Core-IV-Molecular-Biology-and-Microbial-Genetics-Sem-IV

KAMARAJ COLLEGE
SELF FINANCING COURSES

(Reaccredited with “A+” Grade by NAAC)
(Affiliated to Manonmaniam Sundaranar University,Tirunelveli)
THOOTHUKUDI 628 003
STUDY MATERIAL FOR B.Sc., MICROBIOLOGY

MOLECULAR BIOLOGY AND
MICROBIAL GENETICS
IV - SEMESTER
ACADEMIC YEAR 2022-23
PREPARED BY,
DEPARTMENT OF MICROBIOLOGY (SF)
KAMARAJ COLLEGE,
THOOTHUKUDI.
STUDY MATERIAL FOR B.Sc. MICROBIOLOGY
MOLECULAR BIOLOGY AND MICROBIAL GENETICS
IV-SEMESTER, ACADEMIC YEAR 2022 – 2023

UNIT- I
Basic Concepts in Molecular Biology – Introduction – Scope – Applications –. Nucleus:
Nucleus structure, Nucleoid, Chromatin and Chromosomes, allele, loci, gene. Nucleic acids as
genetic material –Discovery of genetic material-Structure, organization and types of DNA and
RNA–Extra chromosomal DNA(Plasmid), DNA supercoiling, DNA replication in prokaryotes,
Mechanism and enzymology of replication, Rolling circle replication.
UNIT –II
Gene structure and expression Genes in prokaryotes &Eukaryotes – organization, Molecular
mechanism and Enzymology of Transcription in prokaryotes and Eukaryotes, Post-
transcriptional modifications, Genetic code, Molecular mechanism and Enzymology of
Translation of proteins in prokaryotes and Eukaryotes, Posttranslational modifications.
Regulation of gene expression in prokaryotes– Operon concept– lac &trp Operon.
UNIT – III
Mutations: Spontaneous and induced, mutagens, base pair changes, frame shifts, deletions,
inversions and duplications, insertions, useful phenol types (auxotrophic, conditional lethal,
resistant),reversion vs. suppression, Ames test. DNA damage and repair mechanism.
UNIT IV
Bacterial and Viral genetics: Bacterial plasmids: structure and properties, replication,
incompatibility, plasmid amplification. Transposition: structure of bacterial transposons, IS
elements, types of bacterial transposons. Organization of viral genome – DNA(Polio Virus)
and RNA (Influenza) Retro viral genome, Bacteriophage genome – T4 and T7 life cycle.
UNIT V
Recombination and Gene Transfer mechanisms: Molecular basis of recombination in bacteria.
Recombination types, Gene transfer mechanisms-Transformation: natural transformation,
competence, DNA uptake, role of natural transformation, artificially induced competence,
electroporation. Transduction (generalized and specialized). Conjugation: self-transmissible
plasmids, F factor, tra genes, on T, F' and Hfr strains, steps in conjugation, chromosome
mobilization.
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UNIT- I
BASIC CONCEPTS IN MOLECULAR BIOLOGY
Introduction
Molecular biology is a specialized branch of biology and biochemistry, which is
specifically concerned with the study of various biological activities at the molecular level.
Molecular biology includes different biomolecules like amino acids, nucleic acids (DNA and
RNA) proteins, carbohydrates, and lipids, along with their compositions, interactions,
structure, and functions in the life processes.
The study of molecular biology will establish a strong foundation on the fundamental
importance of macromolecular mechanisms such as replication, transcription, translation and
other cellular functions. The more commonly used molecular biology techniques include-
Polymerase Chain Reaction, Electrophoresis, Restriction Digestion, Blotting, Cloning, etc.
The term “Molecular Biology” was coined by an American scientist, Warren Weaver
in the year 1938. As per the records, the discovery of molecular biology began in the early
1940s and its fundamental development took place in the year 1953, during the invention of
the double-helical structure of the DNA molecule by the two molecular biologists named James
Watson and Francis Crick.
Scope
Molecular genetics is a field of biology that studies the structure and functions of genes at a
molecular level, and their influence in determining the overall makeup of an organism.
 The discipline focuses on the molecular mechanism of genetic processes so that genes
can be used for genetic engineering to isolate, modify, and sequence genes.
 Initial studies in molecular biology were involved in the determination of three-
dimensional structures of proteins in order to understand their structure and mechanism
of action and expression.
 Initial studies in molecular biology were based on the rapid growth and readily mani
pulable genetics of simple bacteria, such as E. coli, and their viruses.
 More recently, not only the fundamental principles but also many of the experimental
approaches first developed in prokaryotes have been successfully applied to eukaryotic
cells.
 Current advances in recombinant DNA technology have made even the determination
of the complete sequence of the human genome a feasible project.
 Complete genome sequencing of various living beings, including humans, has been
done with modern approaches like DNA sequencing techniques and PCR.
 Studies of molecular biology have also been applied in clinical research and medical
therapies, both of which are covered under gene therapy.
 Molecular biology also plays a vital role in studies related to regulations of various parts
of a cell, which can then be used to target new drugs and diagnose diseases efficiently.
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HISTORY
1866- Genetics start to get attention when Mendel Experimented with green peas and
publish his finding 1910- Morgan revealed that the units of heredity are contained with
chromosome, 1944- It is confirmed through studies on the bacteria that it was DNA that carried
the genetic information. 1953-Franklin and Wilkins study DNA by X-ray crystallography
which subsequently lead to unrevealing the double helical structure of DNA by Watson and
Crick 1960s- Smith demonstrate that the DNA can be cleaved by restriction enzymes. During
1962–1964, through the use of conditional lethal mutants of a bacterial virus, fundamental
advances were made in our understanding of the functions and interactions of the proteins
employed in the machinery of DNA replication, DNA repair, DNA recombination, and in the
assembly of molecular structures. 1966 -Gene transcription become reality 1975- Southern blot
was invented 1977- DNA sequencing methodology discovered 1981-Genetic diagnosis of
sickle cell disease was first shown to be feasible by Kan and Chang 1985- PCR developed by
Kary Mullis and Co-workers 2001-Draft of Human genome sequence was revealed.
APPLICATIONS
Molecular biology applications are expected to be a mainstay in the manufacture of
chemicals, energy, medical, consumer products, agriculture and food, industry, cosmetics and
environmental technologies. Molecular biology methods have tremendous value not only in
the investigation of basic scientific questions, but also in application to a wide variety of
problems affecting the overall human condition. Disease prevention and treatment, generation
of new protein products, and manipulation of plants and animals for desired phenotypic traits
are all applications that are routinely addressed by the application of molecular biology
methods. Disease prevention and treatment, generation of new protein products, and
manipulation of plants and animals for desired phenotypic traits are all applications that are
routinely addressed by the application of molecular biology methods.
Advances in molecular biology and genetic engineering technology, microbial genetic
manipulations have promoted the application of microorganisms in in ecological and
environmental research. Genetically engineered microorganisms are being developed and
assessed for their beneficial uses in environmental monitoring, toxic chemicals pollution
control and genetically engineered microorganisms.
1. Hybridization probe
Due to the stringent specificity and high sensitivity of nucleic acid hybridization,
hybridization probes are used on a broad level in microbial ecology, such as microbial
detection, qualitative and quantitative analysis of microbial, distribution, abundance and
adaptability of microbes.
2. PCR based technologies:

The polymerase chain reaction (PCR) is a technique used in molecular biology to amplify
DNA template, generating thousands to millions of copies of a particular DNA sequence
in vitro. This technique can be used to analyze mRNA expression profile among different
growth stages.
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3. Electrophoresis:
The interaction between DNA double helix are disrupted during denaturing gradient gel
electrophoresis(DGGE)， temperature gradient gel electrophoresis(TGGE) and other
special forms of electrophoresis, thus the DNA fragments consist of different sequences
can be separated on acrylamide gel with superior resolution.
4. Genetic engineering:
New recombinant DNA may be generated by first isolation and amplification the genetic
material of interest using molecular cloning methods, then the chimera DNA sequence or
artificially synthesized DNA maybe inserted into the host organism. This technique is
essential for the construction of microbes with enhanced biodegradability that may be used
in controlling remediation of contaminated environment or fermentation of waste to
produce natural gas.
Nucleus
The nucleus is a spherical-shaped organelle that is present in every eukaryotic cell. The
nucleus is the control centre of eukaryotic cells. It is also responsible for the coordination of
genes and gene expression. The structure of the nucleus includes nuclear membrane,
chromosomes, nucleoplasm, and nucleolus. The nucleus is a pivotal organelle responsible for
regulating almost all forms of cellular activities. Mostly, every type of cell that exists is
categorized on the basis of the absence or presence of the nucleus within its cell (Prokaryotes
and Eukaryotes). A nucleus is a double-membraned eukaryotic cell organelle that contains the
genetic material. Typically, it is the most evident organelle in the cell. The nuclear membranes
is referred to as the nuclear envelope.The membrane distinguishes the cytoplasm from the
contents of the nucleus.The cell’s chromosomes are also confined within it.DNA is present in
the Chromosomes, and they provide the genetic information required for the creation of
different cell components in addition to the reproduction of life.
Functions
 It contains the cell’s hereditary information and controls the cell’s growth and
reproduction. First one, the nucleus is the site of DNA replication.
 Secondly, the nucleus is the site of transcription. Transcription creates different types
of RNA from DNA. Transcription would be a lot like creating copies of individual
pages of the human body’s instructions which may be moved out and read by the rest
of the cell.
 The central rule of biology states that DNA is copied into RNA, and then proteins.
Nucleoid is the regulatory center of the prokaryotic cell. This region regulates the
growth, reproduction, and function of the prokaryotic cell.
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The DNA is packaged by special proteins called histones to form chromatin. The
chromatin further condenses to form chromosomes. Chromatin is a lower order of DNA
organization, whereas chromosomes are a higher order of DNA organization. Chromatin is
located in the nucleus of our cell. It is composed of DNA and proteins that condense to form
chromosomes. It compresses the DNA structure into a compact unit so that it can fit within the
nucleus. The histone proteins organize the DNA into special structures called nucleosomes.
The nucleosome further folds to form a chromatin fibre.
 Chromosome
 Chromatids
 DNA helix
i. Chromosomes
The DNA is packed into thread-like structures called chromosomes. The DNA is tightly
coiled around histone proteins several times to form a chromosome. These are formed by the
condensation of chromatin fibres.
The long string like structure that makes up a chromosome is made up of a
chemical called deoxyribonucleic acid (DNA). Each chromosome contains one DNA
molecule. The DNA is coiled tightly around proteins called histones. These proteins
provide structural support to a chromosome and allow the very long DNA molecule to
form a compact shape and fit inside the nucleus of a cell. Individual chromosomes cannot
be seen very clearly in the periods between cell division. This is because during this phase
i.e. the interphase the chromosomes exist as very loosely coiled, long, thin threads spread
throughout the nucleus. Before the nucleus of a cell divides an exact copy of the DNA
molecule in a chromosome is made so that at nuclear division the chromosome is a double
structure, containing two identical DNA molecules. These two part structure of
chromosomes are called chromatids with each chromatid of the pair containing one of the
two identical DNA molecules. The chromatids are held together at a point called
the centromere. The centromere may occur anywhere along the length of the chromosome.
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An allele is one of two or more versions of DNA sequence (a single base or a segment
of bases) at a given genomic location. An individual inherits two alleles, one from each parent,
for any given genomic location where such variation exists. If the two alleles are the same, the
individual is homozygous for that allele. A pair of alleles determine the same trait, for example,
eye color; one allele codes for black eyes, and another allele codes for brown eyes. All the
alleles found in an organism make up the genotype. If a pair of alleles are similar, the
organism's genotype is called homozygous.
A locus is the specific physical location of a gene or other DNA sequence on a chromosome.
A gene is the basic physical and functional unit of heredity. Genes are made up of DNA.
Some genes act as instructions to make molecules called proteins. However, many genes do
not code for proteins. In humans, genes vary in size from a few hundred DNA bases to more
than 2 million bases. DNA is responsible for building and maintaining your human
structure. Genes are segments of your DNA, which give you physical characteristics that make
you unique.
Proof of DNA as Genetic Material
The experiments of Griffith, in 1928, were one of the first steps toward proof that
nucleic acids are the genetic material. He used different strains of the bacterium pneumococcus
to demonstrate a genetic ‘transformation’ of one strain type into another. Different strains of
pneumococcus can be distinguished by the type of polysaccharide found in the cell capsule.
The capsule type is a constant, inherited characteristic of each strain. Occasionally some cells
may lose the ability to form a capsule, but such un encapsulated cells do not change strain type.
Encapsulated cells are deadly when injected into mice, while un encapsulated cells have lost
their virulence. Griffith carried out a series of experiments on mice injected with various
combinations of pneumococcus strains. He discovered that mice injected with un encapsulated,
avirulent cells mixed with an extract of heat-killed encapsulated cells generally died, just as if
they had received live virulent cells. Clearly, the extract of heat killed cells was somehow
conferring virulence to the live encapsulated cells. Griffith noted that bacteria recovered from
the dead animals proved to be encapsulated with the genetically determined polysaccharide
characteristic of the heat-killed strain.
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Griffith experiment or Transformation

Introduction
 In 1928, Griffith proved that the genetic material of bacteria is DNA by transformation
experiment in Streptococcus pneumoniae/ Diplococcus pneumoniae.
 DNA contains the genetic information of all living organisms.
Phenotypic characteristics of Streptococcus pneumoniae.
Presence or absents of capsule
1. Type II rough strain - small, non capsulated (Non virulent)

2. Type III Smooth strain - Large, Capsulated (Virulant)
Genotypic characteristics:
Genotype of the Diplococcus has different antigenic types, they are type II and type III.
These two types mainly depending on the molecular composition of the polysaccharide in
capsule.
Characteristics of Diplococcus pneumoniae grown on blood agar.
Colony Capsule Virulence Reaction with Antiserum

Morphology
Type II R Type III S
Type II R small Absent non virulent None None
colonies
Type II S large Present Virulent agglutination None
colonies
Type III R small Absent non virulent None None
Colonies
Type III S large Present Virulent None Agglutination
colonies
Procedure
 Live type II Rough strain is injected into the mice, and the mice was alive.
 When live type III smooth strain is injected into mice, mice was dead. Due to the
presents of capsule in type III smooth strain.
 After, heat killed type III smooth strain injected into mice, and the mice remain alive.
 When heat killed type III smooth stain is mixed with live type II rough strain, then
injected into mice, the mice was dead.
Conclusion
The results showed that non virulent Type II R cells converted to virulent Type III S
cells. This cannot be explained by Griffith. Now, that is component of the dead Type III S cells
must transferred to non-virulent Type II R cells, that convert living type II R cells into Type III
S cells. This experiment is known as Griffith transformation.
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Purification of the transforming material

The experiments of Griffith were subsequently confirmed by other investigators, and
in 1944, Avery, MacLeod and McCarty identified DNA as the agent responsible for bacterial
transformation. Their experiments utilized encapsulated (S) type III pneumococci, from which
they isolated a very highly purified DNA fraction. That fraction was capable of transforming
unencapsulated (R) variants of type II cells into fully encapsulated cells of type III, from which
the DNA was isolated. One key to eliminating any role of molecules other than DNA in the
transformation process was the use of the enzyme deoxyribonuclease (DNAase), which
depolymerizes DNA. Avery et al. showed that treating the active transforming fraction with
the nuclease destroyed the structure of DNA and the ability of that fraction to transform
pneumococcus cells. Treatments with enzymes that attack other cellular components had no
effect on transformation activity. The demonstration that DNA is the genetic material of a
Bacteria.
Hershey and Chase experiment
Introduction
In 1952, A.D Hershey, M Chase proved that DNA is the genetic material in T2
Bacteriophage.
Bacteriophage
Virus which affect bacteria is known as bacteriophage, it is also known as acellular
obligate parasites.It can reproduce only appropriate Host cells. Mainly, T2 bacteriophage
grown within the Colon Bacteria (E.Coli). Hershey and Chase knew that the phages attached
to the surface of a host bacterial cell and injected some substance (either DNA or protein) into
the host. To establish whether the phage injected DNA or protein into host bacteria, Hershey
and Chase prepared two different batches of phage. In each batch, the phage were produced in
the presence of a specific radioactive element, which was incorporated into the macromolecules
(DNA and protein) that made up the phage.
One sample was produced in the presence of S35(instead of S32), a radioactive isotope of
sulphur. Sulphur is found in many proteins and is absent from DNA, so only phage proteins
were radioactively labelled by this treatment. The other sample was produced in the presence
of P32(instead of P31) a radioactive isotope of phosphorous. Phosphorous is found in DNA and
not in proteins, so only phage DNA (and not phage proteins) was radioactively labelled by this
treatment.
Each batch of phage was used to infect a different culture of bacteria. After infection
had taken place, each culture was whirled in a blender, removing any remaining phage and
phage parts from the outside of the bacterial cells. Finally, the cultures were centrifuged, or
spun at high speeds, to separate the bacteria from the phage debris.
Centrifugation causes heavier material, such as bacteria, to move to the bottom of the
tube and form a lump called a pellet. Lighter material, such as the medium (broth) used to grow
the cultures, along with phage and phage parts, remains near the top of the tube and forms a
liquid layer called the supernatant.
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Conclusion:
When Hershey and Chase measured radioactivity in the pellet and supernatant from
both of their experiments, they found that a large amount of P32 appeared in the pellet, whereas
almost all of the S35 appeared in the supernatant. Based on this and similar experiments,
Hershey and Chase concluded that DNA, not protein, was injected into host cells and made up
the genetic material of the phage.
RNA as a genetic material in small virus (Reconstitution experiment)

Many viruses have been shown to contain ribonucleic acid (RNA) as their genetic
material. One of the early examples is the Tobacco mosaic virus (TMV), shown in the 1930s
to be composed of protein and RNA. No DNA is found in the particle. The protein can be easily
separated from the RNA by mild alkali treatment. That it is the RNA that acts as the genetic
material in such particles was demonstrated directly by Gierer and Schramm, in 1956, who
showed that tobacco plants could be infected by inoculation with the RNA alone. Frankel-
Conrat and his associates determined that infective virus particles could be reconstituted by
mixing together the protein and RNA. Using this technique, he and Singer, in 1957,
reconstituted viruses by mixing protein produced by a mutant strain together with the RNA
from another strain, or vice versa.
 Viruses contain RNA and protein but no DNA.

 One of the first experiments that concluded RNA as the genetic material in RNA
viruses.
 In 1957 H. Frankel Conrat & Singer published reconstitution experiment was done with
Tobacco mosaic virus (TMV)
 TMV is a small virus composed of a single molecule of RNA encapsulated in a protein
coat. Different strains of TMV can be identified on the basis of differences in the
chemical composition of their protein coat.
 By using appropriate chemical treatments, one can separate protein coats of TMV from
RNA this process is reversible by mixing the “Proteins and RNA under appropriate
condition. This process is called as reconstitution” that will occur, yielding infective
TMV Particles.
 They took two strains of TMV, one is strain A and another strain B. Both strain is
separated the RNA from protein coat & reconstituted viruses by mixing the proteins of
one strain with the RNA of second strain under appropriate condition.
 When the one type mixed viral strain contain strain A genetic material and strain B
protein coat, that allowed to infect tobacco leaves and produced the progeny viruses,
that always found to be phenotypically and genotypically identical to strain A, not the
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strain of B, from which the strain A RNA had been coated with strain A protomers and
strain A, RNA genetic material, not strain B RNA.
 Thus, the experiment proved that the genetic information of TMV is stored in RNA,
not in Protein.
Nucleic acids are the organic materials present in all organisms in the form of DNA or RNA.
These nucleic acids are formed by the combination of nitrogenous bases, sugar molecules and
phosphate groups that are linked by different bonds in a series of sequences. The DNA structure
defines the basic genetic makeup of our body. In fact, it defines the genetic makeup of nearly
all life on earth.
DNA/ Deoxyribonucleic acid
Deoxyribonucleic acid, or DNA, is a molecule that contains the instructions an
organism needs to develop, live and reproduce. These instructions are found inside every cell,
and are passed down from parents to their children. The genetic information of all living
organisms, except RNA virus, is stored in DNA. Nucleic Acids, first called “Nuclein”, they
were isolated from cell nuclei by F. Miescher in 1869, are macromolecules composed of
repeating subunits called nucleotides.
DNA is made up of molecules called nucleotides. Each nucleotide contains a phosphate
group, a sugar group and a nitrogen base. The four types of nitrogen bases are adenine (A),
thymine (T), guanine (G) and cytosine (C). The order of these bases is what determines DNA's
instructions, or genetic code. Nucleotides joined together by phosphodiester bond . Nitrogen
base and sugar molecules are joined together by glycosidic linkage. Nucleotides are attached
together to form two long strands that spiral to create a structure called a double helix.
Watson & Crick – DNA Double helix Structure

 The correct structure of DNA was first proposed by J.D. Watson and F.H.C. Crick in
1953.
 When the composition of DNA from many different organisms was analyzed by E.
Chargaff. It was observed that “Concentration of thymine was always equal to the
concentration of adenine and the concentration of cytosine was always equal to the
concentration of guanine.
 When X-rays are focused through isolated macromolecules recorded on x-ray sensitive
film.
 The DNA was a highly ordered double stranded structure with repeating subunits of
nucleotide, spaced every 3.4A0 of the DNA molecule.
 Watson & Crick proposed that DNA exist as a double helix in which the two
polynucleotide chains are complementary to one another.
 Each polynucleotide chain consists of sequence of nucleotide linked together by
phospho di ester bonds. Each nucleotide is composed of
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 Phosphate group
 a Five Carbon Sugar.
 a cyclic nitrogen base.
 In DNA, the sugar is 2-deoxyribose , which is a cyclic pentose (5-carbon sugar) and are
four different bases found in DNA
 Adenine
 Guanine
 Thymine
 Cytosine
Adenine & Guanine are double ringed bases called purine. Cytosine, thymine & uracil
are single ring bases called pyri midines.
 DNA contain two purines and two pyrimidine bases

 The two polynucleotide strands are held together in their helical configuration by
hydrogen bond between bases in opposing strands, base pairs being stacked between
the two chains perpendicular to the axis to the molecule.
 The base pairing is specific adenine is always paired with thymine and guanine is
always paired with cytosine.
 The specificity of base pairing results from the hydrogen – bonding capacities of the
bases in their normal configurations.
 In common structural configuration of DNA, adenine & thymine form 2 hydrogen
bonds, guanine & cytosine form 3 hydrogen bonds. Hydrogen bonding between
cytosine & adenine is not possible except when they exist in their rare structural states.
 The two strands of a DNA double helix are thus said to be complementary.
 The Sugar Phosphate backbones of the two complementary strands are antiparallel, they
have opposite chemical polarity.
 The high degree of stability of DNA double helix results in part of hydrogen bonds
between the base pairs and hydrophobic bonds.
 In the Watson – crick double helix form of DNA is the “B” form of DNA.
 The diameter of the DNA double helix in 20 A0
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 The length of the each turn of DNA double helix is 34A0. The space between the two
nucleotide is 3.4A0.
 Helical nature of the DNA has 2 grooves. The narrow grooves are called minor groove.
The wider grooves are called major grooves. The grooves are mainly used for the
binding of proteins on DNA.
 Polynucleotides are made of the successive addition of monomeres in a general 5' -> 3'
configuration.
DNA Types
There are three different DNA types:
 A-DNA: It is a right-handed double helix similar to the B-DNA form. Dehydrated DNA
takes an A form that protects the DNA during extreme conditions such as desiccation.
Protein binding also removes the solvent from DNA, and the DNA takes an A form.
 B-DNA: This is the most common DNA conformation and is a right-handed helix. The
majority of DNA has a B type conformation under normal physiological conditions.
 Z-DNA: Z-DNA is a left-handed DNA where the double helix winds to the left in a
zig-zag pattern. It was discovered by Andres Wang and Alexander Rich. It is found
ahead of the start site of a gene and hence, is believed to play some role in gene
regulation.
 The bases are at the outer surface. Phosphates are closer together than in B-DNA.
 Z-DNA cannot form nucleosomes. A high G-C content favours Z conformation- DNA
sequences can flip from a B form to a Z form and vice versa.
 Z-DNA formation occurs during transcription of genes, at transription start sites near
promoters of actively transcribed genes. The negative supercoiling upstream favours Z-
DNA formation. At the end of transcription, topoisomerase relaxes DNA back to B
conformation. Certain proteins bind to Z-DNA.
RNA Structure
RNA is typically single stranded and is made of ribonucleotides that are linked by
phosphodiester bonds. A ribonucleotide in the RNA chain contains ribose (the pentose sugar),
one of the four nitrogenous bases (A, U, G, and C), and a phosphate group. The subtle structural
difference between the sugars gives DNA added stability, making DNA more suitable for
storage of genetic information, whereas the relative instability of RNA makes it more suitable
for its more short-term functions. The RNA-specific pyrimidine uracil forms a complementary
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base pair with adenine and is used instead of the thymine used in DNA. Even though RNA is
single stranded, most types of RNA molecules show extensive intramolecular base pairing
between complementary sequences within the RNA strand, creating a predictable three-
dimensional structure essential for their function.
Structure and Function of RNA
mRNA rRNA tRNA
Short (70-90
nucleotides),
stable RNA
Longer, stable with extensive
Short, unstable, single-
RNA molecules intramolecular
stranded RNA corresponding
Structure composing 60% base pairing;
to a gene encoded within
of ribosome’s contains an
DNA
mass amino acid
binding site
and an mRNA
binding site
Ensures the
proper alignment Carries the
Serves as intermediary
of mRNA, correct amino
between DNA and protein;
tRNA, and acid to the site
Function used by ribosome to direct
ribosome during of protein
synthesis of protein it
protein synthesis in
encodes
synthesis; the ribosome
catalyzes peptide
bond formation
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Structure and Function of RNA
mRNA rRNA tRNA
between amino
acids
Plasmid DNA
Bacterial plasmids are closed circular molecules of double-stranded DNA that
range in size from 1 to >200 kb. They are found in a variety of bacterial species, where
they behave as additional genetic units inherited and replicated independently of the
bacterial chromosome. However, they rely upon enzymes and proteins provided by the
host for their successful transcription and replication.
Plasmids often contain genes that code for enzymes that can be advantageous to
the host cell in some circumstances. The encoded enzymes may be involved in resistance
to, or production of, antibiotics, resistance to toxins found in the environment (e.g.,
complex organic compounds), or the production of toxins by the bacteria itself.
Their ability to replicate independently makes plasmid a cloning vector in the recombinant
DNA technology for transferring and manipulating genes.
 Many antibiotic-resistant genes in bacteria are present in plasmids.

 The size of plasmid varies from a few base pairs to thousands of bp.
 Plasmids also get transferred from one bacterial cell to another by the process of
conjugation.
 Plasmids carrying a specific gene are introduced into bacterial cells, which multiply
rapidly and the required DNA fragment is produced in larger quantities.
 Plasmids are used to prepare recombinant DNA with the desired gene to transfer genes
from one organism to another. This is known as genetic engineering.
 Joshua Lederberg coined the term plasmid.
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Plasmid Structure
 Plasmids are extrachromosomal and not essential. They are useful but not necessarily
present in every organism of the species
 Plasmids are not a part of the genome and the same plasmid can exist in different species
and gets transferred from one another
 Plasmids have their own origin of replication (ORI) and they replicate along with the
cell so that each daughter cell possesses a copy of the plasmid also
 Apart from the origin of replication, often it contains genes for antibiotic resistance, for
the production of toxins and other useful genes, that may be required for the survival of
cells.
DNA supercoiling
DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an

expression of the strain on that strand. Supercoiling is important in a number of biological
processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases
are able to change DNA topology to facilitate functions such as DNA replication or
transcription. Mathematical expressions are used to describe supercoiling by comparing
different coiled states to relaxed B-form DNA.
The DNA of most organisms is negatively supercoiled. In a “relaxed” double-helical

segment of B-DNA, the two strands twist around the helical axis once every 10.4 to 10.5 base
pairs of sequence. Adding or subtracting twists, as some enzymes can do, imposes strain. If a
DNA segment under twist strain were closed into a circle by joining its two ends and then
allowed to move freely, the circular DNA would contort into a new shape, such as a simple
figure-eight. Such a contortion is a supercoil.
The simple figure eight is the simplest supercoil, and is the shape a circular DNA
assumes to accommodate one too many or one too few helical twists. The two lobes of the
figure eight will appear rotated either clockwise or counterclockwise with respect to one
another, depending on whether the helix is over or underwound. For each additional helical
twist being accommodated, the lobes will show one more rotation about their axis.
Extra helical twists are positive and lead to positive supercoiling, while subtractive
twisting causes negative supercoiling. Many topoisomerase enzymes sense supercoiling and
either generate or dissipate it as they change DNA topology. In part because chromosomes may
be very large, segments in the middle may act as if their ends are anchored. As a result, they
may be unable to distribute excess twist to the rest of the chromosome or to absorb twist to
recover from under winding—the segments may become supercoiled, in other words. In
response to supercoiling, they will assume an amount of writhe, just as if their ends were joined.
Supercoiled DNA forms two structures; a plectoneme or a toroid, or a combination of

both. A negatively supercoiled DNA molecule will produce either a one-start left-handed helix,
the toroid, or a two-start right-handed helix with terminal loops, the plectoneme. Plectonemes
are typically more common in nature, and this is the shape most bacterial plasmids will take.
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The Importance of DNA Supercoiling
DNA supercoiling is important for DNA packaging within all cells. Because the length
of DNA can be thousands of times that of a cell, packaging this genetic material into the cell
or nucleus (in eukaryotes ) is a difficult feat. Supercoiling of DNA reduces the space and allows
for much more DNA to be packaged. In prokaryotes, plectonemic supercoils are predominant,
because of the circular chromosome and relatively small amount of genetic material. In
eukaryotes, DNA supercoiling exists on many levels of both plectonemic and solenoidal
supercoils, with the solenoidal supercoiling proving the most effective in compacting the DNA.
Solenoidal supercoiling is achieved with histones to form a 10 nm fiber. This fiber is further
coiled into a 30 nm fiber, and further coiled upon itself numerous times more.
DNA Replication in Prokaryotes

DNA replication in prokaryotes is the process of copying of prokaryote genome the
same as that of its original genome that is passed on to the daughter cells.DNA replication
employs a large number of proteins and enzymes, each of which plays a critical role during the
process. One of the key players is the enzyme DNA polymerase, also known as DNA pol,
which adds nucleotides one by one to the growing DNA chain that are complementary to the
template strand. The addition of nucleotides requires energy; this energy is obtained from the
nucleotides that have three phosphates attached to them, similar to ATP which has three
phosphate groups attached. When the bond between the phosphates is broken, the energy
released is used to form the phosphodiester bond between the incoming nucleotide and the
growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA
pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA
synthesis; DNA pol I and DNA pol II are primarily required for repair.
There are specific nucleotide sequences called origins of replication where replication
begins. In E. coli, which has a single origin of replication on its one chromosome (as do most
prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin
of replication is recognized by certain proteins that bind to this site. An enzyme
called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous
base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped
structures called replication forks are formed. Two replication forks are formed at the origin of
replication and these get extended bidirectionally as replication proceeds. Single-strand
binding proteins coat the single strands of DNA near the replication fork to prevent the single-
stranded DNA from winding back into a double helix. DNA polymerase is able to add
nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this
direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a
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phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This
essentially means that it cannot add nucleotides if a free 3'-OH group is not available. Then
how does it add the first nucleotide? The problem is solved with the help of a primer that
provides the free 3'-OH end. Another enzyme, RNA primase, synthesizes an RNA primer that
is about five to ten nucleotides long and complementary to the DNA. Because this sequence
primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now
extend this RNA primer, adding nucleotides one by one that are complementary to the template
strand.
Fig: A replication fork is formed when helicase separates the DNA strands at the origin
of replication. The DNA tends to become more highly coiled ahead of the replication fork.
Topoisomerase breaks and reforms DNA’s phosphate backbone ahead of the replication fork,
thereby relieving the pressure that results from this supercoiling. Single-strand binding proteins
bind to the single-stranded DNA to prevent the helix from re-forming. Primase synthesizes an
RNA primer. DNA polymerase III uses this primer to synthesize the daughter DNA strand. On
the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is
synthesized in short stretches called Okazaki fragments. DNA polymerase I replaces the RNA
primer with DNA. DNA ligase seals the gaps between the Okazaki fragments, joining the
fragments into a single DNA molecule.
The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase
can only extend in the 5' to 3' direction, which poses a slight problem at the replication fork.
As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5' to 3' direction
and the other is oriented in the 3' to 5' direction. One strand, which is complementary to the 3'
to 5' parental DNA strand, is synthesized continuously towards the replication fork because the
polymerase can add nucleotides in this direction. This continuously synthesized strand is
known as the leading strand. The other strand, complementary to the 5' to 3' parental DNA, is
extended away from the replication fork, in small fragments known as Okazaki fragments, each
requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese
scientist who first discovered them. The strand with the Okazaki fragments is known as
the lagging strand.
The leading strand can be extended by one primer alone, whereas the lagging strand needs a
new primer for each of the short Okazaki fragments. The overall direction of the lagging strand
will be 3' to 5', and that of the leading strand 5' to 3'. A protein called the sliding clamp holds
the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-
shaped protein that binds to the DNA and holds the polymerase in
place. Topoisomerase prevents the over-winding of the DNA double helix ahead of the
replication fork as the DNA is opening up; it does so by causing temporary nicks in the
DNAhelix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA.
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The primers are removed by the exonuclease activity of DNA pol I, and the gaps are filled in
by deoxyribonucleotides. The nicks that remain between the newly synthesized DNA (that
replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme
DNA ligase that catalyzes the formation of phosphodiester linkage between the 3'-OH end of
one nucleotide and the 5' phosphate end of the other fragment.
Once the chromosome has been completely replicated, the two DNA copies move into two
different cells during cell division.
DNA replication steps

1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication forks; these are extended
bidirectionally.
3. Single-strand binding proteins coat the DNA around the replication fork to prevent
rewinding of the DNA.
4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
5. Primase synthesizes RNA primers complementary to the DNA strand.
6. DNA polymerase starts adding nucleotides to the 3'-OH end of the primer.
7. Elongation of both the lagging and the leading strand continues.
8. RNA primers are removed by exonuclease activity.
9. Gaps are filled by DNA pol by adding dNTPs.
10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the
formation of phosphodiester bonds.
Replication in prokaryotes starts from a sequence found on the chromosome called the
origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double
helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to
the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes
an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only in
the 5' to 3' direction. One strand is synthesized continuously in the direction of the replication
fork; this is called the leading strand. The other strand is synthesized in a direction away from
the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is
known as the lagging strand. Once replication is completed, the RNA primers are replaced by
DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds
between the 3'-OH of one end and the 5' phosphate of the other strand.
Enzymes involved in Replication

Prokaryotic DNA Replication: Enzymes and Their Function
Enzyme/protein Specific Function
DNA pol I Exonuclease activity removes RNA primer and replaces with newly
synthesized DNA
DNA pol II Repair function
DNA pol III Main enzyme that adds nucleotides in the 5'-3' direction
Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous
bases
Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand
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Primase Synthesizes RNA primers needed to start replication

Sliding Clamp Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase Helps relieve the stress on DNA when unwinding by causing breaks and
then resealing the DNA
Single-strand binding proteins (SSB) Binds to single-stranded DNA to avoid DNA rewinding
back.
1. Topoisomerase is responsible for initiation of the unwinding of the DNA. The tension
holding the helix in its coiled and supercoiled structure can be broken by nicking a single
strand of DNA.
2. Helicase accomplishes unwinding of the original double strand, once supercoiling has been
eliminated by the topoisomerase.
3. DNA polymerase (III) proceeds along a single-stranded molecule of DNA, recruiting
free dNTP's (deoxy-nucleotide-triphosphates) to hydrogen bond with their appropriate
complementary dNTP on the single strand (A with T and G with C), and to form a covalent
phosphodiester bond with the previous nucleotide of the same strand. DNA
polymerases cannot start synthesizing de novo on a bare single strand. It needs a primer
with a 3'OH group onto which it can attach a dNTP.
DNA polymerase also has proofreading activities, so that it can make sure that it inserted
the right base, and nuclease (excision of nucleotides) activities so that it can cut away any
smistakes it might have made.
4. Primase: This enzyme attaches a small RNA primer to the single-stranded DNA to act as
a substitute 3'OH for DNA polymerase to begin synthesizing from. This RNA primer is
eventually removed and the gap is filled in by DNA polymerase (I).
5. Ligase can catalyze the formation of a phosphodiester bond given an unattached but
adjacent 3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA
primer is removed and filled in.
6. Single-stranded binding proteins are important to maintain the stability of the replication
fork. Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it
remains single stranded and keep it from being degraded.
Rolling circle Replication
In 1968, the rolling circle model of DNA replication was first proposed. This model
describes the DNA replication mechanism in circular plasmids and single-stranded circular
DNA of viruses.
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 Numerous plasmids replicate independently of chromosomal replication. Numerous

circularly closed plasmids reproduce autonomously using a process known as rolling
circle replication. Unidirectional replication is another name for rolling circle
replication.
 Certain circular DNAs reproduce not by the theta (θ) mode we have just studied, but by
the rolling circle replication process.
 The intermediates gave this method the name “rolling circle” because the double-
stranded portion of the replicating DNA can be compared to a roll of toilet paper
unwinding as it moves across the floor.
 This mechanism is sometimes referred to as the θ mode, to differentiate it from the
mode, because this intermediate resembles an upside-down Greek letter σ (sigma).
 The rolling circle mechanism is not limited to single-stranded DNA synthesis. This
technique is utilised by certain phages (e.g., λ) to replicate double-stranded DNA.
 During the initial step of λ DNA replication, the phage replicates using the θ mode to
produce multiple copies of circular DNA.
 These circular DNAs are not packed into phage particles; instead, they act as templates
for the rolling circle synthesis of packaged linear λ DNA molecules.
 Here, the replicating fork resembles that of E. coli DNA replication considerably more,
with continuous synthesis on the leading strand (the one moving around the circle) and
discontinuous synthesis on the trailing strand.
 Before it is packed, the progeny DNA in λ reaches lengths that are many genomes long.
 Multiple-length DNAs are referred to as concatemers. In order to provide each phage
head with one genome’s worth of linear DNA, the concatemer is cut enzymatically at
the cos sites bordering each full λ genome on the concatemer.
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UNIT- II
GENES STRUCTURE AND EXPRESSION

Prokaryotic genomes organisation
Most of the well-characterized prokaryotic genomes consist of double-stranded DNA
organized as a single circular chromosome 0.6-10 Mb in length and one or more circular
plasmid species of 2 kb-1.7 Mb. The past few years, however, have revealed some major
variations in genome organization. Each bacterial chromosome is made by a single circular
DNA molecule (rarely linear). • Usually each cell contain one single copy of each chromosome.
• The genetic material can be seen as a fairly compact clump (or series of clumps) that occupies
about a third of the volume of the cell named NUCLEOID. • The DNA of these loops is not
found in the extended form of a free duplex, but instead is compacted by association with
proteins.
Eukaryotic genome organization
 Each eukaryotic chromosome is made by a single linear DNA molecule.

 Chromosomes are made of chromatin, some other proteins and are located on the
nucleus.
 The cell can have one single copy (haploid), two (diploid) or multiple (polypoid) copies
of each chromosome.
 They can be directly seen only during cell mitosis. Single chromosomes can be seen
only during mitosis.
 In interphase chromatin is present as:  Euchromatin (less dens): expressed genes (not
all)  Heterochromatin (more dens): constitutive.
 Single chromosomes can be seen only during mitosis.
 In interphase chromatin is present as:  Euchromatin (less dens): expressed genes (not
all)  Heterochromatin (more dens): constitutive.
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Gene Structure
Prokaryotes vs. Eukaryotes
1. In prokaryotes, genes tend to be clustered in coordinately-regulated groups called operons.

The genes are transcribed together on a single transcript and each protein within the cluster
is translated separately. Prokaryotes can "couple" transcription and translation - i.e. a
mRNA being transcribed can begin being translated even before transcription is complete.
2. In eukaryotes, genes are not clustered in operons. In addition, eukaryotic genes often contain
non-coding introns ("intervening sequences") interspersed among the coding regions
(exons).During RNA processing, introns are removed from RNA transcripts and the exons
are spliced together. Mature mRNA, after being transcribed and processed in the nucleus,
is transported into the cytoplasm where translation occurs. Because transcription and
translation occur in different "compartments" in eukaryotes, "coupling" of these two
processes is not possible.
Transcription in Prokaryotes and Eukaryotes.
 Transcription involves the construction of an RNA copy of the genetic information in

a DNA template
 Eukaryotic transcription is generally more complex than prokaryotic transcription.
 mRNA synthesis and processing in eukaryotes
Capping - a "cap" is added to the 5' end of eukaryotic RNA transcripts. A methyl group is
added to the 2' hydroxyl group of the 5' terminal base of the transcript and attach a 7-methyl-
guanosine "cap" to the 5' triphosphate end via a 5'--->5' linkage.
Addition of a poly-A tail (hundred adenosine) residues to the 3' end of the transcript.
a. Suppose poly-A is not added - without a poly-A tail, intron splicing will not occur, and
the mRNA will not be transported out of the nucleus.
Removal of introns - introns are cut out of mRNA and the remaining exons are spliced
together.
Export to the cytoplasm - processed transcripts travel out of the nucleus and enter the
cytoplasm where they will be translated.
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Transcription in Prokaryotes
 The process of synthesis of RNA by copying the template strand of DNA is called
transcription.
 During replication entire genome is copied but in transcription only the selected portion
of genome is copied.
 The enzyme involved in transcription is RNA polymerase. Unlike DNA polymerase it
can initiate transcription by itself, it does not require primase. More exactly it is a DNA
dependent RNA polymerase.
The steps of transcription

Transcription is an enzymatic process. The mechanism of transcription completes in three
major steps
1. Initiation:
 closed complex formation
 Open complex fromation
 Tertiary complex formation
2. Elongation
3. Termination
Initiation:
 The transcription is initiated by RNA polymerase holoenzyme from a specific point
called promotor sequence.
 Bacterial RNA polymerase is the principle enzyme involved in transcription.
 Single RNA polymerase is found in a bacteria which is called core polymerase and it
consists of α, β, β’ and ω sub units.
 The core enzyme bind to specific sequence on template DNA strand called promotor.
The binding of core polymerase to promotor is facilitates and specified by sigma (σ)
factor. (σ70 in case of E. coli).
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 The core polymerase along with σ-factor is called Holo-enzyme ie. RNA polymerase
holoenzyme.
 In case of e. coli, promotor consists of two conserved sequences 5’-TTGACA-3’ at -35
element and 5’-TATAAT-3’ at -10 element. These sequence are upstream to the site
from which transcription begins. Binding of holoenzyme to two conserve sequence of
promotor form close complex.
 In some bacteria, the altered promotor may exist which contain UP-element and some
may contain extended -10 element rather than -35 element.
2.
Elongation:
 After synthesis of RNA more than 10 bp long, the σ-factor is ejected and the enzyme
move along 5’-3’ direction continuously synthesizing RNA.
 The synthesized RNA exit from RNA exit channel.
 The synthesized RNA is proof reads by Hydrolytic editing. For this the polymerase
back track by one or more nucleotide and cleave the RNA removing the error and
synthesize the correct one. The Gre factor enhance this proof reading process.
 Pyrophospholytic editing another mechanism of removing altered nucleotide.
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3. Termination:
There are two mechanism of termination.
Rho independent:
 In this mechanism, transcription is terminated due to specific sequence in terminator
DNA.
 The terminator DNA contains invert repeat which cause complimentary pairing as
transcript RNA form hair pin structure.
 This invert repeat is followed by larger number of TTTTTTTT (~8 bp) on template
DNA. The uracil appear in RNA. The load of hair pin structure is not tolerated by A=U
base pair so the RNA get separated from RNA-DNA heteroduplex.
 ii. Rho dependent: In this mechanism, transcription is terminated by rho (ρ) protein.
 It is ring shaped single strand binding ATpase protein.
 The rho protein bind the single stranded RNA as it exit from polymerase enzyme
complex and hydrolyse the RNA from enzyme complex.
 The rho protein does not bind to those RNA whose protein is being translated. Rather
it bind to RNA after translation.
 In bacteria transcription and translation occur simultaneously so the rho protein bind
the RNA after translation has completed but transcription is still ON.
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Eukaryotic Transcription
 Transcription is the process by which the information in a strand of DNA is copied into
a new molecule of RNA.
 It is the first step of gene expression, in which a particular segment of DNA is copied
into RNA (especially mRNA) by the enzyme RNA polymerase.
 It results in a complementary, antiparallel RNA strand called a primary transcript.
Transcription in Eukaryotes
Transcription occurs in eukaryotes in a way that is similar to prokaryotes with reference to the
basic steps involved. However, some major differences between them include:
 Initiation is more complex.
 Termination does not involve stem-loop structures.
 Transcription is carried out by three enzymes (RNA polymerases I, II and III).
 The regulation of transcription is more extensive than prokaryotes.
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Enzyme(s) Involved in Eukaryotic Transcription

Unlike prokaryotes where all RNA is synthesized by a single RNA polymerase, the nucleus of
a eukaryotic cell has three RNA polymerases responsible for transcribing different types of
RNA.
 RNA polymerase I (RNA Pol I) is located in the nucleolus and transcribes the 28S,
18S, and 5.8S rRNA genes.
 RNA polymerase II (RNA Pol II) is located in the nucleoplasm and transcribes
protein-coding genes, to yield pre-mRNA, and also the genes encoding small nucleolar
RNAs (snoRNAs) involved in rRNA processing and small nuclear RNAs (snRNAs)
involved in mRNA processing, except for U6 snRNA.
 RNA polymerase III (RNA Pol III) is also located in the nucleoplasm. It transcribes
the genes for tRNA, 5S rRNA, U6 snRNA, and the 7S RNA associated with the signal
recognition particle (SRP) involved in the translocation of proteins across the
endoplasmic reticulum membrane.
 Each of the three eukaryotic RNA polymerases contains 12 or more subunits and so
these are large complex enzymes.
 The genes encoding some of the subunits of each eukaryotic enzyme show DNA
sequence similarities to genes encoding subunits of the core enzyme of E. coli RNA
polymerase.
 However, four to seven other subunits of each eukaryotic RNA polymerase are unique
in that they show no similarity either with bacterial RNA polymerase subunits or with
the subunits of other eukaryotic RNA polymerases.
Features of Eukaryotic Transcription

 Transcription in eukaryotes occurs within the nucleus and mRNA moves out of the
nucleus into the cytoplasm for translation.
 The initiation of RNA synthesis by RNA polymerase is directed by the presence of a
promoter site on the 5’ side of the transcriptional start site.
 The RNA polymerase transcribes one strand, the antisense (-) strand, of the DNA
template.
 RNA synthesis does not require a primer.
 RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase catalyzing a
nucleophile attack by the 3-OH of the growing RNA chain on the alpha-phosphorus
atom on an incoming rib nucleoside 5-triphosphate.
 mRNA in eukaryotes is processed from the primary RNA transcript, a process called
maturation.
Process of Eukaryotic Transcription

The basic mechanism of RNA synthesis by these eukaryotic RNA polymerases can be divided
into the following phases: Initiation Phase
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 During initiation, RNA polymerase recognizes a specific site on the DNA, upstream
from the gene that will be transcribed, called a promoter site and then unwinds the
DNA locally.
 Most promoter sites for RNA polymerase II include a highly conserved sequence
located about 25–35 bp upstream (i.e. to the 5 side) of the start site which has the
consensus TATA(A/T)A(A/T) and is called the TATA box.
 Since the start site is denoted as position +1, the TATA box position is said to be located
at about position -25.
 The TATA box sequence resembles the -10 sequence in prokaryotes (TATAAT) except
that it is located further upstream.
 Both elements have essentially the same function, namely recognition by the RNA
polymerase in order to position the enzyme at the correct location to initiate
transcription.
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 The sequence around the TATA box is also important in that it influences the efficiency
of initiation. Transcription is also regulated by upstream control elements that lie 5′ to
the TATA box.
 Some eukaryotic protein-coding genes lack a TATA box and have an initiator element
instead, centered around the transcriptional initiation site.
 In order to initiate transcription, RNA polymerase II requires the assistance of several
other proteins or protein complexes, called general (or basal) transcription factors,
which must assemble into a complex on the promoter in order for RNA polymerase to
bind and start transcription.
 These all have the generic name of TFII (for Transcription Factor for RNA polymerase
II).
 The first event in initiation is the binding of the transcription factor IID (TFIID) protein
complex to the TATA box via one its subunits called TBP (TATA box binding protein).
 As soon as the TFIID complex has bound, TFIIA binds and stabilizes the TFIID-TATA
box interaction. Next, TFIIB binds to TFIID.
 However, TFIIB can also bind to RNA polymerase II and so acts as a bridging protein.
Thus,
 RNA polymerase II, which has already complexed with TFIIF, now binds.
 This is followed by the binding of TFIIE and H. This final protein complex contains at
least 40 polypeptides and is called the transcription initiation complex.
 Those protein-coding genes that have an initiator element instead of a TATA box
appear to need another protein(s) that binds to the initiator element.
 The other transcription factors then bind to form the transcription initiation complex in
a similar manner to that described above for genes possessing a TATA box promoter.
Elongation Phase
TFIIH has two functions:
1. It is a helicase, which means that it can use ATP to unwind the DNA helix, allowing
transcription to begin.
2. In addition, it phosphorylates RNA polymerase II which causes this enzyme to change its
conformation and dissociate from other proteins in the initiation complex.
 The key phosphorylation occurs on a long C-terminal tail called the C-terminal domain
(CTD) of the RNA polymerase II molecule.
 Interestingly, only RNA polymerase II that has a non-phosphorylated CTD can initiate
transcription but only an RNA polymerase II with a phosphorylated CTD can elongate
RNA.
 RNA polymerase II now starts moving along the DNA template, synthesizing RNA,
that is, the process enters the elongation phase.
 RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase catalyzing a
nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus
atom on an incoming ribonucleoside 5-triphosphate.
 The RNA molecule made from a protein-coding gene by RNA polymerase II is called
a primary transcript.
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Termination Phase
 Elongation of the RNA chain continues until termination occurs.

 Unlike RNA polymerase in prokaryotes, RNA polymerase II does not terminate
transcription at a specific site but rather transcription can stop at varying distances
downstream of the gene.
 RNA genes transcribed by RNA Polymerse II lack any specific signals or sequences
that direct RNA Polymerase II to terminate at specific locations.
 RNA Polymerase II can continue to transcribe RNA anywhere from a few bp to
thousands of bp past the actual end of the gene.
 The transcript is cleaved at an internal site before RNA Polymerase II finishes
transcribing. This releases the upstream portion of the transcript, which will serve as
the initial RNA prior to further processing (the pre-mRNA in the case of protein-
encoding genes.)
 This cleavage site is considered the “end” of the gene. The remainder of the transcript
is digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed
by the RNA Polymerase II.
 When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the
overhanging RNA, it helps disengage the polymerase from its DNA template strand,
finally terminating that round of transcription.
Post transcriptional Modification

RNA processing
The primary eukaryotic mRNA transcript is much longer and localised into the nucleus,
when it is also called heterogenous nuclear RNA (hnRNA) or pre- mRNA.
It undergoes various processing steps to change into a mature RNA:
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Cleavage
 Larger RNA precursors are cleaved to form smaller RNAs.
 Primary transcript is cleaved by ribonuclease-P (an RNA enzyme) to form 5-7 tRNA
precursors.
Capping and Tailing

 Initially at the 5′ end a cap (consisting of 7-methyl guanosine or 7 mG) and a tail of
poly A at the 3′ end are added.
 The cap is a chemically modified molecule of guanosine triphosphate (GTP).
Splicing
 The eukaryotic primary mRNAs are made up of two types of segments; non-coding
introns and the coding exons.
 The introns are removed by a process called RNA splicing where ATP is used to cut
the RNA, releasing the introns and joining two adjacent exons to produce mature
mRNA.
Translation in Prokaryotes
 It is the process of synthesis of protein by encoding information on mRNA.
 Protein synthesis requires mRNA, tRNA, aminoacids, ribosome and enzyme aminoacyl
tRNA synthase
Various protein factors involved in protein synthesis
Factors Translation steps Functions
IF-1 Initiation Helps to stabilize 30S ribosomal subunit
Binds fmet-tRNA with 30S subunit mRNA

IF-2 Initiation complex; bind GTP and hydrolyse
IF-3 Initiation Binds 30S subunit with mRNA
Binds GTP; bring Aminoacyl-tRNA to A-site

EF-TU Elongation of ribosome
EF-TS Elongation Generates EF-TU
EF-G Elongation Helps in translocation of ribosome
Helps to dissociates polypeptide from tRNA

ribosome complex; specific for UAA and
RF-1 Termination UAG
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Helps to dissociates polypeptide; specific for

RF-2 Termination UGA and UAA
RF-3 Termination Stimulates RF-1 and RF-2
Steps in translation:
1. Activation of aminoacids:
 The activation of aminoacids take place in cytosol.

 The activation of aminoacids is catalyzed by their aminoacyl tRNA synthetases.
 All the 20 aminoacids are activated and bound to 3’ end of their specific tRNA in the
presence of ATP and Mg++.
 The N-formylated methionine is chain initiating aminoacid in bacteria whereas
methionine is chain initiating aminoacid in eukaryotes.
 Methionine is activated by methionyl-tRNA synthetase. For N-formylmethionine two
types of tRNA are used ie. tRNAmet and tRNAfmet.
 Similarly, all 2o aminoacids are activated (amino acyl-AMP enzyme complex) and then
bound to their specific tRNA forming Aminoacyl tRNA.
2. Initiation:
 In the first step, initiation factor-3 (IF-3) binds to 30S ribosomal unit.
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 Then mRNA binds to 30S ribosomal subunit in such a way that AUG codon lie on the
peptidyl (P) site and the second codon lies on aminoacyl (A) site.
 The tRNA carrying formylated methionine ie. FMet–tRNAFMet is palced at P-site.
This specificity is induced by IF-2 with utilization of GTP. The IF-1 prevent binding
of FMet–tRNAFMet is in A-site.
 Shine dalgarno sequence in the mRNA guide correct positioning of AUG codon at P-
site of 30S ribosome.
 After binding of FMet–tRNAFMet on P-site, IF-3, IF-2 and IF-1 are released so that 50S
ribosomal unit bind with 30S forming 70S sibosome. The exit site is located in 50S.
3. Elongation:
i. Binding of AA-tRNA at A-site:
 The 2nd tRNA carrying next aminoacid comes into A-site and recognizes the codon on
mRNA. This binding is facilitated by EF-TU and utilizes GTP.
 After binding, GTP is hydrolysed and EF-TU-GDP is releasd
 EF=TU-GDP then and enter into EF-TS cycle.
ii. Peptide bond formation:
 The aminoacid present in t-RNA of P-site ie Fmet is transferred to t-RNA of A-site
forming peptide bond. This reaction is catalyzed by peptidyltransferase.
 Now, the t-RNA at P-site become uncharged
iii. Ribosome translocation:
 After peptide bond formation ribosome moves one codon ahead along 5’-3’ direction
on mRNA, so that dipeptide-tRNA appear on P-site and next codon appear on A-site.
 The uncharged tRNA exit from ribosome and enter to cytosol.
 The ribosomal translocation requires EF-G-GTP (translocase enzyme) which change
the 3D structure of ribosome and catalyze 5’-3’ movement.
 The codon on A-site is now recognized by other aminoacyl-tRNA as in previous.
 The dipeptide on P-site is transferred to A-site forming tripeptide.
 This process continues giving long polypeptide chain of aminoacids.
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4. Termination:
 The peptide bond formation and elongation of polypeptide continues until stop codon
appear on A-site.
 If stop codon appear on A-site it is not recognized by t-RNA carrying aminoacids
because stop codon donot have anticodon on mRNA.
 The stop codon are recognized by next protein called release factor (Rf-1, RF-2 and
RF-3) which hydrolyses and cause release of all component ie 30s, 50S, mRNA and
polypeptide separates.
 RF-1 recognisaes UAA and UAg while RF-2 recognises UAA and UGA while RF-3
dissociate 30S and 50S subunits.
 In case of eukaryotes only one release actor eRF causes dissociation.
Post translational modifications
The newly formed polypeptide may not be biologiy functional so it undergoes several
folding and processing known as post translation modification.
1. Amino terminal and carboxyl terminal modification:

The N-formylmethionine in case of bacteria is removed from polypeptide chain and some
carboxyl terminal are also removed by enzymatic action to make functional protein. In case
of eukaryotic protein, amino terminal is N- acetylated.
2. Loss of signal sequences:
In some protein the amino terminal end is cleaved by specific peptidase so that protein loss
its signaling property.
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3. Modification of individual aminoacids:

The aminoacids may be phosphorylated, acetylated for modification
4. Attachment of carbohydrate side chain:
Carbohydrate side chain is added to make protein functional. Eg, glycoprotein. Lipoprotein
5. Addition of isoprenyl group:
In some protein, isoprenyl group is added so to make protein active.
Post-translational modifications (PTMs) mainly occur in the endoplasmic reticulum of

the cell. After synthesis is completed, proteins can be modified by various methods such as
phosphorylation, glycosylation, ADP ribosylation, hydroxylation, and addition of other groups.
1. Proteolysis
As the newly synthesized protein is released in the lumen of the ER, signal peptidases
cleave peptide sequence. Apart from signal peptide, some polypeptide sequence of the protein
is also cleaved resulting in the final sequence.
Example:
Insulin is synthesized in the cells in its inactive form which cannot perform its function.
Post translational modifications ensure proper function which involves the removal of the part
of protein to convert it into a three dimensional and fully active form.
2. Phosphorylation
Phosphorylation is the addition of one or more phosphate groups to the protein. Post
Translational Phosphorylation is one of the most common protein modifications that occur in
animal cells. Majority of phosphorylation occurs as a mechanism to regulate the biological
activity of a protein. In animal cells Serine, tyrosine and thereonine are the amino acids that
subjected to the phosphorylation.
3. Glycosylation
Glycosylation is the addition of carbohydrate molecules to the polypeptide chain and
modifying it into glycoproteins. Many of the proteins that are destined to become a part of
plasma membrane or to be secreted from the cell, have carbohydrate chains attached to the
amide nitrogen of asparagine(N linked) or the hydroxyl groups of serine, threonine(O linked).
N glycosylation occurs in ER and O glycosylation occurs in the golgi complex.
4. Sulfation
Sulfate modidication takes place by the addition of sulphate molecules and these
modifications of proteins occurs at tyrosine residues. Tyrosine sulfation accomplished via the
activity of tyrosylproteinsulfotransferases (TPST) which are membrane associated enzymes of
trans-Golgi network. There are two known TPSTs. TPST-1 TPST-2 The universal phosphate
donor is 3’-phosphoadenosyl- 5’-phosphosulphate (PSPA). phosphoadenosyl- 5’-
phosphosulphate (PSPA).
5. Methylation
The transfer of one-carbon methyl groups to nitrogen or oxygen to amino acid side
chains increases the hydrophobicity of the protein and can neutralize a negative amino acid
charge when bound to carboxylic acids. Methylation is mediated by methyltransferases and S-
adenosyl methionine (SAM) is the primary methyl group donor.
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6. Hydroxylation
The biological process of addition of a hydroxy group to a protein amino acid is called
Hydroxylation. Protein hydroxylation is one type of PTM that involves the conversion of –CH
group into –COH group and these hydroxylated amino acids are involved in the regulation of
some important factors called transcription factors. Among the 20 amino acids, the two amino
acids regulated by this method are proline and lysine.
Eukaryotic Translation
 Eukaryotic translation is the biological process by which messenger RNA is translated
into proteins in eukaryotes.
 It consists of four phases: gene regulation, elongation, termination, and recycling.
Eukaryotic initiation factor
Eukaryotic initiation factors (eIFs) are proteins or protein complexes involved in the
initiation phase of eukaryotic translation. These proteins help stabilize the formation of
ribosomal preinitiation complexes around the start codon and are an important input for post-
transcription gene regulation.
Steps of Eukaryotic Translation
The eukaryotic translation is completed in the following steps;

 Initiation of Translation
 Elongation of peptide chain
 Termination of peptide chain
Initiation of Translation
The initiation of translation in eukaryotes is complex, involving at least ten eukaryotic initiation
factors (eIFs). Some of the eIFs contain multiple (3-8) subunits. The process of translation
initiation can be divided into four steps.
 Ribosomal dissociation.
 Formation of 43S preinitiation complex.
 Formation of 48S initiation complex.
 Formation of 80S initiation complex.
Ribosomal dissociation
 The 80S ribosome dissociates to form 40S and 60S subunits.
 Two initiating factors namely eIF3 and eIF-1A bind to the newly formed 40S subunit,
and thereby block its reassociation with 60S subunit.
Formation of 43S preinitiation complex

 A ternary complex containing met-tRNAi and eIF-2 bound to GTP attaches to 40S
ribosomal subunit to form 43S preinitiation complex.
 The presence of eIF-3 and eIF-1A stabilizes this complex (Note : Met-tRNA is
specifically involved in binding to the initiation condon AUGs; hence the superscript
is used in met tRNAi).
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Formation of 48S initiation complex

 The binding of mRNA to 43S preinitiation complex results in the formation of 48S
initiation complex through the intermediate 43S initiation complex. This, however,
involves certain interactions between some of the eIFs and activation of mRNA.
 eIF-4F complex is formed by the association of eIF-4G, eIF-4A with eIF-4E.
 The so formed eIF-4F (referred to as cap binding protein) binds to the cap of mRNA.
 Then elF-4A and elF-4B bind to mRNA and reduce its complex structure.
 This mRNA is then transferred to 43S complex.
 For the appropriate association of 43S preinitiation complex with mRNA, energy has to
be supplied by ATP.
Recognition of initiation codon:

 The ribosomal initiation complex scans the mRNA for the identification of appropriate
initiation codon. 5c-AUG is the initiation codon and its recognition is facilitated by a
specific sequence of nucleotides surrounding it.
 This marker sequence for the identification of AUG is called as Kozak consensus
sequence.
 In case of prokaryotes the recognition sequence of initiation codon is referred to as
Shine- Dalgarno sequence.
Formation of 80S initiation complex

 48S initiation complex binds to 60S ribosomal subunit to form 80S initiation complex.
 The binding involves the hydrolysis of GTP (bound to eIF-2). This step is facilitated by
the involvement of eIF-5.
 As the 80S complex is formed, the initiation factors bound to 48S initiation complex
are released and recycled.
 The activation of eIF-2 requires eIF-2B (also called as guanine nucleotide exchange
factor) and GTP.
 The activated eIF-2 (i.e. bound to GTP) requires eIF2C to form the ternary complex.
Regulation of initiation
 The eIF-4F, a complex formed by the assembly of three initiation factors controls
initiation, and thus the translation process.
 eIF4E, a component of eIF-4F is primarily responsible for the recognition of mRNA
cap. And this step is the rate-limiting in translation.
 eIF-2 which is involved in the formation of 43S preinitiation complex also controls
protein biosynthesis to some extent.
Elongation of Translation
Ribosomes elongate the polypeptide chain by a sequential addition of amino acids. The
amino acid sequence is determined by the order of the codons in the specific mRNA. Elongation,
a cyclic process involving certain elongation factors (EFs), may be divided into three steps.
 Binding of aminoacyl t-RNA to A-site.
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 Peptide bond formation.

 Translocation.
Binding of aminoacyl—tRNA to A-site
 The 80S initiation complex contains met-tRNAi in the P-site, and the A-site is free.
 Another aminoacyl-tRNA is placed in the A-site. This requires proper codon
recognition on the mRNA and the involvement of elongation factor 1a (EF-Ia) and
supply of energy by GTP.
 As the aminoacyl-tRNA is placed in the A-site, EF-1D and GDP are recycled to bring
another aminoacyl-tRNA.
Peptide bond formation

 The enzyme peptidyltransferase catalyses the formation of peptide bond.
 The activity of this enzyme lies on 28S RNA of 60S ribosomal subunit. It is therefore
the rRNA (and not protein) referred to as ribozyme that catalyses the peptide bond
formation.
 As the amino acid in the aminoacyl-tRNA is already activated, no additional energy is
required for peptide bond formation. The net result of peptide bond formation is the
attachment of the growing peptide chain to the tRNA in the A-site.
Translocation
 As the peptide bond formation occurs, the ribosome moves to the next codon of the
mRNA (towards 3c-end). This process called translocation, basically involves the
movement of growing peptide chain from A-site to P-site.
 Translocation requires EF-2 and GTP.
 GTP gets hydrolysed and supplies energy to move mRNA. EF-2 and GTP complex
recycles for translocation.
 In recent years, another site namely exit site (E-site) has been identified in eukaryotes.
The deacylated tRNA moves into the E-site, from where it leaves the ribosome.
 In case of prokaryotes, the elongation factors are different, and they are EF-Tu, EF-Ts
(in place of of EF-1a) and EF-G (instead of EF-2).
Termination of Translation
Termination is a simple process when compared to initiation and elongation. After

several cycles of elongation, incorporating amino acids and the formation of the specific protein/
polypeptide molecule, one of the stop or termination signals (UAA, UAG and UCA) terminates
the growing polypeptide.
The termination codons which act as stop signals do not have specific tRNAs to bind.
As theb termination codon occupies the ribosomal A-site, the release factor namely eRF
recognizes the stop signal. eRF-GTP complex, in association with the enzyme
peptidyltransferase, cleaves the peptide bond between the polypeptide and the tRNA occupying
P-site. In this reaction, a water molecule, instead of an amino acid is added. This hydrolysis
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releases the protein and tRNA from the P-site. The 80S ribosome dissociates to form 40S and
60S subunits which are recycled. The mRNA is also released.
Gene Regulation in Prokaryotes

Gene regulation in prokaryotes is most extensively observed at the initiation of transcription.
Thus, the gene expression during transcription initiation is affected by regulation. The
regulation usually takes place in the expression of the RNA polymerase at the promoter site.
This affects the accessory proteins which bind to the recognition sites. These accessory proteins
can regulate the promoter site in two ways:
 Positive regulation by activators

 Negative regulation by repressors
In Operons, the operator is situated right next to the promoter where the regulator binds to
control its entire functioning.
Lac Operon
“Lac operon is an operon or a group of genes with a single promoter that encode genes
for the transport and metabolism of lactose in E.coli and other bacteria.”
Lac Operon Concept
Gene regulation in prokaryotes can be explained with the help of the Lac Operon model.
Here the alteration in physiological and environmental conditions can be observed leading to
an alteration in expression in prokaryotes. It was observed by Jacob and Monod. The lac operon
consists of:
 Regulatory gene i – It codes for the repressor protein.

 Z gene – It codes for beta-galactosidase which catalyzes the hydrolysis of lactose into
glucose and galactose.
 Y gene – It codes for permease which regulates the lactose permeability in the cell.
 A gene – It codes for transacetylase which assists the enzyme beta-galactosidase.
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Hence, all these genes help in lactose metabolism. In lac operon, lactose acts as an
inducer. If lactose is provided in the medium for the bacteria, the regulatory gene is activated.
The inducer will bind to the repressor protein and render it inactive which allows transcription
of the operon. Thus, the lac operon is negatively regulated in this case.
In bacteria the expression of genes is controlled by extracellular signals often present
in the medium in which bacteria are grown. These signals are carried to the genes by regulatory
proteins. Regulatory proteins are of two types. They are positive regulators called activators
and negative regulators called repressors. These activators and repressors are DNA binding
proteins.
Negative Regulators or Repressors:

The repressor or inhibitor protein binds to the target site (operator) on DNA. These
block the RNA polymerase enzyme from binding to the promoter, thus preventing the
transcription. The repressor binds to the site where it overlaps the polymerase enzyme. Thus,
activity of the genes is turned off. It is called negative control mechanism.
An anti-repressor or anti-inhibitor called inducer is needed to inactivate the repressor

and thereby activating the genes. Thus, the genes are switched on. This is demonstrated by
lactose operon.
The lac operon in E. coli has more complex regulation, involving both a repressor and
an activator. E. coli uses glucose for food, but is able to use other sugars, such as lactose, when
glucose concentrations are low. Three proteins are needed to break down lactose; they are
encoded by the three genes of the lac operon.
When lactose is not present, the proteins to digest lactose are not needed. Therefore, a
repressor binds to the operator and prevents RNA polymerase from transcribing the operon.
When lactose is present, lactose binds to the repressor and removes it from the operator.
RNA polymerase is now free to transcribe the genes necessary to digest lactose (Figure 17.4)
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Figure 17.4 Transcription of the lac operon only occurs when lactose is present. Lactose
binds to the repressor and removes it from the operator.
Positive Regulators or Activators:
To activate the transcription by the promoter, the activator helps polymerase enzyme to bind
to the promoter.Genes under positive control mechanism are expressed only when an activator
or stimulator or active regulator is present.
Lac operon also shows positive control by catabolic repression. This is an additional
control system, which binds the repressor-operator. In E. coli, in the presence of both glucose
and lactose, the glucose in first fully utilized and then lactose is taken up for production of
energy. Glucose is richest and more efficient source of energy. Glucose has an inhibitory effect
on the expression of lac operon. The mechanism of positive control enables E. coli to adapt
more efficiently to the changing environment of its natural habitat, which is the human
intestine. In the presence of glucose, synthesis of β-galactosidase enzyme becomes suppressed.
The inhibitory effect of glucose is due to the marked drop in the level of a nucleotide called
cyclic AMP (c-AMP), which inhibits the transcription of mRNA.
Lactose operon transcription requires not only cyclic AMP but also another protein
called catabolic activator protein (CAP). The cAMP and CAP form a complex called cAMP-
CRP complex, which is necessary for the functioning of lactose operon.
A catabolic breakdown product of glucose, called glucose catabolite, prevents the

activation of lac operon by lactose. This effect is called catabolic repression. When glucose
concentration increases, the cAMP concentration decreases and vice versa. High concentration
of cAMP is necessary for the activation of lac operon.
Normally in the presence of glucose, the lactose operon remains inactive.Glucose

catabolite prevents the formation cAMP-CRP complex. When glucose levels drop, cyclic AMP
(cAMP) begins to accumulate in the cell. cAMP binds to CAP and the complex binds to
the lac operon promoter (Figure). This increases the binding ability of RNA polymerase to the
promoter and ramps up transcription of the genes.
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Figure 17.5 When there is no glucose, the CAP activator increases transcription of the lac
operon. However, if no lactose is present, the operon is not activated.
Tryptophan Operon:
It is also a negative control system but forms a biosynthetic pathway. It is known as
repressible system. It works on the principle that when the amino acid tryptophan is present,
there is no need to activate the tryptophan operon.
Repressor protein is activated by the co-repressor (tryptophan-the end product) and it

binds the operator to switch it “off’. Tryptophan is synthesized in five steps, each step requiring
a particular enzyme. The genes for encoding these enzymes lie adjacent to one another, called
trp E, trp D, trp C, trp B and trp A.
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Tryptophan operon codes for five enzymes that are required for the synthesis of amino
acid tryptophan. In repressible system, the regulatory gene produces a repressor protein, which
is normally inactive and unable to bind to operator on DNA. The repressor upon joining the
co-repressor (which is the end product tryptophan in this case) undergoes conformational
changes that activate it and enable it to bind to the operator. This prevents the binding of RNA
polymerase enzyme to the promoter. This is opposite to the situation of lac operon in which
the repressor is active on its own and loses the affinity for the operator when bound to the
inducer.Here the availability of tryptophan which is the end product regulates the expression
of this operon and represses the synthesis of tryptophan. In this way the synthesis of enzymes
of a metabolic pathway is stopped by the end product of the metabolic chain. This mechanism
enables the bacteria to synthesize enzymes only when they are required. This is known as feed
back repression.
The trp operon

It is an operon of anabolic metabolism—a group of genes that is transcribed, together—
those codes for the components for production of tryptophan. The trp operon is present in many
bacteria, but was first characterized in Escherichia coli. The operon is regulated so that when
tryptophan is present in the environment, the genes for tryptophan synthesis are not expressed.
If the tryptophan absents in the medium, the genes for tryptophan synthesis are expressed.
Trp operon contains five structural genes: trpE, trpD, trpC, trpB, and trpA, which
encode enzymatic parts of the pathway. It also contains a repressive regulator gene called trpR.
trpR has a promoter where RNA polymerase binds and synthesizes mRNA for a regulatory
protein. The protein that is synthesized by trpR then binds to the operator which then causes
the transcription to be blocked. In the trp operon, tryptophan binds to the repressor protein
effectively blocking gene transcription. In this situation, repression is that of RNA polymerase
transcribing the genes in the operon. The trp operon contains a leader peptide and an attenuator
sequence which allows for graded regulation.
It is an example of repressible negative regulation of gene expression.

Trp operon contains five structural genes. Their roles are:
 TrpE: Anthranilate synthase produces anthranilate.
 TrpD: Cooperates with TrpE.
 TrpC: Phosphoribosyl anthranilate isomerase domain first turns N anthranilate into 1-
deoxy-D-ribulose 5-phosphate. The Indole-3-glycerol-phosphate synthase on the same
protein then turns the product into glycerol 3-phosphate.
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 TrpA, TrpB : two subunits of tryptophan synthetase. Combines TrpC's product with
serine to produce tryptophan.
Repression
The operon operates by a negative repressible feedback mechanism. The repressor for
the trp operon is produced upstream by the trpR gene, which is constitutively expressed at a
low level. Synthesized trpR monomers associate into dimers. When tryptophan is present,
these tryptophan repressor dimers bind to tryptophan, causing a change in the repressor
conformation, allowing the repressor to bind to the operator to form allorepressor. This
prevents RNA polymerase from binding to and transcribing the operon, so tryptophan is not
produced from its precursor.
When tryptophan is not present, the repressor is in its inactive conformation and
cannot bind the operator region, so transcription is not inhibited by the repressor.
Attenuation is a second mechanism of negative feedback in the trp operon. The

repression system targets the intracellular trp concentration whereas the attenuation responds
to the concentration of charged tRNAtrp. Thus, the trpR repressor decreases gene expression by
altering the initiation of transcription, While the TrpR repressor decreases transcription by a
factor of 70, attenuation can further decrease it by a factor of 10, thus allowing accumulated
repression of about 700-fold.
 Once RNA polymerase has started transcribing the operon, a ribosome can attach to the
still-forming transcript and begin translating the leader region. The polypeptide
encoded by the leader is short, just 141 amino acids long, and it includes two tryptophan
(Trp) residues^11start superscript, 1, end superscript. The tryptophans are important
because:
 If there is plenty of tryptophan, the ribosome won't have to wait long for a tryptophan-
carrying tRNA, and will rapidly finish the leader polypeptide.
 If there is little tryptophan, the ribosome will stall at the Trp codons (waiting for a Trp-
carrying tRNA) and will be slow to finish translation of the leader.
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At the beginning of the transcribed genes of the trp operon is a sequence of at least 130
nucleotides termed the leader transcript (trpL); the attenuation efficiency is correlated with the
stability of a secondary structure embedded in trpL, and the 2 constituent hairpins of the
terminator structure. This transcript includes four short sequences designated 1–4, each of
which is partially complementary to the next one. Thus, three distinct secondary structures
(hairpins) can form: 1–2, 2–3 or 3–4. However if the 1–2 hairpin were to form it would prevent
the formation of the 2–3 structure (but not 3–4). The formation of a hairpin loop between
sequences 2–3 prevents the formation of hairpin loops between both 1–2 and 3–4. The 3–4
structure is a transcription termination sequence (abundant in G/C and immediately followed
by several uracil residues), once it forms RNA polymerase will disassociate from the DNA and
transcription of the structural genes of the operon cannot occur.
If the ribosome attempts to translate this peptide while tryptophan levels in the cell are
low, it will stall at either of the two trp codons. While it is stalled, the ribosome physically
shields sequence 1 of the transcript, preventing the formation of the 1–2 secondary structure.
Sequence 2 is then free to hybridize with sequence 3 to form the 2–3 structure, which then
prevents the formation of the 3–4 termination hairpin, which is why the 2–3 structure is called
an anti-termination hairpin. In the presence of the 2–3 structure, RNA polymerase is free to
continue transcribing the operon. If tryptophan levels in the cell are high, the ribosome will
translate the entire leader peptide without interruption and will only stall during translation
termination at the stop codon. At this point the ribosome physically shields both sequences 1
and 2. Sequences 3 and 4 are thus free to form the 3–4 structure which terminates transcription.
This terminator structure forms when no ribosome stalls in the vicinity of the Trp tandem (i.e.
Trp codon): either the leader peptide is not translated or the translation proceeds smoothly
along the strand 1 with abundant charged tRNAtrp. The end result is that the operon will be
transcribed only when tryptophan is unavailable for the ribosome, while the trpL transcript is
constitutively expressed.
In attenuation, where the translating ribosome is stalled determines whether the

termination hairpin will be formed. To ensure that the ribosome binds and begins translation
of the leader transcript immediately following its synthesis, a pause site exists in the trpL
sequence. Upon reaching this site, RNA polymerase pauses transcription and apparently waits
for translation to begin. This mechanism allows for synchronization of transcript translation, a
key element in attenuation.
If the ribosome translates quickly, it will fall off the mRNA after translating the leader
peptide. This allows the terminator hairpin and an associated hairpin to form, making RNA
polymerase detach and ending transcription.
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UNIT III
MUTATION
Mutations mean any heritable changes occurs in the genetic materials. Mutations result
from errors during DNA replication or other types of damage to DNA (such as may be caused
by exposure to radiation, or error-prone repair.
Mutation refers to any change in the sequence of Nucleotides in DNA, which leads to changes
in the genotype and phenotype of an organism. Mutations can be induced or spontaneous.
Mutations can either be beneficial or harmful. Mutations lead to variation, which is one of the
driving forces of evolution.
Mutations occur due to deletion, insertion or substitution of one or more nucleotides.
When a purine nucleotide is replaced by a purine nucleotide it is known as transition, and when
a purine is replaced by a pyrimidine, it is known as transversion.
It may also result from insertion or deletion of segments of DNA due to transposable elements.
Mutations may or may not changes in the observable characteristics (phenotype) of an
organism. Mutations play a part in both normal and abnormal biological processes including:
evolution, cancer, and the development of the immune system.
Spontaneous Mutation
Spontaneous mutations occur naturally in the genome. They generally occur due to error
during replication, mitosis, meiosis, etc. Mutations may also occur due to mobile genetic
elements or transposons. The main causes of spontaneous mutations are:
 Replication errors
 Slipped strand mispairing
 Wobble base pairing
 Depurination or deamination
 Tautomerism
 Unequal crossing over
 DNA DAMAGE
Spontaneous Mutation It is a kind of alteration that happened in a DNA sequence called

DNA damage. It is a mutation, so mutation is the permanent alteration of the gene means it can
be inherited. Any permanent change, which is in a genetic material could be a substitution,
deletion of a nucleotide or it may be deletion of a chromosome fragment or it could be deletion
of the complete chromosome. These are all referred to as mutations. DNA damage occurs due
to two reasons: Spontaneous Mutation and Induce Mutation
 Spontaneous Mutation: Spontaneous Mutation occurs in the absence of any mutagens.
If there is no mutagen but still DNA got damaged then it is S Spontaneous Mutation.
How these damages could have happened when there is no mutagen. So, there is a
variety of reasons. The most important reason is an error occurs during DNA
replication.
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 Tautameric shift In DNA replication DNA pol makes 1 error after adding 105
nucleotides but it has proofreading activity so the error can be removed. After the
addition of 107 nucleotides, there could be 1 error solved by the DNA damage repair
system. So, 1 in 105 or 1 in 107 or 1 in 1010 there could be an error. So that tautomeric
shift occurs. Due to this, the base-pairing properties of nitrogenous base get changed.
 Slippage error This occurs on the site where repetitive sequences are present. When
DNA pol replicates repetitive sequence microsatellite or minisatellite which are
variable tendon repeated DNA pol gets slipped from the strand and due to this slippage
error occurs. In chromosome centromere and telomere are a hot spot for mutation
because of slippage error. If a mutation occurs in a repetitive sequence that causes an
autosomal disorder named Huntington disorder.
 Spontaneous deamination It makes changes in base-pairing properties. If Adenine
deamination occurs then it gets converted into Hypoxanthin. So, deamination is always
oxidative. Due to deamination in adenine =o comes then it could be referred to as
Hypoxanthin. Here the donor group changed into acceptor and the acceptor group
changed into the acceptor. In replication, if Adenine is in sequence, on adenine thymine
gets attached, but here adenine is deaminated so it converts into Hypoxanthin. Guanine
converts into Xanthin and this xanthin will not participate in hydrogen bonding with
anyone so it is a problem. If cytosine gets deaminated then it will convert into uracil.
So uracil will start base-pairing with adenine. d. Spontaneous Depurination In the
template DNA, phosphate sugar presents with these bases get attached. They are purine
on pyrimidine. There is the formation of the glycosidic bond between bases and sugar
which is the weakest bond. In comparison to pyrimidine, purine forms the weakest
glycosidic bond with sugar.
 So, when the temperature gets increases these bonds get broken and purin was removed
from sugar and Apurin site occurs. These are referred to as depurination.
Induced Mutation
Induced mutations do not occur spontaneously. They are induced through various chemical
and physical agents known as mutagens. Mutagens greatly enhance the frequency of
mutation.Some of the mutagens are:
 Alkylating agents (Ethyl methanesulfonate or EMS, N-ethyl-N-nitrosourea or ENU)

 Base analogue (5-Bromouracil, Bromodeoxyuridine)
 Hydroxylamine modifies bases
 Deamination by nitrous acid
 DNA intercalating agents (ethidium bromide, proflavine)
 Oxidative damage (Reactive oxygen species, e.g. superoxide radical, hydrogen
peroxide)
 Ionising and non-ionising radiations (gamma radiations, ultraviolet radiations, X-rays,
etc.)
DNA DAMAGE- spontaneous Mutation

It is a kind of alteration that happened in a DNA sequence called
DNA damage.
It is a mutation, so mutation is the permanent alteration of the gene
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means it can be inherited.

Any permanent change, which is in a genetic material could be a
substitution, deletion of a nucleotide or it may be deletion of a
chromosome fragment or it could be deletion of the complete
chromosome.
These are all referred to as mutations.
DNA damage occurs due to two reasons:
Spontaneous Mutation and Induce Mutati
DNA damage.
chromosome.
Spontaneous Mutation and Induce MutatiInduced mutation
Induced mutations are induced by known factor- such as-physical (ionizing irradiation,
ultraviolet light), chemical and biological mutagens (bacteria and viruses).
Mechanism of induced mutation:
Induced mutations occurs by at least three different mechanisms. They are-

 By replacing nitrogenous base with base analogs
 By base alteration-altering a base so that it specifically mispair with another base
 By distortion of DNA molecule- damaging a base so that it no longer pair with another
base
I. Incorporation of base analogs:
 Some chemical compounds are sufficiently similar to the normal nitrogenous bases of
DNA known as base analogs and they can incorporated into DNA in place of normal
bases.
 These analogs can base pair with other nitrogenous bases but they induce insertion of
incorrect nucleotide during replication causing mutation.
 Uracil is halogenated in the carbon-5 position to give 5-bromouracil, 5-chlorouracil,
and 5-iodouracil which can be incorporated into DNA in the place of thymine.
 5-bromouracil (5-BU) bromine is formed by bromination at the carbon-5 position of
uracil. The resulting structure of 5-bromouracil is similar to thymine but thymine has
CH3 group at C5 .
 5-Bromouracil is most effective analog to thymine because size of bromine has same
van der Waals radius as the methyl group in thymine.
 5-bromouracil is highly mutagenic and it pairs with Adenine in normal condition. In 5-
BU, the bromine atom is not in a position in which it can hydrogen-bond during base
pairing, so the keto form of 5-BU pairs with adenine.
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 However the frequency of tautomeric shift of 5-bromouracil is much higher than

Thymine. It changes from keto form to either enol form or an ionized from amino form
to keto form when protonated. Now, 5-bromouracil form hydrogen bond with Guanine
instead of complementary base Adenine which result in base pair transition from T=A
to C=G in subsequent replication cycle.
 Another commonly used base is 2-Aminopurine which is analog to adenine and pairs
with thymine by two hydrogen bond. After a tautomeric shift due to protonation, it can
form pair with cytosine.
 Therefore, 2-aminopurine can induce base pair transition from A=T to G=C in
subsequent replication cycle.
II. Base alteration: alkylation, depurination, deamination and hydroxylation

 In this case, the mutagens do not incorporate with the DNA, however they can causes
base alteration in a specific way.
 Some of the base alteration are- alkylation, depurination, deamination, hydroxylation
etc.
Alkylation:
 Alkylating agents are most widely used mutagens which carry one, two or more alkyl
groups in reactive form.
 Alkylating agents transfer their alkyl group (methyl or ethyl) group to nitrogenous bases
of DNA and to phosphate group (alkylation).
 The most commonly used alkylating agents are- ethyl-methane sulfonate (EMS),
Dimethyl sulphonate (DMS), Diethyl sulphonate (DES) etc , nitrosoguanidine (NG)
Mechanism of alkylating agents:

Transfer alkyl group to phosphate group of DNA and produces unstable phosphate
trimester which hydrolyses to give alkyl group. But some alkyl group may remain attached to
phosphate which interfere with DNA replication cycle as well as cause breakage of sugar
phosphate back bone.
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Transfer alkyl group to 6-oxygen and 7-nitrogen of DNA bases. Among these alkylation
of nitrogenous base, 7-ethylguanine is most common derivative. 7-ethylguanine is base analog
to cytosine and can form base pair with thymine which ultimately cause base transition from
G=C to AT.
Some difunctional or polyfunctional alkylating agents can crosslink with DNA strand and
interfere DNA replication leading to chromosome breakage.
Depurination:
 Alkylation of purine base (Adenine and Guanine) give rise to unstable quaternary
nitrogenous base which interfere with glycosidic bond between nitrogenous base and
deoxyribose sugar.
 The loss of purine bases from DNA is termed as Depurination.
 Once the base is depurinated, it cannot specify complimentary base to the original
purine during replication which result in incorporation of wrong base.
 Therefore, this causes base pair substitution.
Deamination:
 Some mutagens react with nitrogenous base containing amino group and remove it with
another functional group.
 Nitrous acid (HNO2) a chemical which reacts with amino group containing nitrogenous
bases (Adenine, cytosine and guanine) and replace it.
 Deamination of adenine by nitrous acid yields hypoxanthine (H) which can base pair
with cytosine. This deamination at the position of adenine result in base transition from
A:T to G:C in successive replication.
 Similarly, deamination of cytosine by HNO2 results in uracil. Now uracil can base pair
with adenine. Therefore, change of base from cytosine to uracil results in base transition
from G:C to A:U and then A:T in successive replication cycle.
 Other example of deamination: conversion of 5-methylcytosine to thymine.
Hydroxylation:
 The mutagen hydroxylamine (NH2OH) reacts with amino group of cytosine causing
hydroxylation to yield hydroxylcytosine.
 Now the hydroxylcytosine can form base pair with adenine instead of guanine. This
results in base pair transition.
Distortion of DNA molecule:

 Some of the mutagens directly reacts with DNA molecules causing distortion and
modify the structure.
i. Intercalating agents:
 Certain florescent acridine dyes such as acridine orange, proflavin causes DNA
mutation by insertion or deletion of nitrogenous bases.
 Acridine dyes are planar (flat) molecule that mimic nitrogenous bases and at low
concentration it can inserts or intercalates between subsequent nitrogenous bases in
DNA molecule.
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 Insertion of the agent stretches the distance between adjacent base pair by 0.68nm
which is twice the normal distance. This distortion result in single nucleotide deletion
or insertion at this position during recombination.
Intercalating agents result in insertion of bases:

 Intercalation of acridine dye between two nitrogenous bases in template strand result in
stretch of DNA molecule.
 During DNA replication, a new base can be inserted in the newly synthesized strand
opposite of acridine molecule.
 Now during replication of newly synthesized strand, a complimentary base in added
opposite to newly added base.
Intercalating agents result in deletion of bases:
 Insertion of acridine molecule in DNA strand may block the base in the template strand
and does not allow to base pair.
 During replication, one base is deficient in newly synthesized strand.
ii. DNA damage: by radiation
 Various kinds of radiations can induce mutation. Mutagenic radiations are two types-
non-ionizing radiation (UV rays) and ionizing radiation (X-rays, gamma rays):
a) Mutation caused by UV rays: Pyrimidine dimer formation
 The most effective wavelength of UV rays for inducing mutation is 260nm.

 UV rays are non-ionizing radiation and have a penetration power lower than X-rays.
 Exposure of DNA to UV rays causes nitrogenous bases to become highly reactively
unstable free radicals.
 One of the consequences of UV rays exposure is photochemical fusion of two
pyrimidine that are adjacent to each other on a same polynucleotide chain.
 Pyrimidine dimer formation is the primary effect of UV rays on DNA molecule.
 In case of two thymine, the fusion is called thymine dimer. UV rays causes adjacent
thymine on the same DNA strand to bond together by covalent linkage between carbon
5 and 6 of adjacent thymine molecule forming cyclobutane ring.
 In case of thymine adjacent to a cytosine, the resulting fusion is a thymine-cytosine
dimer in which the thymine is linked via its carbon atom 6 to the carbon atom 4 of
cytosine.
 This dimer structure cannot fit into DNA helix and cause distortion of DNA molecule
which result in failure of DNA replication and sometime leads to lethal effect.
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DNA damage.
chromosome.
Spontaneous Mutation and Induce Mutation
Spontaneous Mutation:
Spontaneous Mutation occurs in the absence of any mutagens.
If there is no mutagen but still DNA got damaged then it is S
Spontaneous Mutation.
How these damages could have happened when there is no
mutagen.
So, there is a variety of reasons. The most important reason is an
error occurs during DNA replication.
b) Mutation caused by Ionizing radiation (IR):
 In direct effect, IR breaks the phosphate ester bond in DNA. The breakage may take
place at one or more points. As a result of breakage, segment of DNA either get lost or
rearranged during repair. Sometimes the effect is fatal.
 In indirect effect, the ionizing radiation ionize water producing free radicals which is
extremely reactive.
 The free radicals react with DNA molecule to alter its structure.
Base pair changes: A point mutation is a genetic mutation where a single nucleotide base is
changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point
mutations have a variety of effects on the downstream protein product—consequences that are
moderately predictable based upon the specifics of the mutation.
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A substitution is a mutation in which there is an exchange between two bases (i.e. a

change in a single "chemical letter" such as switching a T to a C). Such a substitution could
change a codon to another one that encodes a different amino acid and cause a change in the
protein produced. Sometimes substitutions may not affects the protein structure, such mutations
are called silent mutations and sometimes they may change an amino-acid-coding codon to a
single "stop" codon and cause an incomplete protein. This can seriously affect the protein
structure which may completely change the organism.
Example of Substitution Mutation: Sickle Cell Anaemia is caused by substitution mutation,

where in codon (GAG mutates to --> GTG) and leads to (Glu --> Val) change.
Insertion
An insertion (also called an insertion mutation) is the addition of one or more
nucleotide base pairs into a DNA sequences.
Insertions are mutations in which extra base pairs are inserted into a new place in the DNA.
The number of base pairs inserted can range from one to thousands.
GGCCAUGGG
GGACCAUGGG← addition of A nucleotide
Example of Insertion Mutation: Huntington's disease and the fragile X syndrome are examples
of insertion mutation wherein trinucleotide repeats are inserted into the DNA sequence leading
to these diseases.
Deletions
Deletions are mutations in which a section of DNA is lost, or deleted. The number of
base pairs deleted can again range from one to thousands.
GGCCAUGGG
GGC↑AUGGG← deletion of C nucleotide
Example of Deletion Mutation: 22q deletion syndrome is caused by the deletion of some bases
of chromosome 22. This disease is characterized by cleft palate, heart defects, autoimmune
disorders etc.
Frameshift Mutation
A frameshift mutation (also called a framing error or a reading frame shift) is a
genetic mutation caused by insertions or deletions of a number of nucleotides in a DNA
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sequence that is not divisible by three. Due to the triplet nature of gene expression by codons,
the insertion or deletion can change the reading frame (the grouping of the codons), resulting
in a completely different translation from the original.
A frameshift mutation will in general cause the reading of the codons after the mutation
to code for different amino acids. The frameshift mutation will also alter the first stop codon
("UAA", "UGA" or "UAG") encountered in the sequence. The polypeptide being created could
be abnormally short or abnormally long, and will most likely not be functional.
Examples of Frameshif Mutation: Tay-Sachs Disease, Cancers of many types, Crohn's Disease,
cystic fibrosis.
Duplication : any duplication of a region of DNA that contains a gene. Duplication refers to a
type of mutation in which one or more copies of a DNA segment (which can be as small as a
few bases or as large as a major chromosomal region) is produced. Duplications occur in all
organisms.
One example of a rare genetic disorder of duplication is called Pallister Killian
syndrome, where part of the #12 chromosome is duplicated.
Transition mutation: one in which a single purine is substituted for another purine or vice
versa. E.g. AT to GC, CG to TA.
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Transversion mutation
It is a specific kind of point mutation, one in which a single purine is substituted for a
pyrimidine or vice versa. As the result of a transversion mutation, the mutated position in the
gene may for example have an adenine where it had a thymine or cytosine.
A trans version can be spontaneous, or can be caused by ionizing radiation or alkylating
agents. It can only be reversed by a spontaneous reversion. For example 8-oxo-2'-
deoxyguanosine (8-oxodG) is an oxidized derivative of deoxyguanosine, and is one of the
major products of DNA oxidation. During DNA replication in the germ line of mice, the
oxidized base 8-oxoguanine (8-oxoG) causes spontaneous and heritable G to T
G-C------------»T-C ------------»AG
Next replication T can base pair with A so, GC base pair converted to A-T, that cause mutation.
Base analogs are bases that are similar to the bases normally found in DNA. Like normal bases,
base analogs exist in normal and rare tautomeric states. In each of the two states, the base
analog pairs with a different normal base in DNA. Because base analogs are similar to the
normal nitrogen bases, they may be incorporated into DNA in place of normal bases. E.g. 5 –
bromouracil (5-BU). If 5-BU is incorporated in its more common normal state, it pairs with
adenine. If it changes into its rare state during replication, it pairs with guanine instead. In the
next round of replication, the 5-BU – G base pair is resolved into a C – G base pair instead of
the T – A base pair. By this process a transition mutation is produced, from TA to CG.
Intercalating agents
Acridine, proflavin, ethidium bromide are examples few examples of intercalating
agents. These insert (intercalate) themselves between adjacent bases in one or both strands of
the DNA double helix.
Phenotype is the collection of observable traits; genotype is the genetic endowment of

the individual. Genotype is also used to refer to the pair of alleles present at a single locus.
With alleles 'A' and 'a' there are three possible genotypes AA, Aa and aa. Not all pairs of alleles
will have the same phenotype: dominance when AA = Aa in phenotype, A is dominant, a is
recessive. An allele can be dominant over one allele but recessive to another allele. An
organism's phenotype results from two basic factors: the expression of an organism's genetic
code, or its genotype, and the influence of environmental factors, which may interact, further
affecting phenotype on of that genotype, and the environment can affect the phenotype.
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Useful Phenotypes
Auxotrophs are a group of organisms that lost the ability to synthesize certain
substances required for their growth owing to the presence of mutations. Compared to the wild
type strain, the auxotrophic mutants cannot grow in minimal medium if the corresponding
nutrients are not supplied.
Conditional lethal mutation is that kind of mutation where the viability of the mutant
produced depends on the conditions of the growth. It grows normally in favourable conditions
(temperature or media) but does not grow in restrictive conditions. Conditional lethal mutations
are lethal in one the permissive condition. These allow geneticists to identify and study
mutations in essential genes that result in complete loss of gene-product.
Activity even in haploid organisms. Mutants carrying conditional lethals can be propagated
under permissive conditions, and information about the functions of the gene products can be
inferred by studying the consequences of their absence under the restrictive conditions.
The three major classes of mutants with conditional lethal phenotypes are (1)
auxotrophic mutants, (2) temperature-sensitive mutants, and (3) suppressor sensitive mutants.
Auxotrophs are mutants that are unable to synthesize an essential metabolite (amino
acid,purine, pyrimidine, vitamin, and so forth) that is synthesized by wild-type or prototrophic
organisms of the same species. The auxotrophs will grow and reproduce when the metabolite
is supplied in the medium (the permissive condition); they will not grow when the essential
metabolite is absent (the restrictive condition).
Temperature-sensitive mutants will grow atone temperature but not at another. Most
temperature-sensitive mutants are heat-sensitive; however, some are cold-sensitive. The
temperature sensitivity usually results from the increased heat or cold lability of the mutant
gene product—for example, an enzyme that is active at low temperature but partially or totally
inactive at higher temperatures. Occasionally, only the synthesis of the gene product is sensitive
to temperature, and once synthesized, the mutant gene product may be as stable as the wild-
type gene product.
Resistants: Through mutation and selection, bacteria can develop defense mechanisms
against antibiotics. For example, some bacteria have developed biochemical “pumps” that can
remove an antibiotic before it reaches its target, while others have evolved to produce enzymes
to inactivate the antibiotic. Antimicrobial resistance mechanisms fall into four main
categories: (1) limiting uptake of a drug; (2) modifying a drug target; (3) inactivating a drug;
(4) active drug efflux. Antibiotic resistance can be achieved by horizontal acquisition of
resistance genes (carried by plasmids or transposons), by recombination of foreign DNA into
the chromosome, or by mutations in different chromosomal loci.
Reversion and Suppression

Reversion mutation is a mutation that restores the function of a mutant gene while
suppression mutation is a mutation that suppresses the phenotype of another mutation
Mutations that convert the phenotype from wild-type to mutant are called forward
mutations, and mutations that change the phenotype from mutant back to wild-type are called
reverse mutations (reversions). Bacterial strains that contain reverse mutations are called
revertants. Analysis of mutations that cause phenotypic reversion yields useful information.
Reverse mutations that restore the exact nucleotide sequence of the wild-type DNA are true
reversions. True revertants are identical to wild-type strains both genotypically and
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phenotypically. Reverse mutations that do not restore the exact nucleotide sequence of the
wild-type DNA are called suppressor mutations (suppressors). Some revertants that harbor
suppressor mutations are phenotypically indistinguishable from wild-type strains.
Suppressor mutations can be intragenic or extragenic.

Intragenic suppressors are located in the same gene as the forward mutations that they
suppress. The possible locations and nature of intragenic suppressors are determined by the
original forward mutation and by the relationships between the primary structure of the gene
product and its biologic activity.
Extragenic suppressors are located in different genes from mutations whose effects they
suppress. The ability of extragenic suppressors to suppress a variety of independent mutations
can be tested. Some extragenic suppressors are specific for particular genes, some are specific
for particular codons, and some have other specificity patterns. Extragenic suppressors that
reverse the phenotypic effects of chain-terminating codons have been well characterized and
found to alter the structure of specific tRNAs.
Ames test
The Ames test is a widely employed method that uses bacteria to test whether a given
chemical can cause mutations in the DNA of the test organism. It is one of a biological assay to
assess the mutagenic potential of chemical compounds. A positive test indicates that the
chemical is mutagenic and therefore may act as a carcinogen, because cancer is often linked
to mutation. The test serves as a quick and convenient assay to estimate the carcinogenic
potential of a compound because standard carcinogen assays on mice and rats are time-
consuming and expensive. The procedure was described in 1970s by Bruce Ames and his co-
workers.
Mutagens identified via Ames test are also possible carcinogens, and early studies by
Ames showed that 90% of known carcinogens may be identified via this test. The Ames test is
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often used as one of the initial screens for potential drugs to weed out possible carcinogens,
Salmonella typhimurium is a prokaryote, and therefore it is not a perfect model for humans.
Rat liver S9 fraction is used to mimic the mammalian metabolic conditions so that the
mutagenic potential of metabolites formed by a parent molecule in the hepatic system can be
assessed; however, there are differences in metabolism between human and rat that can affect
the mutagenicity of chemicals being tested. The test may therefore be improved by the use of
human liver S9 fraction; its use was previously limited by its availability, but it is now available
commercially and therefore may be more feasible.
Principle
Ames test is developed by Bruce N. Ames in 1970s to test for determining if the
chemical is mutagens. This test is based on the principle of reverse mutation or back
mutation. So, the test is also known as bacterial reverse mutation assay.
Test organism:
Ames test uses several strains of bacteria (Salmonella, E.coli) that carry mutation. Eg
A particular strain of Salmonella typhimurium carry mutation in gene that encodes histidine.
So it is an auxotrophic mutant which loss the ability to synthesize histidine (an amino acid)
utilizing the ingredients of culture media. Those strains are known as His- and require histidine
in growth media.
Culturing His- Salmonella is in a media containing certain chemicals, causes mutation

in histidine encoding gene, such that they regain the ability to synthesize histidine (His+) This
is the reverse mutation. Such chemicals responsible to revert the mutation is actually a
mutagen. So, this Ames test is used to test mutagenic ability of varieties of chemicals.
 Isolate an auxotrophic strain of Salmonella typhimurium for histidine. (ie. His-ve)
 Prepare a test suspension of his-ve Salmonella typhimurium in a plain buffer with test
chemical (2-aminofluorene). Also add small amount of histidine.
 Small amount of histidine is required for initial growth of bacteria. Once histidine is
depleted only those bacteria mutated to gain the ability to synthesize histidine form
colony.
 Also prepare a control suspension of His- Salmonella typhimurium, but without test
chemicals.
 Incubate the suspensions at 37°C for 20 minutes
 Prepare the two-agar plate and spread the suspension on agar plate.
 Incubate the plates at 37°C for 48 hours.
 After48 hours count the number of colonies in each plate.
The mutagenicity of chemicals is proportional to number of colonies observed. If large
number of colonies on test plate is observed in comparison to control, then such chemicals are
said to be mutagens.
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DNA Damage
Types of DNA Damage: 1. Replication errors Repair Pathway followed. 2. Base tautomers
Base tautomerism may result in either Transition or Transversion Transition: GC - AT; AT -
GC (Order of Purines and Pyrimidine is conserved) ♥ Transversion: GC - TA or CG; AT - CG
or TA (Order of Purines and Pyrimidines is reversed)
Cytosine Deamination ♥ Cytosine in DNA spontaneously deaminates to form Uracil. ♥
Deamination of Cytosine is mutagenic because Uracil pairs with Adenine and so one of the
daughter strands will contain a U-A base pair rather than the original C-G base pair. 3. Covalent
modification
Oxidative Damage Oxidative damage to Guanine ♥ Mutagens include reactive oxygen species,
such as hydroxyl radical. ♥ Hydroxyl radical reacts Guanine to form 8-Oxoguanine. ♥ 8-
Oxoguanine is mutagenetic because it often pairs with Adenine rather than Cytosine in DNA
replication. ♥ Repair Pathway followed.
UV- -damaging
Such a pyrimidine dimer cannot fit into a double helix, and so replication and gene expression
-link is a Thymine
ss-links
(Compounds produced by a Chinese herb), forms such Interstrand Cross-Links. Interstrand

cross-links disrupt replication since they prevent strand separation.Chemical Damage
(including alkylation) Deamination of Adenine ♥ Adenine can also be deaminated by Nitrous
Acid (HNO2) to form Hypoxanthine. ♥ The process is mutagenic because Hypoxanthine pairs
with Cytosine rather than Thymine. ♥ Repair Pathway followed: BER Alkylation ♥ Nucleotide
bases are subject to Alkylation. ♥ Electrophilic centers can be attacked by nucleophiles such
as N-7 of Guanine and Adenine to form alkylated adducts.
DNA REPAIR MECHANISM

Repair system play a significant role in mutation process. As a result of repair,
potentially lethal changes in DNA may be eliminated. If the repair system function in error free
manner, potentially mutagenic lesions are eliminated before they can be converted into final
mutation.Following are the repair mechanism
1. Photoreactivation
2. Excision repair system
3. Post replicative recombination repair
4. Adaptive repair
5. SOS repair
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1. Photo reactivation
 Ultraviolet light is a physical mutagen and can induce mutation. Ultra violet radiation
(254 nm) causes formation of pyrimidine dimers (cyclobutane ring), when two
pyrimidine bases occurs together in single strand of DNA.
 Thymine dimer is most common one but cytosine dimer as well as thymine-cytosine
may also occurs. Thymine dimer is a state in which two adjacent thymine molecules
are chemically joined distorting the structure of DNA, so that impeding transcription
and replication process.
 This pyrimidine dimer formation is lethal to the cell unless it is corrected. A repair
mechanism known as photo reactivation can repair this mutation.
 When UV radiated population of bacteria is subsequently exposed to visible light of
wave length of 300-450nm, the survival rate increases and frequency of mutation
decreases. This is due to activation of photo reactivating enzyme photolyase, which
splits thymine dimer.

 In the dark, the enzyme bind with thymine dimer and in presence of visible light the
enzyme split the thymine dimers.
 Upto 80% of thymine dimers existing in genome can be photoreactivated.
2. Excision repair system
 The repair system remove and replace the altered bases from damaged DNA. Excision
repair system involves nucleotide repair and base excision repair.
i. Base excision repair:
 In this mechanism modified bases are recognized and cut out. Mutation causes
alkylation and deamination of bases which are recognized by special DNA glycosylase
enzyme.
 Glycosylase recognizes and remove the damaged bases by hydrolyzing the glycosidic
bond and cut out the damaged base creating AP site (a purinic or pyrimidinic site).
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 The AP site is recognized by AP endonucleases which split the phosphodiester bond on

DNA strand at AP site and removes the AP sugar.
 After the damaged nucleotide is removed, the gap is repair by DNA polymerase I and
ligated by DNA ligase.
figure: base excision repair

ii. Nucleotide excision repair:
 In nucleotide excision repair mechanism, the defective nucleotide are cut out and
replaced.
 Unlike base excision repair, the enzymes in nucleotide excision repair recognizes the
distortion in shape of double stranded DNA structure caused by thymine dimers or
intercalating agents.
 The multi sub unit enzyme excinucleases (endonuclease and exonuclease activity)
hydrolyses two phosphodiester bond one on either side of distortion caused by lesion
creating 3’-OH group and 5’-P group.
 The excised nucleotide is removed and the resulting gap is filed by DNA polymerase-I
in E. coli and DNA polymerase E in Human and finally joined by DNA ligase.
3. Post replicative recombination repair:
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 It is a double strand break (DSB) repair mechanism. If a DNA strand contain lesions
which prevent base pairing by creating gap in the daughter strand during replication.
This causes break in both strand.
 In this repair mechanism, the gap is filled by sequence information from parent strand
of sister chromosome by homologous recombination process. The repair of parent
strand is done by DNA polymerase I
4. SOS repair:
 Photoreactivation, excision repair and post reactive recombination repair are generally
error free repair mechanism. However, there also exist an error prone and mutation
inducing repair called SOS repair.
 Error in both complementary strand of DNA would be lethal for cell. Thus SOS repair
reconstruct the chemical structure of DNA but the heredity information is lost. Hence
SOS repair causes mutation along with repair of DNA. DNA polymerase V help in SOS
repair.
 It is also known as inducible repair. It involves more than 40 gene which encodes
protein responsible for protection and replication of DNA as well as repair and
mutation.
 SOS response have been found in E. coli, Salmonella Typhimurium, Mycobacterium
tuberculosis etc, but not in eukaryotic cell.
 It is not a single discrete mechanism but includes diverse responses such as the ability
to repair thymine dimers, to induce various prophages, to shut off respiration, to delay
septum formation during cell division. These all responses are regulated coordinately.
Mechanism of SOS response:

 SOS response is induced when DNA is damaged or when replication of DNA stops and
single stranded DNA accumulates.
 Agents such as UV radiations, methyl methane sulphonate, as well as chemicals that
damages DNA induces SOS response. Rec A and Lex A gene are major in SOS
response induction.
 lexA encodes lexA protein which is repressor. It binds to SOS box near promotor of
SOS gene and prevents gene expression.
 RecA protein is a nucleoprotein and is always high in bacterial cell. It form
nucleoprotein filaments on single stranded DNA and protects further damage of DNA.
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Rec A protein acquire proteases activities and activates self-cleavage of lexA protein
from SOS box.
 Now the operator and promoter site become free and facilitates gene expression of SOS
box gene.
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UNIT IV
BACTERIAL AND VIRAL GENETICS
Bacterial plasmids: Plasmids are the extrachromosomal genetic elements found in

bacteria. They are circular pieces of DNA that are extra genes. About 1-20 copies of plasmids
are present in one bacterial cell. Episomes are the type of plasmid that can be inserted into the
bacterial chromosome and can replicate with it.
 Plasmids are replicons that are maintained as discrete, extrachromosomal genetic
elements in bacteria.
 They are usually much smaller than the bacterial chromosome, varying from less than
5 to more than several hundred kbp. though larger plasmids as 2 Mbp occur in some
bacteria.
 Plasmids usually encode traits that are not essential for bacterial viability, and replicate
independently of the chromosome.
 Most plasmids are supercoiled, circular, double-stranded DNA molecules, but linear
plasmids have also been demonstrated in Borrelia and Streptomyces.
 Closely related or identical plasmids demonstrate incompatibility; they cannot be stably
maintained in the same bacterial host.
 Some hybrid plasmids contain more than one replicon.
 Conjugative plasmids code for functions that promote transfer of the plasmid from the
donor bacterium to other recipient bacteria, but nonconjugative plasmids do not.
Conjugative plasmids that also promote transfer of the bacterial chromosome from the
donor bacterium to other recipient bacteria are called fertility plasmids.
 Large plasmids (>40 kilobase pairs) are often conjugative, have small copy numbers (1
to several per chromosome), code for all functions required for their replication, and
partition themselves among daughter cells during cell division in a manner similar to
the bacterial chromosome.
 Plasmids smaller than 7.5 kilobase pairs usually are nonconjugative, have high copy
numbers (typically 10–20 per chromosome), rely on their bacterial host to provide some
functions required for replication, and are distributed randomly between daughter cells
at division.
COPY NUMBER:
Determines how many copies of the plasmid will be present in a cell e.g. different types
of plasmid types:High copy number (e.g. pUC): ~500-700 copies/cell
Medium copy number (e.g. pET): ~40 copies/cell
Low copy number (e.g. pACYC): ~10
Ultra-low (e.g. RP4): ~1-2•
Two different plasmids need to have different (and therefore compatible) origins in order for
both to maintain compatibility
Plasmid incompatibility refers to the inability for two plasmids to coexist stably over a
number of generations in the same bacterial cell line. Origin Incompatibility: When different
plasmids have the same (or closely related) origins of replication, they won’t both be stably
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maintained •If you have two different plasmids with the same replication origin, replication
will occur randomly (as normal) and since the replication machinery is the same it will pick
one plasmid for duplication and when again picking up another cell randomly it may choose to
pick the blue plasmid that had already been replicated again.
In other words..Compatibility refers to the ability of two different plasmids to coexist

stably in the same cell. Incompatibility refers to inability of two plasmids with similar replicon
or segregation system to coexist in the same cell. Whenever two different plasmids have similar
replication or partitioning system, there will be competition between the plasmids and
whichever is able to utilize the system better will prevail over the other. All plasmids belong
to only one of the approximately 30 compatibility groups.
Types of plasmids: Naturally occurring plasmids are wild plasmid found naturally in
bacteria. Recombinant plasmids are altered plasmids introduced into the bacterium for genetic
studies. Cryptic plasmids are those that serve no known functions. They may be present for
possible exclusion of plasmids that are incompatible with the resident plasmid. Integrative
plasmids: These are plasmids that can occasionally integrate into chromosome (previously
called episomes). In Bacteroides uniformis, certain sections of chromosome separate
themselves from chromosome and become plasmids, which are capable of conjugation.
Metabolic plasmids carry some genes that help in cells metabolism. ƒ Virulence plasmids carry
one or several genes that confer virulence properties on the bacterial cell. Conjugative plasmids
are those that are able to mobilize self transfer. These tend to be larger because they have to
accommodate genes coding for self transfer. Conjugative plasmids in Gram positive bacteria
tend to be smaller than those in Gram-negative bacteria. Mobilizable plasmids are those
plasmids that lack genes to initiate self transfer but do encode the functions needed specifically
for transfer of their own DNA. The initiation function is provided by other conjugative plasmid
present in the same cell. Suicide plasmids are referred to those plasmids which get transferred
to another bacterial cell but does not replicate further. These are actually mobilizable plasmids.
ƒ R (resistance) plasmids are large conjugative plasmids that carry one or more antibiotic
resistance genes. Resistance plasmid can be conjugative or mobilizable. Usually, R plasmids
code for their own replication, and they are known to have mobile genetic elements. Colicin
plasmids are small plasmids which encode the genes to synthesize colicins (bacteriocines). F
(fertility) plasmids are those that have complete gene set to mediate self transfer by
conjugation. Cells possessing it (donor) are termed F+ and those lacking it (recipient) are
termed F- .
 Conjugative plasmids contain a set of transfer or tra genes which promote sexual
conjugation between different cells. In the complex process of conjugation, plasmid
may be transferred from one bacterium to another via sex pili encoded by some of
the tra genes.

 Non-conjugative plasmids are incapable of initiating conjugation, hence they can be
transferred only with the assistance of conjugative plasmids. An intermediate class of
plasmids are mobilizable, and carry only a subset of the genes required for transfer.
They can parasitize a conjugative plasmid, transferring at high frequency only in its
presence.
 Plasmids can also be classified into incompatibility groups.
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 A microbe can harbour different types of plasmids, but different plasmids can only exist
in a single bacterial cell if they are compatible. If two plasmids are not compatible, one
or the other will be rapidly lost from the cell. Different plasmids may therefore be
assigned to different incompatibility groups depending on whether they can coexist
together.
 Incompatible plasmids (belonging to the same incompatibility group) normally share
the same replication or partition mechanisms and can thus not be kept together in a
single cell.
F Plasmids
 The fertility factor (first named F by one of its discoverers Esther Lederberg; and also
called the sex factor in E. coli .Now also called F-plasmid.
 F plasmid plays an important role in reproduction given that they contain genes that
code for the production of sex pilus as well as enzymes required for conjugation. F
plasmid also contains genes that are involved in their own transfer. Therefore, during
conjugation, they enhance their own transfer from one cell to another.
 Whereas the cells that process the F plasmids are referred to as donors, those that lack
this factor are the recipients. On the other hand, the plasmids that enhance the ability of
the host cell to behave like a donor are known as the transfer factor.
 During conjugation, the donor cell (bacteria) with sex pili (1-3 sex pili) binds to a
specific protein on the outer membrane of the recipient thus initiating the mating
process.
 Following the initial binding, the pili retract thus allowing the two cells to bind together.
This is then followed by the transfer of DNA from the donor to the recipient and
consequently the transfer of the F plasmid. As a result, the recipient acquires the F
factor and gains the ability to produce sex pilus involved in conjugation.
 During conjugation, only DNA is passed from the donor to the recipient. Therefore,
cytoplasm and other cell material are not transferred.
 Sexual pili (sex pili) are tiny rod-like structures that allow the F-positive (cells that have
the F factor) bacterial cells to attach to the F-negative (cells lacking the pili) female to
promote conjugative transfer.
Structure
Fertility plasmids (F plasmid) have a circular structure and measures about 100 kb. The
F-plasmid is an episome with a length of about 100 kb. It carries its own origin of replication,
the oriV, and an origin of transfer, or oriT. Bacteria that possess a copy of F plasmid are
called F-positive or F-plus (denoted F+). Cells that lack F plasmids are called F-negative or F-
minus(F−) and as such can function as recipient cells. Among other genetic information, the F-
plasmid carries a tra locus, which together are about 33 kb long and consist of about 40 genes.
The tra locus includes the pilin gene and regulatory genes, which together form pili on the cell
surface.
The most common functional segments constituting F factors are:

 OriT (Origin of Transfer): The sequence which marks the starting point of conjugative
transfer.
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 OriC (Origin of Replication): The sequence starting with which the plasmid-DNA will
be replicated in the recipient cell.
 tra-region (transfer genes): Genes coding the F-Pilus and DNA transfer process.
 IS (Insertion Elements) composed of one copy of IS2, two copies of IS3, and one copy
of IS1000: so-called "selfish genes.
 Transposable element (IS2, 1S3, and Tn1000)
 Replication sites (RepFIA, RepFIB, and RepFIC)

 When an F+ cell conjugates/mates with an F− cell, the result is two F+ cells, both capable
of transmitting the plasmid to other F− cells by conjugation.
 The F-plasmid belongs to a class of conjugative plasmids. The tra operon includes
genes required for conjugation and plasmid transfer. F+ bacteria can always act as a
donor cell.
 F+ cells also have the surface exclusion proteins TraS and TraT on the bacterial surface.
These proteins prevent secondary mating events involving plasmids belonging to the
same incompatibility (Inc) group. Thus, each F+ bacterium can host only a single
plasmid type of any given incompatibility group.
 In the case of Hfr transfer, the resulting transconjugates. The result of
Hfr/F− conjugation is a F− strain with a new genotype(recombination).
Col Plasmids
 Col plasmids confer to bacteria the ability to produce toxic proteins known as colicines.
Such bacteria as E. coli, Shigella and Salmonella use these toxins to kill other bacteria
and thus thrive in their respective environments.
 There are different types of Col plasmids in existence that produce different types of
colicines/ colicins. A few examples of Col plasmids include Col B, Col E2 and E3.
Their differences are also characterized by differences in their mode of action.
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 For instance, whereas Col B causes damage to cell membrane of other bacteria (lacking
the plasmid) Col E3 has been shown to induce degradation of the nucleic acids of the
target cells.
 The Col plasmids can be transferred from one cell (donor) to another (recipient). As a
result, the recipient acquires the ability to produce toxins that kill or inhibit the growth
of the target bacteria lacking the plasmid.
 Colicins/colicines belong to a group of toxins known as bacteriocins.
 These toxins affect the target bacteria by affecting such processes as replication of
DNA, translation and energy metabolism among others.
Functions of colicins
These colicins contain at least three domains: an N-terminal translocation domain
responsible for movement across the outer membrane and periplasmic space; a central domain
responsible for receptor recognition and a C-terminal cytotoxic domain responsible for channel
formation in the cytoplasmic membrane. One domain regulates the target and binds to the
receptor on the sensitive cell. The second is involved with translocation, co-opting the
machinery of the target cell. The third is the 'killing' domain and may produce a pore in the
target cell membrane, or act as a nuclease to chop up the DNA or RNA of the target cell.
Virulence Plasmids
 Bacteria that tend to be pathogenic in nature carry genes for virulence factors that allow
them to invade and infect their respective hosts.
 For some of these bacteria, the virulence factors are the result of the organisms' own
genetic material. However, for others, this is as a result of genetic elements from extra-
chromosomal DNA. Although there are other sources of such elements, e.g.
transposons, plasmids are some of the most common mobile genetic elements.
 With regards to pathogenicity, virulence plasmids play an important role given that they
can help bacteria effectively adapt to their respective environments. This is because the
virulence plasmid can enable the organism to express an array of virulence-associated
functions thus providing the organism with more advantageous characteristics to thrive
in their environment.
 Like other types of plasmids, virulence plasmids can also be transmitted from one
bacterium to another. Apart from the virulence gene, plasmids have also been shown to
carry other important elements that enhance transmission and maintenance.
 For this reason, they are larger in size but low in numbers. This ensures that they do not
cause additional burden to the organism during cell division.
 Typically, cell division and cell maintenance require the use of energy. By having low
numbers of virulence plasmids, the cells are spared significant metabolic burden that
would be required for maintenance and genome duplication of numerous plasmids.
Resistance Plasmids (R-plasmids)

 It also referred to as antimicrobial resistance plasmids. Resistance plasmids are a type
of plasmids that carry genes that play an important role in antibiotic resistance.
 They are also highly involved in bacterial conjugation by producing pili during
conjugation, which transfer the R plasmid from one bacterium to another.
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 Resistance factors or resistance plasmids are a group of conjugative plasmids which

confer to their bacterial host resistance to specific antibiotics, such as, chloramphenicol,
sulphonamide, streptomycin, tetracycline etc. These factors cannot get inserted into the
bacterial chromosome.
 They are self-replicating, small, circular DNA elements; they were first discovered in
Japan in 1959 when strains of Shigella were found to become resistant to several
antibiotics used during a dysentery epidemic. Some R factor by itself is non-
transmissible.
 Structure of Resistance Plasmid: R plasmid conains two factors. One is resistance
transfer factor and r determinant. The resistance transfer factor (RTF) is about 80 kb in
length and carries genes for autonomous replication, conjugation and resistance to
ampicillin. However, it also contains a fin 0 gene that represses the function of transfer
operon (tra) while the F factor does not contain the fin 0 gene.
 The r factors (r determinant) vary in size and in the content of genes for drug resistance.
The R determinant is smaller than the RTF. Both the RTF and R determinant combine
to form one unit; they are separated from each other by one IS 1 element on either side
.
Resistance plasmids are divided into two main groups that include:
 Narrow-host-range group - Often replicated within a single species.
 Broad-host-range group - Easily transferred between bacteria species. This group of
resistance plasmids has been shown to carry a range of antibiotic resistance genes.
Degradative plasmids
 It enables the host organism to degrade/break down xenobiotic compounds. Also
referred to as recalcitrant substances, xenobiotic compounds include a range of
compounds released into the environment as a result of human actions and are therefore
not naturally occurring or common in nature.
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 Hosts of degradative plasmids are found in groups IncP-1, IncP-7, and IncP-9 and
include such species as Rhizobium sp, Ochrobactrum anthropi, Burkholderia hospita,
Escherichia coli, and Pseudomonas fluorescens among many others.
 The use of biodegradative microorganisms for the purposes of removing xenobiotic
compounds from contaminated environments is known as Bioaugmentation.
 Whereas IncP-1 plasmids have a broad range of hosts, IncP-7 has been shown to have
a narrow host range. On the other hand, IncP-9 has an intermediate host range.
 Plasmids may be classified in a number of ways. Plasmids can be broadly classified
into
 Conjugative plasmids and non-conjugative plasmids.
Plasmid replication mechanisms

 Plasmid replication requires host DNA replication machinery.
 Most wild plasmids carry genes needed for transfer and copy number control.
 All self-replication plasmids have a oriV: origin of replication
 Some plasmids carry and oriT: origin of transfer. These plasmids will also carry
functions needed to be mobilized or mob genes.
 Plasmid segregation is maintained by a par locus-a partition locus that ensures each
daughter cells gets on plasmid. Not all plasmids have such sequences.
 There are 5 main “incompatibility” groups of plasmid replication. Not all plasmids can
live with each other.
 Agents that disrupt DNA replication destabilize or cure plasmids from cells.
 There are three types of plasmid replication namely rolling circle, Col E1 type and teron
contain replication.
Rolling circle replication

 Rolling circle replication mechanism is specific to bacteriophage family m13 and the
fertility F factor which encodes for sex pili formation during conjugation.
 It allows the transfer of single stranded replication product at a faster rate to the
recipient cell through pilus. Rolling circle occurs to a covalently closed circular piece
of double-stranded DNA.
 A nick is produced in one of the strands by enzyme nickases creating a 5’ phosphate
and a 3’ hydroxyl.
 Free 3’ hydroxyl will be used by DNA polymerase to make new DNA pushing the old
nicked strand off of the template DNA.
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Figure 1: Mechanism of rolling circle replication.
Col E1 type replication:

 Col E1 replication is a negative regulation mechanism which enables the plasmid to
control its own copy numbers by involving RNA type I, RNA type II, Rom protein, and
the plasmid itself.
 Col E1 replication is initiated by means of RNA-RNA interactions and does not rely on
replication initiation protein encoded by the plasmid to regulate its copy number.
 Mechanism: RNA type II that originates 555 base pairs upstream from the replication
origin of Col E1 plasmid is transcribed which marks the start of Col E1 replication.
 A determined hybrid with the DNA strand is formed by a loop enriched in G nucleotide
positioned 290 of RNAII and a C-rich region on the template strand positioned
20 nucleotides upstream from the origin.
 Several stems and loops are exhibited by the newly formed secondary structure.
 A DNA/RNA hybrid is recognized by enzyme RNase and dissociates the RNA hybrid
to the 3' end of RNAII.
 The resultant RNA primer is linked to the plasmid with a free 3' hydroxyl group.
 This RNA enables replication of DNA to begin by providing DNA polymerase a
specific site to initiate nucleotides synthesis.
 Consequently DNA synthesis is commenced with the leading strand is happening (Fig).
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Amplification is used for enhancing the copy number of a plasmid. Some plasmids
have capability to replicate even in the absence of protein synthesis. This is incompatible with
the primary bacterial chromosome, which are not able to replicate in the given environment.
Transposable elements in Prokaryotes (Bacteria)
Transposons
Transposons (transposable elements or "jumping genes" ) are small pieces of DNA that
encode enzymes that transpose the transposon, that is, move it from one DNA location to
another, either on the same molecule of DNA or on a different molecule. Transposons may be
found as part of a bacterium's nucleoid (conjugative transposons) or in plasmids and are usually
between one and twelve genes long. A transposon contains a number of genes, coding for
antibiotic resistance or other traits, flanked at both ends by insertion sequences coding for an
enzyme called transpoase. Transpoase is the enzyme that catalyzes the cutting and resealing of
the DNA during transposition. Thus, such transposons are able to cut themselves out of a
bacterial nucleoid or a plasmid and insert themselves into another nucleoid or plasmid and
contribute in the transmission of antibiotic resistance among a population of bacteria.
Plasmids can also acquire a number of different antibiotic resistance genes by means of
integrons. Integrin’s are transposons that can carry multiple gene clusters called gene cassettes
that move as a unit from one piece of DNA to another. An enzyme called integrase enables
these gene cassettes to integrate and accumulate within the integron. In this way, a number of
different antibiotic resistance genes can be transferred as a unit from one bacterium to another.
Plasmids and conjugative transposons are very important in horizontal gene transfer in
bacteria. Horizontal gene transfer , also known as lateral gene transfer, is a process in which an
organism transfers genetic material to another organism that is not its offspring. The ability
of Bacteria and Archaea to adapt to new environments as a part of bacterial evolution most
frequently results from the acquisition of new genes through horizontal gene transfer rather
than by the alteration of gene functions through mutations.
Transposons are mobile genetic elements (MGE) that can carry additional genetic cargo
nonessential for their own transposition. This cargo can include antibiotic or heavy metal
resistance genes or those increasing metabolic plasticity.
There are two main type of transposable elements in bacteria having different size and
structure. They are;
 Insertion sequences (IS elements)
 Prokaryotic Transposons (Tn): Composite and non-composite transposons
1. Insertion sequences (IS element):
 IS elements are the simplest type of bacterial transposable sequences that can insert at
different location of bacterial chromosome and plasmid through illegitimate
recombination.
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 They are typically short sequences and contains only one gene that encode the enzyme
for transposition.
 IS elements were first identified as spontaneous insertion in certain lac operon
mutations of E. coli which inactivate the gene and inhibit transcription and translation.
The mutation of Lac operon gene was found to be unstable and molecular analysis
reveals the presence of extra copies of DNA sequences near the lac gene. When the
mutated E. coli undergoes reverse mutation, the extra DNA sequence is lost.
 A bacterial chromosome may contain several copies of a particular type of IS element.
For example, 6 to 10 copies of IS1 are found in the E. coli chromosome.
 Plasmids may also contain IS elements
Characteristics of Insertion sequences (IS element)
 IS elements are compactly organized and containing about 1000 nucleotide pairs and
contain only genes (open reading frame) which encode for enzyme for regulating
transposition.
 Many distinct types of IS elements have been identified. The smallest IS element is
IS1 which is 768 nucleotide pairs long.
 Each type of IS element contains inverted terminal repeats at both end and a transposon
sequence in between those inverted repeats. Transposon is the only gene that code for
transposition of IS element.
 The inverted terminal repeats is 9-40 base pair long and is the characteristics of most
IS element
 IS element have the capacity to duplicate the inserted sequence at the site of insertion;
known as target site duplication.
Transposition of insertion sequence in bacteria:

 IS element contains single open reading frame (ORF) which encodes for the enzyme
transposase, catalyzing its own transposition.
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 The enzyme transposase is like restriction endonuclease which binds to terminal

inverted repeats (IR) of IS element which is the restriction site. Then the enzyme cut
and excise IS elements from chromosome or plasmid.
 The excised IS element is mobile in nature and moves along the length of chromosome
to recognizes the target site for insertion on same or different chromosome or plasmid.
 Once recognizing the target site, it generate staggered cleavage (cut the single strand of
DNA) generating sticky and itself get inserted.
 As IS element get inserted, the proofreading mechanism of DNA results in duplication
of the DNA sequence at the target site of the insertion such that one copy of target DNA
is located on each side of IS element.
 Thus IS elements helps in target site duplication
Prokaryotic Transposons (Tn):
 Prokaryotic Transposons are similar to IS element but they are larger and also contains
other genes (mostly antibiotic resistance gene) in addition to gene that encode
transposase.
 Transposons are several thousand base pairs long and contains inverted terminal
repeats.
 There are two types of prokaryotic transposons- composite and non-composite
transposons.
 The composite transposons and Tn3-like elements are more complex than IS elements,
containing some genes that encode products unrelated to the transposition process.
i. Composite transposons:
 Composite transposon are created when two IS elements insert near each other and the
region between the two IS elements can then be transposed when the elements act
jointly.
 For example: Tn10 is a composite transposons of 9.3kbp which contains 1.4 kbp
terminal inverted repeats and in between them is gene for transposase and gene for
antibiotic resistance.
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ii. Non-composite transposons:

 The non-composite transposons is a sequence of DNA containing gene for trasnposase
and multiple other gene in between terminal inverted repeats.
 Unlike composite transposons, it does not contains IS elements at each end but instead
it contains simple inverted repeats 0f 38-40 nucleotide pairs at each end.
 For example; Tn3 is a non-composite transposons of 5kbp which contains three gene
for beta-lactamase (bla), transposase (tnpA) and resolvase (tnpB).
 The beta lactamase provide resistance to the antibiotic ampicillin, and the other two
enzymes play important roles in transposition and recombination.
Transposable Element # 7. Transposing Bacteriophage Mu:

Bacteriophage Mu is a temperate phage of E. coli having a double-stranded 38 kb linear
DNA genome. Like other temperate phages, it can either have a lytic cycle or a lysogenic
pathway. The phage possesses the ability to integrate its genome into the bacterial chromosome
without any site-specificity. Such random insertion of the phage genome into the bacterial
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chromosome causes mutation and, hence, the name of the phage (Mu stands for mutator). The
phage Mu is, therefore, considered as a transposable agent.
It may be remembered that a temperate phage, on infecting a host bacterium, can

integrate its genome into the chromosome of the host cell making the host lysogenic. The phage
genome may persist as a prophage in the host chromosome for many generations.
On induction, either spontaneously or by an external agent like UV, the prophage is

excised and begins the lytic cycle by production of phage genome and phage proteins. In case
of phage Mu, induction does not result in excision of the prophage.When the phage Mu infects
an E. coli cell, the phage DNA is integrated into the host DNA randomly, but with a low
frequency. However, when the prophage is induced to enter into a lytic cycle, the frequency of
integration into the host DNA increases significantly. This has been designated as replicative
transposition.It appears that copies of phage DNA produced by replication can integrate with
higher efficiency into the host chromosome at multiple sites resulting in mutation of host genes.
Eventually, phage-coded proteins are synthesized and new phage particles are assembled and
released by lysis of the host cells to complete the lytic cycle.
The exact mechanism of transposition by phage Mu is not yet well understood.

Transposition- induced mutations by Mu can be of different types including deletions and
inversions of DNA segments of the host chromosome.
The structure of bacteriophage Mu is shown in Fig. 9.87:
Deletions or inversions of the host chromosome occur when two Mu genomes are integrated
into the same chromosome and homologous recombination between the Mu genomes takes
place.
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These are diagrammatically shown in Fig. 9.88:
Types of bacterial transposons
Transposons are divided into two main groups: retrotransposons (class І) and DNA
transposons (class ІІ). Retrotransposons are often found in eukaryotes. DNA transposons can
be found in both eukaryotes and prokaryotes.
Organization of viral genome
Organization of viral genome Polio, or poliomyelitis, is a disabling and life-threatening

disease caused by the poliovirus. The virus spreads from person to person and can infect a
person's spinal cord, causing paralysis. A poliovirus, the causative agent of polio, is a serotype
of the species Enterovirus C, in the family of Picornaviridae. There are three poliovirus
serotypes: types 1, 2, and 3. Poliovirus is composed of an RNA genome and a protein capsid.
Polio is caused by 1 of 3 types of the poliovirus. It often spreads due to contact with infected
feces. This often happens from poor handwashing. It can also happen from eating or drinking
contaminated food or water.
Structure of polio virus

The viral particle is about 30 nm in diameter with icosahedral symmetry. Because of its
short genome and its simple composition—only RNA and a nonenveloped icosahedral protein
coat that encapsulates it.
Poliovirus was first isolated in 1909 by Karl Landsteiner and Erwin Popper. The structure of
the virus was first elucidated in 1958 using x-ray diffraction by a team at Birkbeck College led
by Rosalind Franklin.
 Virions are spherical in shape with a diameter of about 27nm.
 The particles are simple in that they are composed of a protein shell surrounding the
naked RNA genome.
 The genome is monopartite, linear ssRNA(+) genome of 7.2-8.5 kb, polyadenylated,
composed of a single ORF encoding a polyprotein.
 The capsids are composed of four structural proteins: VP1, VP2, VP3, and VP4.
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 The basic building block of the picornavirus capsid is the protomer, which contains one
copy each of VP1, VP2, VP3, and VP4.
 The shell is formed by VP1 to VP3, and VP4 lies on its inner surface.
 The virus particles lack a lipid envelope, and their infectivity is insensitive to organic
solvents.
Genome organization
Poliovirus is a positive-stranded RNA virus. Thus, the genome enclosed within the viral
particle can be used as messenger RNA and immediately translated by the host cell.
The RNA molecule is 7,433 nucleotides long, polyadenylated at the 3' terminus, and covalently
linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2,207 consecutive
triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein
NCVPOO.
 Polio virus genome can be divided into three parts

 a 5′ noncoding region (NCR) that comprises approximately 10% of the genome, is
uncapped, and is covalently linked at the 5′ terminus to viral protein VPg
 a single open reading frame that appears to encode all of the viral proteins, with regions
designated as P1 for capsid proteins and P2 and P3 for nonstructural proteins
 a short 3′ NCR terminating in a polyA tail.
 The genomes vary in length from 7,209 to 8,450 bases.
 The 5′-noncoding region contains the internal ribosome entry site (IRES), an element
that directs translation of the mRNA by internal ribosome binding.
 The regions P1 contains four segments for structural proteins which make up the capsid
protein; 1A-VP4, 1B- VP2, 1C-VP3, 1D-VP1.
 P2 comprises of three non structural proteins; 2A, 2B, 2C which play a role in viral
replication.
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 P3 makes up four non structural proteins

 3A- anchors the replication complex to cell membrane
 3B- it is VPg protein
 3C- it is cysteine protease that cleaves the protein from polypeptides
 3D- it is RNA dependent RNA Polymerase.
Influenza virus
 The family Orthomyxoviridae contains a single genus Influenza virus with three types-
A, B and C. Influenza viruses are classic respiratory viruses. They cause influenza, an
acute infections disease of the respiratory tract that occurs in sporadic, epidemic and
pandemic forms
 A unique feature of influenza virus is its ability to undergo antigenic variations. The
surface glycoprotein (Haemagglutinin and Neuraminidase) show variations and are
primarily responsible for antigenic variations.
Structure of Influenza virus
 Shape:
 The influenza virus particle is typically spherical with a diameter of about 80-120 mm
but pleomorphism is common.
 Filamentous forms upto several micrometers in length
 Symmetry:
 The virus is core consists of ribonucleoprotien in helical symmetry.
 The nucleoprotein (NP) associates with the viral RNA to form a ribonucleoprotein
(RNP) which is a structure of 9 mm in diameter that assumes a helical configuration
and forms the viral nucleocapsid.
 Genome and protein:
 Influenza virus contains negative sense single stranded RNA (-ssRNA) genome which
is segmented.
 Type A and B influenza virus consist of 8 pieces of segmented RNA (while type C
influenza virus contains 7 segments), which encode for 11 proteins (HA, NA, NP, M1,
M2, NS1, NEP, PA, PB1, PB1, PB2).
 Also contains a viral RNA-dependent RNA polymerase that transcribes the negative
polarity genome into mRNA.
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 Three large proteins (PB1, PB2, and PA) are bound to the viral Ribonucleoprotein and
are responsible for RNA transcription and replications.
 The matrix (M1) protein which forms a shell underneath the viral lipid envelope is
important in particle morphogenesis and is a major competent of the virion.
 Envelope and glycoprotein spikes:
 The nucleocappsid is surrounded by an envelope which has an inner membrane of
protein known as matrix or M protein which is virus encoded and outer lipid layer
derived from infected host cell membrane during the process of replication by budding.
 Two virus encoded glycoproteins, the haemagglutinin (HA) and the neuraminidase
(NA) are inserted into the envelope and are exposed as spikes about 10mm long on the
surface of the surface of the particles.
These glycoproteins are triangular and mushroom shaped

respectively. Haemagglutinin (HA):Haemagglutin derives its name from its ability to
agglutinate erythrocytes under certain conditions.Haemagglutinin is a glycoprotein composed
of two polypeptides- HA 1 and HA2, responsible for hemadsorption and haemagglutination.
These two polypeptides are joined together by disulfide bond.
 Influenza A virus (IAV) is responsible for common respiratory infection in human,
spreading as seasonal epidemics and sporadic pandemics. IAV belongs to the
family Orthomyxoviridae. Its viral genome is organized in eight, negative-sense RNA
segments. The viral RNA (vRNA) sizes range from ~0.9 to 2.3 kb, while the total
genome size is about 13.5 kb. All vRNAs show the same organization: A central open
reading frame that encodes one or more protein (in the antisense orientation) flanked
by two short untranslated regions (UTRs). Segments 1–8 are named according to the
encoded protein: polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1),
polymerase acidic protein (PA), hemagglutinin (HA), nucleoprotein (NP),
neuraminidase (NA), matrix protein (M), and nonstructural protein (NS). IAVs can be
classified into antigenic subtypes, based on their surface glycoproteins: HA proteins
fall into classes H1 to H18 and NA proteins fall into classes N1 to N11. Only a limited
number of these HAs and NAs have been isolated from viruses known to infect humans.
 The vRNA segments in the virion are organized in ribonucleoprotein complexes

(vRNPs) . Each segment, through base-pairing of the highly conserved 13 nucleotides
at the 5΄ end and 12 at 3΄, forms a partially double-stranded promoter structure, which
undergoes substantial structural rearrangements at certain stages of the viral replication
cycle to perform distinct functions . It is bound by the viral-encoded trimeric RNA-
dependent RNA polymerase complex. The rest of the vRNA associates with multiple
copies of NP. vRNA has been shown to fold into secondary structure motifs in both in
vitro and in virio conditions . Eight vRNPs (containing each of vRNA segments) are
required for the production of complete infectious progeny virions.
 A retrovirus is a virus that uses RNA as its genetic material. When a retrovirus infects
a cell, it makes a DNA copy of its genome that is inserted into the DNA of the host cell.
There are a variety of different retroviruses that cause human diseases such as some
forms of cancer and AIDS. Retrovirus, any of a group of viruses that belong to the
family Retroviridae and that characteristically carry their genetic blueprint in the form
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of ribonucleic acid (RNA). Retroviruses are named for an enzyme known as reverse
transcriptase, which was discovered independently in 1971 by American
virologists Howard Temin and David Baltimore. Reverse transcriptase transcribes RNA
into deoxyribonucleic acid (DNA), a process that constitutes a reversal of the usual
direction of cellular transcription (DNA into RNA). The action of reverse transcriptase
makes it possible for genetic material from a retrovirus to become permanently
incorporated into the DNA genome of an infected cell; the enzyme is widely used in
the biological sciences to synthesize genes.
Retroviruses cause tumour growth and certain cancers in animals and are associated
with slow infections of animals, such as equine infectious anemia. In humans, a retrovirus
known as human T-cell lymphotropic virus type 1 (HTLV-1) causes a form of cancer called
adult T-cell leukemia (ATL). The retrovirus known as human immunodeficiency virus (HIV)
causes acquired immunodeficiency syndrome (AIDS) in humans.
HIV-1 is composed of two copies of noncovalently linked, unspliced, positive-
sense single-stranded RNA enclosed by a conical capsid composed of the viral protein p24,
typical of lentiviruses. The two copies of RNA strands are vital in contributing to HIV-1
recombination, which occurs during reverse transcription of viral replication. The containment
of two copies of single-stranded RNA within a virion but the production of only a single DNA
provirus is called pseudodiploidy. The RNA component is 9749 nucleotides long and bears a 5’
cap (Gppp), a 3’ poly(A) tail, and many open reading frames (ORFs). Viral structural proteins
are encoded by long ORFs, whereas smaller ORFs encode regulators of the viral life cycle:
attachment, membrane fusion, replication, and assembly. The single-strand RNA is tightly
bound to p7 nucleocapsid proteins, late assembly protein p6, and enzymes essential to the
development of the virion, such as reverse transcriptase and integrase. Lysine tRNA is the
primer of the magnesium-dependent reverse transcriptase. The nucleocapsid associates with
the genomic RNA (one molecule per hexamer) and protects the RNA from digestion
by nucleases. Also enclosed within the virion particle are Vif, Vpr, Nef, and viral protease.
The envelope of the virion is formed by a plasma membrane of host cell origin, which is
supported by a matrix composed of the viral p17 protein, ensuring the integrity of the virion
particle. At the surface of the virion can be found a limited number of the
envelope glycoprotein (Env) of HIV, a trimer formed by heterodimers of gp120 and gp41. Env
is responsible for binding to its primary host receptor, CD4, and its co-
receptor(mainly CCR5 or CXCR4), leading to viral entry into its target cell.
The third variable loop or V3 loop is a part or region of the Human Immunodeficiency
Virus. The V3 loop of the viron's envelope glycoprotein, gp120, allows it to infect human
immune cells by binding to a cytokine receptor on the target human immune cell, such as
a CCR5 cell , depending on the strain of HIV. The envelope glycoprotein (Env) gp 120/41 is
essential for HIV-1 entry into cells. Env serves as a molecular target of a medicine treating
individuals with HIV-1 infection, and a source of immunogen to develop AIDS vaccine.
Several conserved secondary structure elements have been identified within the HIV
RNA genome. The 5'UTR structure consists of series of stem-loop structures connected by
small linkers. These stem-loops (5' to 3') include the trans-activation region (TAR) element,
the 5' polyadenylation signal [poly(A)], the PBS, the DIS, the major SD and the ψ hairpin
structure located within the 5' end of the genome and the HIV Rev response element (RRE)
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within the env gene. Another RNA structure that has been identified is gag stem loop 3 (GSL3),
thought to be involved in viral packaging. RNA secondary structures have been proposed to
affect the HIV life cycle by altering the function of HIV protease and reverse transcriptase,
although not all elements identified have been assigned a function.
RNA secondary structure OF HIV
T4 bacteriophage genome
Bacteriophage T4 is classified as a member in the Myoviridae family of the
Caudovirales order because it has a contractile tail. The head, the tail and the long tail fibers
(LTFs) of T4 are assembled independently before they are joined together to produce a mature
phage. Phage T4 has provided countless contributions to the paradigms of genetics and
biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products.
T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle. The T4
Phage initiates an E. coli infection by recognizing cell surface receptors of the host with its
long tail fibers (LTF).
Genome
 The double helical DNA genome is almost 169 kbp long and encodes 289 proteins.
 The genome is terminally redundant and possesses intron sequences like eukaryotes.
 The DNA undergoes replication by a rolling circle mechanism.
 In the Shine-Dalgarno sequence, GGAG is found abundantly in the early T4 genes.
 The DNA genome is packed in an icosahedral head that is known as a capsid.
 This virus can only undergo a lytic life cycle, not the lysogenic life cycle.
T-4 bacteriophage is a bacteriophage that infects E. coli bacteria. Its double-stranded

DNA genome is about 169 kbp long and is held in an icosahedral head, also known as a capsid.
T4 is a relatively large phage, at approximately 90 nm wide and 200 nm long (most phages
range from 25 to 200 nm in length). Its tail fibres allow attachment to a host cell, and the T4’s
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tail is hollow so that it can pass its nucleic acid to the cell it is infecting during attachment. T4
is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle.
The T4 Phage initiates an E. coli infection by recognizing cell surface receptors of the
host with its long tail fibers (LTF). A recognition signal is sent through the LTFs to the
baseplate. This unravels the short tail fibers (STF) that bind irreversibly to the E. coli cell
surface. The baseplate changes conformation and the tail sheath contracts causing GP5 at the
end of the tail tube to puncture the outer membrane of the cell. The lysozyme domain of GP5
is activated and degrades the periplasmic peptidoglycan layer. The remaining part of the
membrane is degraded and then DNA from the head of the phage can travel through the tail
tube and enter the E. coli.
The lytic lifecycle (from entering a bacterium to its destruction) takes approximately 30
minutes (at 37 °C) and consists of:
 Adsorption and penetration (starting immediately)

 Arrest of host gene expression (starting immediately)
 Enzyme synthesis (starting after 5 minutes)
 DNA replication (starting after 10 minutes)
 Formation of new virus particles (starting after 12 minutes)
After the life cycle is complete, the host cell bursts open and ejects the newly built
viruses into the environment, destroying the host cell. T4 has a burst size of approximately
100-150 viral particles per infected host. Complementation, deletion, and recombination tests
can be used to map out the rII gene locus by using T4. These bacteriophage infect a host cell
with their information and then blow up the host cell, thereby propagating themselves.
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Bacteriophage T7 (or the T7 phage) is a bacteriophage, a virus that infects bacteria. It infects
most strains of Escherichia coli and relies on these hosts to propagate. Bacteriophage T7 has
a lytic life cycle, meaning that it destroys the cell it infects.
Virion structure
The virus has complex structural symmetry, with a capsid of the phage that
is icosahedral (twenty faces) with an inner diameter of 55 nm and a tail 19 nm in diameter and
28.5 nm long attached to the capsid. The ejection of proteins from the capsid upon infection
causes the virus to change structure when it enters the cell.
Genome
The genome of phage T7 was among the first completely sequenced genomes and was
published in 1983. The head of the phage particle contains the roughly 40 kbp dsDNA genome
which encodes 55 proteins. The genome features numerous overlapping genes that were
partially removed through 'refactoring' the genome to produce T7.1.
Life cycle of T7
T7 has a life cycle of 17 min at 37˚C, i.e. the time from infection to the lysis of the host
cell when new phage are released. Due to the short latent period, most physiological studies
are conducted at 30˚C where infected cells lyse after 30 min. However, high-fitness strains of
T7 have been isolated with a latent period of only ~11 min at 37˚C growing under optimal
conditions in rich media results. This adapted phage can undergo an effective expansion of its
population by more than 1013 in one hour of growth
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The T7 phage recognizes certain receptors on the surface of E.coli cells, and binds to
the cell surface by its viral tail fibers. In some strains of T7, the tail fibers are replaced with
tail-spikes that degrade the O- or K-antigens on the cell surface by way of enzymatic activity.
The adsorbtion and penetration process use lysozymes to create an opening within
the peptidoglycan layer of the bacterial cell wall, allowing transfer of the viral DNA into the
bacterium. The short, stubby tail of the T7-like phage is too short to span the cell envelope and,
in order to eject the phage genome into the cell at the initiation of infection, virion proteins
must first make a channel from the tip of the tail into the cell cytoplasm. The phage also releases
five proteins needed to begin replication of the viral genome and cleave the host genome. T7
bacteriophage has been evolved to override several of the host bacteria's defenses including the
peptidoglycan cell wall. Once the T7 phage has inserted the viral genome, the process of DNA
replication of the host genome is halted and replication of viral genome begins. Under optimal
conditions, the T7 phage can complete the lytic process within 25 minutes, leading to the death
of the E. coli host cell. At the time of lysis, the virus can produce over 100 progeny.
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UNIT V
RECOMBINATION AND GENE TRANSFER MECHANISMS
GENE TRANSFER MECHANISMS

BACTERIAL CONJUGATION
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct
cell-to-cell contact or by a bridge-like connection between two cells. This takes place through
a pilus. During conjugation, one bacterium serves as the donor of the genetic material, and the
other serves as the recipient. The donor bacterium carries a DNA sequence called the fertility
factor, or F-factor. The genetic information transferred is often beneficial to the recipient.
Benefits may include antibiotic resistance, xenobiotic tolerance etc.

 Donor cell produces pilus (if the presence of F plsmid).
 Pilus attaches to recipient cell and brings the two cells together.
 The mobile plasmid is nicked and a single strand of DNA is then transferred to the
recipient cell.
 Both cells synthesize a complementary strand to produce a double stranded circular
plasmid and also reproduce pili
The F-plasmid is an episome with a length of about 100 kb. It carries its own origin of
replication, the oriV, and an origin of transfer, or oriT. Bacteria that possess a copy of F plasmid
are called F-positive or F-plus (denoted F+). Cells that lack F plasmids are called F-
negative or F-minus(F−) and as such can function as recipient cells. Among other genetic
information, the F-plasmid carries a tra locus, which together are about 33 kb long and consist
of about 40 genes. The tra locus includes the pilin gene and regulatory genes, which together
form pili on the cell surface.
Where the pilus are allowed to make contact, conjugation is initiated by a signal
the relaxase enzyme creates a nick in one of the strands of the conjugative plasmid at the oriT.
The nicked strand is then unwound from the unbroken strand and transferred to the recipient
cell in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated either
independent of conjugative action (rolling circle replication of lambda phage).
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High Frequency of Recombination(Hfr strains)

A high-frequency recombination cell (Hfr cell) (also called an Hfr strain) is a bacterium with
a the F-plasmid integrated into its chromosomal DNA. The integration of the plasmid into the
cell's chromosome is through homologous recombination. The amount of chromosomal DNA
that is transferred depends on how long the two conjugating bacteria remain in contact. In
common laboratory strains of E. coli the transfer of the entire bacterial chromosome takes about
100 minutes. The transferred DNA can then be integrated into the recipient genome
via homologous recombination.
The E. coli genome was originally mapped by interrupted mating experiments in which
various Hfr cells in the process of conjugation were sheared from recipients after less than 100
minutes (initially using a Waring blender). The genes that were transferred were then
investigated. Transfer of bacterial chromosome by Hfr cells.
An Hfr cell can transfer a portion of the bacterial genome. Despite being integrated into
the chromosomal DNA of the bacteria, the F factor of Hfr cells can still initiate conjugative
transfer, without being excised from the bacterial chromosome first. Due to the F factor's
inherent tendency to transfer itself during conjugation, the rest of the bacterial genome is
dragged along with it. Due to the large size of bacterial chromosome, it is very rare for the
entire chromosome to be transferred into the F − cell as time required is simply too long for the
cells to maintain their physical contact. Therefore, as the conjugative transfer is not complete.
1. The Hfr cell forms sex pilli

2. A pilus and attaches to a recipient F- cell.
3. A nick in one strand of the Hfr cell’s chromosome is created.
4. DNA begins to be transferred from the Hfr cell to the recipient cell while the second
strand of its chromosome is being replicated.
5. The pilus detaches from the recipient cell and retracts. The Hfr cell ideally wants to
transfer its entire genome to the recipient cell. However, due to its large size and
inability to keep in contact with the recipient cell, it is not able to do so.
6. The F- cell remains F- because the entire F factor sequence was not received. Since no
homologous recombination occurred, the DNA that was transferred is degraded by
enzymes.
7. In very rare cases, the F factor will be completely transferred and the F- cell will
become an Hfr cell.
Transformation
Transformation is the genetic alteration of a cell resulting from the direct uptake and
incorporation of exogenous genetic material from its surroundings through the cell membrane.
Transformation is one of three processes for horizontal gene transfer, in which exogenous
genetic material passes from one bacterium to another. For transformation to take place, the
recipient bacteria must be in a state of competence, which might occur in nature as a time-
limited response to environmental conditions such as starvation and cell density, and may also
be induced in a laboratory.
Transformation experiments
Transformation in bacteria was first demonstrated in 1928 by the British
bacteriologist Frederick Griffith. Griffith was interested in determining whether injections of
heat-killed bacteria could be used to vaccinate mice against pneumonia. However, he
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discovered that a non-virulent strain of Streptococcus HYPERLINK

"https://en.wikipedia.org/wiki/Streptococcus_pneumoniae"pneumoniae could be
made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that
some "transforming principle" from the heat-killed strain was responsible for making the
harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic
by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent
strain of S. pneumonia and using just this DNA were able to make a harmless strain virulent.
They called this uptake and incorporation of DNA by bacteria "transformation."
Competence refers to a temporary state of microorganisms, being able to take up
exogenous DNA from the environment. Competence may be differentiated into natural
competence, a genetically specified ability of bacteria which is thought to occur under natural
conditions as well as in the laboratory, and induced or artificial competence, which arises when
cells in laboratory cultures are treated to make them transiently permeable to DNA.
Competence allows for rapid adaptation and DNA repair of the cell.
Natural competence
Naturally competent bacteria carry sets of genes that provide the protein machinery to
bring DNA across the cell membrane(s). The transport of the exogenous DNA into the cells
may require proteins that are involved in the assembly of type IV HYPERLINK
"https://en.wikipedia.org/wiki/Pilus"pili and type II secretion system, as well as
DNA translocate complex at the cytoplasmic membrane.EgV Gram +ve : Bacillus subtilis,
Streptococcus pneumonae
Gram –ve: Haemphilus infulenzae, Neisseria gonorrahe, Helicobacetr pylori, Acenetobacter
baylyi, Cyanobacteria.
Competence for transformation is typically induced by high cell density and/or

nutritional limitation, conditions associated with the stationary phase of bacterial growth.
Transformation in Haemophilus influenzae occurs most efficiently at the end of exponential
growth as bacterial growth approaches stationary phase. Transformation in Streptococcus
HYPERLINK "https://en.wikipedia.org/wiki/Streptococcus_mutans"mutans, as well as in
many other Streptococci, occurs at high cell density and is associated with biofilm formation.
Most competent bacteria are thought to take up all DNA molecules with roughly equal
efficiencies, but bacteria in the families Neisseriaceae and Pasteurellaceae preferentially take
up DNA fragments containing short DNA sequences, termed DNA uptake sequence (DUS). In
Neisseriaceae and uptake signal sequence (USS) in Pasteurellaceae, that are very frequent in
their own genomes. Neisserial genomes contain thousands of copies of the preferred sequence
GCCGTCTGAA, and Pasteurellaceae genomes contain either AAGTGCGGT or
ACAAGCGGT.
Mechanism of transformation
The DNA first binds to the surface of the competent cells on a DNA receptor, and passes
through the cytoplasmic membrane via DNA translocase. Only single-stranded DNA may pass
through, the other strand being degraded by nucleases in the process. The translocated single-
stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent
process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires
the presence of a channel formed by secretins on the outer membrane. Pilin may be required
for competence, but its role is uncertain. The uptake of DNA is generally non-sequence
specific, although in some species the presence of specific DNA uptake sequences may
facilitate efficient DNA uptake.
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 Bacterial cell DNA

 Bacterial cell plasmids
 Sex pili
 Plasmid of foreign DNA from a dead cell
 Bacterial cell restriction enzyme
 Unwound foreign plasmid
 DNA ligase
I. A plasmid of foreign DNA from a dead cell is intercepted by the sex pili of a naturally
competent bacterial cell.
II. The foreign plasmid is transduced through the sex pili into the bacterial cell, where it
is processed by bacterial cell restriction enzymes. The restriction enzymes break the
foreign plasmid into a strand of nucleotides that can be added to the bacterial DNA.
III. DNA ligase integrates the foreign nucleotides into the bacterial cell DNA.
IV. Recombination is complete and the foreign DNA has integrated into the original
bacterial cell's DNA and will continue to be a part of it when the bacterial cell replicates
next.
 The protein involved in transformation of these Gram +ve bacteria is a product of com
gene
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 In Bacillus subtilis , the com gene are organized into several operons. The product of
com A and com K are involved in regulation of competence and other com E, com F
and com G encodes structural protein for uptake of DNA
 The first gene of com E operon, com EA encodes the protein that directly binds
extracellular double stranded DNA.
 The com F gene encodes the protein that translocate DNA into cell. for example; Com
FA is an ATPase that translocate DNA into cell.
 The com G gene of comG operon encodes protein that form pseudopilus that helps to
move DNA through channel.
 The com E, com F and com G operon are under transcriptional control of com K operon.
 Com K is a transcriptional factor that is regulated by com A, some other genes involved
in transformation are nuc A gene that encodes nuclease enzyme which cuts extracellular
dsDNA to single stranded,
Competence in Gram-ve bacteria:

A variety of Gran Negative bacteria are capable of competence. Some examples are
Acenetobacter calcoaceticus, Helicobacter pylori, Neisseria spp, etc H. pylori and Neisseria
spp require specific DNA sequences for binding of DNA so these species take up DNA from
same species only. Gram-ve bacteria utilizes two different pathway for uptake of DNA
 PSTC transformation pathway
 Type IV secretion related pathway
Type IV secretion related pathway is found in Helicobacter pylori. The DNA is translocated
through cell wall and membrane with the help of protein , Type IV system function transfer of
DNA in two ways-moving in and out of cell.
Artificial transformation
Most of the bacteria are not natural transformable. These bacteria can be made
competence by certain chemical treatment or by strong electrical shock. figure: artificial
transformation Some of the methods are-
Calcium treatment:
Treatment with calcium ion (ca++) make same bacteria eg. E.coli, Salomenlla,
pseudomonas etc competence.The Calcium ion increases the permeability of cell membrane.
Cell treated with calcium can take up both ssDNA as well as dsDNA, no matter circular or
linear.
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Electroporation:
A strong electric shock is applied in the bacterial culture mixed with naked DNA.
The recipient bacteria should be wash with non-ionic (distilled water) solution to prevent
osmotic shock.
The strong electric field creates artificial pore of water lined by phospholipid head
group. The DNA can pass through these artificial hydrophilic pore.
Recombination
Bacterial Recombination:
There are several types of genetic recombination in microorganisms. The most common
recombination is the reciprocal exchange between homologous DNA sequences.
Physical or chemical treatment forces the recipient bacterial cell to receive exogenous
DNA. The foreign DNA is then integrated with the chromosome by homologous
recombi-nation, mediated by Rec A protein. The Rec A protein catalyses the annealing of two
DNA segments and exchange of homologous region.
This involves nick i.e., small cut of DNA strands and rejoining of exchanged parts i.e.,
breakage and reunion. The generally accepted model of the above phenomenon is given below
Page 93 of 97
During genetic recombination usually only a part of the genetic material of a donor cell
is transferred to a recipient cell. The DNA of the recipient cell and the donor pair with each
other and reciprocally exchange DNA strands by crossing over. This gives rise to a new genetic
constitution of the recipient cell with new characters. Subsequent daughter cells that contain
only recombined chromosome.
There are following main methods by which recombination of genetic material takes
place in bacteria.
1. Homologous Recombination:
DNA of the donor cell recombines with recipient DNA by reciprocal exchange of DNA
strands. The recombining DNA molecules have homologus sequences. The DNA
molecules align or pair with each other and undergo crossing over and homologous
recombination. The recombinant DNA has new genetic constitution.
Page 94 of 97
Bacteria can acquire DNA of other closely related bacterial species and can become
transformed. This is known as transformation. Transformation was first demonstrated by
Griffith in Diploccous pneumoniae bacteria to confirm DNA as genetic material.
Homologous recombination is catalysed by rec A protein.
2. Non-homologous Recombination:
Recombination between DNA segments which have no homologous regions is also quite
frequent. Here the addition or insertion of a small DNA sequence in a recipient DNA takes
place. It occurs mainly by jumping genes or transposons. These mobile DNA sequences
regularly jump to a new location anywhere on the genome. Often the transposable elements
replicate to generate two copies. One copy remains at the original site while the other jumps
to the target site.
3. Site-specific Recombination:
When phage λ (lambda) infects E. coli bacterium, DNA of the phage λ is inserted into the
DNA of the bacterium. In this way phage DNA becomes integrated into host DNA and
becomes a part of the host chromosome. Bacterium containing a complete set of phage
DNA is called lysogen. Phage DNA is inserted at a specific site into the host DNA.
The attachment site of both DNAs possesses identical 15 base pair sequence. A phage
encoded protein called integrase catalyses the integration. A host protein called integration
host factor (IGF) is also involved. Integrase is a topoisomerase enzyme which breaks,
rotates and then re-joins the strands of both molecules. In eukaryotes site-specific
recombination produces antibody diversity.
Holliday Junction Model:

Various models to explain homologous genetic recombination have been proposed
based primarily on genetic observation in bacteria and fungi. Now it is known that some amount
of DNA synthesis also takes place during recombination. DNA replication and repair enzymes
are present at the time of genetic recombination.
The key feature of Holliday junction is the cleavage or cutting across the crossover
point which resolves or separates the recombined molecules. To expose two cut sites, the
Holliday junction is rotated by 180° to form a sequare planar structure. Resolution of Holliday
junction occurs by cutting the DNA strands at the site of cross and re-joining them.
Transduction
It is a special method of genetic recombination where genetic material is transferred
from the donor to the recipient cell through a non- replicating bacteriophage — temperate
bacteriophage. This was discovered by Joshua Leaderberg and Nortor Zinder (1952) during
their research with Salmonella typhimurium.
In this process, a small fragment of bacterial DNA is incorporated into an attacking
bacteriophage (i.e., virus which infects bacteria) and when this bacteriophage infects a new
bacterial cell, it transfers the genetic material into it, and thus genetic recombination takes
place.
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Specialised Transduction
In this process, the bacteriophage gets attached to a bacterial cell wall at the receptor
site and the nucleic acid of bacteriophage is transferred into the cytoplasm of the host cell
(Fig.A). The phage does not cause the lysis of the host bacterium. In the bacterial cell, the
phage nucleic acid codes for the synthesis of specific proteins, the repressor proteins.
 The repressor proteins prevent the virus to produce the material require for its
replication. In the bacterial cell, the viral DNA may exist as a fragment in the cytoplasm
or it may attach itself to the chromosome, known as prophage (Fig. B).
 The bacterial cell which carries the prophage is called lysogenic and the phenomenon
where the phage DNA and bacterium exist together is called lysogeny.
 The bacterial cell may remain lysogenic for many generations and during this period
the viral DNA replicates many times together with the bacterial chromosome.
 However, in course of time, the phage stops the synthesis of repressor proteins in the
bacterial cell, and then the synthesis of phage components starts. Now the phage DNA
separates from the bacterial chromosome and starts the synthesis of phage proteins (Fig.
C).
 During this separation, a specific genes (gal and bio gene) from bacteria DNA (eg gal
and bio gene) get attached to it. These attached genes keep on replicating along with
the phage DNA (Fig.D) and later on it develops into phage particles, those come out
from the bacterial cell by bursting (Fig. E).
 When the new phage particle (Fig. F) infects a new bacterial cell (Fig. G, H), the
attached bacterial genes present along with phage particle enters in the chromosome of
the new bacterium and causes recombination (Fig. 2).
 Thus the new bacterial cell contains its own genes and several genes of specific (gal or
bio gene) from the parent bacterial cell. This type of transduction is known as
specialised transduction, which is an extremely rare event.
Generalised Transduction:
This process of transduction is more common than specialized transduction. Here the
prophage particle is present in the cytoplasm of the infected bacterial cell (Fig. J). In this
process, the phage DNA starts synthesising new phages.
During this process chromosome of bacterial cell gets fragmented (Fig. 2K) and some
of the fragments become attached with the DNA of some new phage particle, while others
remain with phase DNA (Fig. L).
When the newly formed phage with any fragment of bacterial chromosome in its DNA
(Fig. M) attacks a new bacterium, the gene of the parent bacterium is transferred to the new
bacterium and causes recombination. This type of transduction is called generalised
transduction. This type of transduction is also rare.
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KAMARAJ COLLEGE

SELF FINANCING COURSES

STUDY MATERIAL FOR B.Sc., MICROBIOLOGY

ACADEMIC YEAR 2022-23

MOLECULAR BIOLOGY AND MICROBIAL GENETICS

2. PCR based technologies:

Griffith experiment or Transformation

1. Type II rough strain - small, non capsulated (Non virulent)

Characteristics of Diplococcus pneumoniae grown on blood agar.

Colony Capsule Virulence Reaction with Antiserum

Purification of the transforming material

RNA as a genetic material in small virus (Reconstitution experiment)

 Viruses contain RNA and protein but no DNA.

Watson & Crick – DNA Double helix Structure

 DNA contain two purines and two pyrimidine bases

Structure and Function of RNA

mRNA rRNA tRNA

Structure and Function of RNA

mRNA rRNA tRNA

 Many antibiotic-resistant genes in bacteria are present in plasmids.

DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an

The DNA of most organisms is negatively supercoiled. In a “relaxed” double-helical

Supercoiled DNA forms two structures; a plectoneme or a toroid, or a combination of

The Importance of DNA Supercoiling

DNA Replication in Prokaryotes

DNA replication steps

Enzymes involved in Replication

Primase Synthesizes RNA primers needed to start replication

Rolling circle Replication

 Numerous plasmids replicate independently of chromosomal replication. Numerous

GENES STRUCTURE AND EXPRESSION

Eukaryotic genome organization

 Each eukaryotic chromosome is made by a single linear DNA molecule.

1. In prokaryotes, genes tend to be clustered in coordinately-regulated groups called operons.

Transcription in Prokaryotes and Eukaryotes.

 Transcription involves the construction of an RNA copy of the genetic information in

The steps of transcription

Enzyme(s) Involved in Eukaryotic Transcription

Features of Eukaryotic Transcription

Process of Eukaryotic Transcription

 Elongation of the RNA chain continues until termination occurs.

Post transcriptional Modification

Capping and Tailing

Various protein factors involved in protein synthesis

Factors Translation steps Functions

IF-1 Initiation Helps to stabilize 30S ribosomal subunit

Binds fmet-tRNA with 30S subunit mRNA

IF-3 Initiation Binds 30S subunit with mRNA

Binds GTP; bring Aminoacyl-tRNA to A-site

EF-TS Elongation Generates EF-TU

EF-G Elongation Helps in translocation of ribosome

Helps to dissociates polypeptide from tRNA

Helps to dissociates polypeptide; specific for

RF-3 Termination Stimulates RF-1 and RF-2

 The activation of aminoacids take place in cytosol.

Post translational modifications

1. Amino terminal and carboxyl terminal modification:

3. Modification of individual aminoacids:

Post-translational modifications (PTMs) mainly occur in the endoplasmic reticulum of

Steps of Eukaryotic Translation

The eukaryotic translation is completed in the following steps;

Formation of 43S preinitiation complex

Formation of 48S initiation complex

Recognition of initiation codon: