of the genes
alter in cells according to the molecular signal it receives.
The product level of these genes rise and fall according to
need of the cell. Such type of control is called regulated
gene expression.

7.10.1 Regulation of gene expression in bacteria

The mechanism of regulation of gene expression was first
studied in bacterial cells. The organisation of functionally
related genes in prokaryotes is different from that of
eukaryotes. In bacteria, genes that have related functions
are clustered and often transcribed together into a single
mRNA molecule. The advantage of clustering related
genes is that a single ‘on-off switch’ can control all the
genes of a cluster. It means all the genes of a cluster are
coordinately controlled. On the other hand, each gene of
eukaryote is transcribed into a separate mRNA. A group
of clustered structural genes that are transcribed together
along with promoter and additional controlling sequences
(that control transcription) is called an ‘operon’.
A typical operon (Fig. 7.39) has a set of structural genes
or cistrons (encode proteins involved in metabolism) at one
end which are transcribed and then translated to produce
different proteins. Upstream to the first structural gene is
‘promoter’ which controls the transcription of structural
genes. RNA polymerase binding site lies in the promoter.
A DNA segment called ‘operator’ positioned within the
promoter or between the promoter and the first structural
gene controls the access of RNA polymerase to the genes. The

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Operon
A model operon
Operator Structural genes
Regulator RNA
gene polymerase Gene a Gene b Gene c

Promoter Promoter

Transcription Transcription

mRNA
mRNA
Translation

Translation
Proteins (enzymes) A B C

Regulator
protein
(repressor)
Biochemical pathway
Precursor X Intermediate Product Y
product

Fig. 7.39: Organisation of an operon

operator is the site of binding of the repressor protein, the

latter binds to the operator forming an operator-repressor
complex. When the repressor binds to the operator,
transcription of the structural genes cannot occur. A
‘regulator gene’ is located upstream to the promoter of
an operon. It is not considered part of the operon although
it regulates the transcription of structural genes. It has its
own promoter and is transcribed to produce a small mRNA
which is then translated into a protein called regulatory
protein (repressor). Repressor may be either an active
repressor or an inactive repressor. The active repressor
protein binds to operator of the operon and prevents
the binding of RNA polymerase to the promoter thereby
interferes with the transcription of structural genes.
The mechanism of regulation of operon was first
described by Francois Jacob and Jacque Monod in 1961
and proposed ‘operon model’ for the genetic control of
lactose metabolism in E. coli cells. There are two types of
operon, inducible operon and repressible operon based
on the nature of their response to an effector molecule. In
case of inducible operons, the effector molecules are called
inducers (substrates) when present bind to active repressor
and inactivates it. The inactive repressor-inducer complex
cannot bind to operator, and transcription of structural
genes in the operon is turned on (induced) and subsequently

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proteins (enzymes) are translated. The enzymes whose

production can be increased by the presence of the
substrate on which it acts are called inducible enzymes
and the genetic system responsible for the synthesis of
such an enzyme is called inducible system. In case of
repressible operons, the effector molecules are called co-
repressors (end products). When co-repressors bind to
inactive repressors, the repressor-corepressor complex
is active, bind to operators and prevent RNA polymerase
from transcribing structural genes. For instance, when no
amino acids are supplied from outside, the E. coli cells
can synthesise all the enzymes needed for the synthesis
of different amino acids. However, if a particular amino
acid, for instance, histidine, is added, the production of
histidine synthesising enzyme falls. In such a system,
the addition of the end product checks the synthesis of
the enzymes needed for the biosynthesis. Such enzymes
whose synthesis can be checked by the addition of the end
product are repressible enzymes and the genetic system
is known as repressible system. There are two types of
transcriptional control: negative and positive control. In
negative control, the regulatory protein is a repressor that
inhibits transcription. In positive control, the regulator
protein is an activator which stimulates transcription.

7.10.2 The Lac operon – an inducible operon

The lactose (milk sugar, a disaccharide) is a β-galactoside is
available to E. coli in the colon when a person drinks milk.
The bacteria uses lactose for energy as well as source of
carbon after it is broken down into glucose and galactose by

Iac Operon
Regulatory sequences Structural gene

DNA
(Pi) lac l (P) (O) lac Z lac Y lac A

Operator
Promoter for Promoter for Structural gene for
regulatory gene structural genes Structural gene -galactoside transacetylase
for  -galactosidase
Gene for regulator
protein Structural gene for
 -galactoside permease

Fig.7.40: Structure of lac operon

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enzyme β-galactosidase. When E. coli are growing in absence

of lactose, few molecules of β-galactosidase are present in
the cells but when lactose is added to the bacterium’s
environment, the number of β-galactosidase molecules in
the cells increases many folds within 2 to 3 minutes.
The lac operon consists of three structural genes; lacZ,
lacY and lacA encoding three different proteins (Fig. 7.40).
The lacZ gene encodes β-galactosidase that break down
lactose into glucose and galactose. This enzyme can also
convert lactose into allolactose which act as an inducer in lac
operon. The gene lacY encodes β-galactoside permease, a
membrane protein which actively transports lactose into the
cell. The lacA encodes β-galactoside transacetylase, but its
function in lactose metabolism is not yet known.

Fig. 7.41: Regulation of lac operon

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The regulator gene called lacI is located upstream

to promoter of lac operon has its own promoter. It is
transcribed into a small mRNA which is then translated
into a regulator protein called repressor protein. The
repressor is an allosteric protein has two binding sites;
one for binding to operator of operon and to the other
binds inducer (allolactose).
In the absence of lactose in E. coli cells, the repressor
protein encoded by lacI is active and binds to lac operator;
physically blocking the binding of RNA polymerase and
transcription of structural genes is prevented (Fig. 7.41).
This is negative control of lac operon. As long as the
repressor is binding with the operator, no proteins are
made. However, when lactose is present, β-galactosidase
converts some of it into allolactose. The allolactose
acts as inducer, binds to active repressor and causes
conformational change by which it becomes inactive. The
inactive repressor fails to bind with operator and binding
of RNA polymerase is no longer blocked. RNA polymerase
now transcribes lacZ, lacY and lacA into a polycistronic
mRNA which translates into three
different enzymes required for lactose RNA
metabolism (Fig. 7.42). The production polymerase

of the enzymes to break down lactose

continues until enough of the lactose
molecules are broken down and then
release repressors to recombine with RNA
the operator to stop production of the polymerase

enzymes. The lac operon is an inducible

operon as presence of lactose induces
the production of β-galactosidase,
Inactive
β-galactoside permease and repressor
β-galactoside transacetylase.
In positive control of lac operon, Lactose
the regulator protein, i.e., an activator
binds to DNA at a site other than
Fig. 7.42: Positive control of lac operon
operator. The activator is produced
in an inactive state and fails to bind to DNA (Fig. 7.42).
The RNA polymerase does not bind to promoter and
transcription is off. When inducer associates with inactive
activator rendering it active, RNA polymerase binds to
promoter and initiates transcription.

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SUMMARY
• DNA was, for the first time, isolated from nuclei of pus
cells by Johann Friedrich Miescher in 1869.
• The phenomenon of transfer of genetic material from
one cell to another that alter the genetic make-up of
the recipient cell is called transformation and this was
discovered by Frederick Griffith in 1928.
• The experiments conducted by Oswald Avery, Colin
Macleod and Maclyn McCarty revealed DNA as the likely
transforming agent.
• The experiments conducted by Hershey and Chase in 1952
provided strong evidence that DNA is the genetic material.
• Gene is the unit of inheritance that controls a specific
trait or character and may also be expressed in alternative
forms known as alleles.
• The expression of DNA through the synthesis of polypeptide
chain via RNA synthesis represents the Central Dogma of
genetics.
• The fact that one gene encodes one polypeptide, the central
dogma also got modified from one gene, one protein to one
gene, one polypeptide.
• Each gene must satisfy the test to be a unit of function,
unit of recombination and unit of mutation.
• The kind of DNA replication in which the parental duplex
DNA form two identical daughter duplex, each of which
consists of one parental stand and one newly synthesised
daughter strand is called semi-conservative replication.
• It was Messelson and Stahl who experimentally
distinguished between the old and new stand of the DNA
after replication using two isotopes of Nitrogen, 14N and 15N
in their experiment.
• Several enzymes and proteins are involved in the replication
of DNA in both prokaryotes and eukaryotes such as DNA
polymerase, primase, helicase, single-strand binding
protein, topoisomerase, DNA ligase, DNA-dependent RNA
polymerase.
• Replication usually starts at a specific site on a DNA
sequence known as origin of replication.
• The new strand that is synthesised continuously in 5'→3'
direction is called the leading strand.
• The DNA strands synthesised discontinuously with the
Okazaki fragments are called the lagging strand.

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• Transcription is a process by which genetic information is

transferred from DNA to mRNA.
• Translation is the process by which information is
transferred from mRNA to polypeptide chain and consists
of four stages such as (i) charging of tRNA (ii) initiation (iii)
elongation, and (iv) termination.
• In case of reverse transcription the information present in
the genetic material which is RNA is first transferred to a
single stranded complementary DNA strand and is then
converted into a double stranded DNA.
• Within a gene, only one of the nucleotide strands is
normally transcribed into RNA. The DNA strand whose
nucleotide sequence is complementary to that of the
mRNA is called the template or antisense strand, while
the other strand whose base sequence is identical to that
of the mRNA (except for T in DNA and U in RNA) is called
the sense or coding strand.
• The major steps which are common to both prokaryotes
and eukaryotes in the process of transcription include
initiation, elongation and termination. In addition to these
steps, in eukaryotes the primary transcripts undergo post-
transcriptional modifications such as capping, splicing,
poly-adenylated tail.
• Genetic codes are triplet codons, i.e., unique combinations
of three bases which codes for a specific amino acid
depending on their combinations. There are 64 codons,
out of which 61 codons code for 20 amino acids.
• Polyribosomes consist of several ribosomes attached to the
same mRNA.
• Mutation is the alteration in the genetic material, i.e.,
DNA (RNA in case of a few viruses) and can be categorised
as addition, deletion and substitution of one or a few
nucleotide.
• High rates of mutation are not observed due to the DNA
repair mechanism which could be through excision repair,
mismatch repair, etc.
• Recombination is a process during which exchange of part
of homologous chromosomes take place.

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 216
Biotechnology

EXERCISES
1. What is the importance of gene expression? What are the
steps involved in it?
2. Describe the process of regulation of gene expression in
prokaryotes by giving example of lac operon.
3. What would be the effect of loss of all proteins from a cell
on DNA replication?
4. How is the structure of DNA affected by UV rays? Discuss
the molecular basis of the type of mutation caused by
this type of radiation and the mechanism used by cells to
correct them.
5. Differentiate between the following
(a) Leading strand and lagging strand
(b) Transcription and translation
(c) Transition and transversion mutation
(d) Codon and anticodon
6. Which of the following types of radiations is least likely to
be harmful to cells?
(a) Gamma rays
(b) Ultraviolet rays
(c) X rays
(d) Alpha rays
7. In which of the following DNA repair mechanism is
apyrimidinic or apurinic (AP) site formed?
(a) Excision repair
(b) Mismatch repair
(c) Both of the above
(d) None of the above

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Chapter [Link] 216 20-Sep-21 11:57:44 AM