MODULE 4

1. What is Recombinant DNA Technology?

The exchange of genetic information between DNA segments of the same species is termed
genetic recombination. Nonetheless, with the advancement of technology, one can transfer
genes of one species to another artificially.
 
The technology of recombinant DNA was developed in 1973 by Boyer and Cohen.
 
It is the technology to produce an artificial DNA molecule by combining two or more
fragments of DNA that are not necessarily associated with each other. Usually, such DNA
fragments are obtained from several biological sources.  
 
Recombinant DNA is DNA from two distinct species injected into a host organism to create
new genetic combinations useful in science, medicine, agriculture, and industry. Laboratory
geneticists' primary purpose is to identify, define, and modify genes, as the gene is the focus
of all genetics. Consider that each human cell has about 2 meters (6 feet) of DNA. As a
result, even a small piece of tissue can contain thousands of kilometers of DNA.
Recombinant DNA technology, on the other hand, has made it possible to isolate a single
gene or other section of DNA, allowing researchers to establish its nucleotide sequence,
investigate its transcripts, alter it in extremely specific ways, and reintroduce the transformed
sequence into a living creature.The said technology is also known as genetic engineering. In a
broader sense, it is created through three different methods – transformation, non-bacterial
transformation, and phage introduction. 
 
Tools of rDNA Technology
Recombinant DNA Technology's Implementation Tools are as follows:
 
Restriction enzymes, for example, aid in cutting, polymerases aid in synthesising, and ligases
aid in binding. In recombinant DNA technology, restriction enzymes play an important role
in deciding where the desired gene is inserted into the vector genome. Endonucleases and
Exonucleases are the two types of endonucleases.
 
Exonucleases remove the nucleotides off the ends of the strands, whereas Endonucleases cut
within the DNA strand. Restriction endonucleases are sequence-specific enzymes that cleave
DNA at specified locations and are frequently palindrome sequences. They examine the
length of DNA and cut it at a specified location known as the restriction site. As a result, the
sequence has sticky ends. The complimentary sticky notes are obtained by cutting the desired
genes and vectors with the same restriction enzymes, enabling the work of the ligases to bind
the desired gene to the vector much easier.
 
The vectors aid in the transport and integration of the desired gene. These are the ultimate
vehicles that transfer the desired gene into the host organism, hence they are a crucial part of
the recombinant DNA technology toolkit. Because of their large copy number, plasmids and
bacteriophages are the most commonly employed vectors in recombinant DNA technology.
The vectors are made up of an origin of replication, which is a nucleotide sequence from
which replication begins, a selectable marker, which are genes that show antibiotic resistance,
such as ampicillin resistance, and cloning sites, which are sites recognised by restriction
enzymes where desired DNAs are inserted.
 
The recombinant DNA is injected into the host organism. The host is the most powerful
instrument in recombinant DNA technology, as it uses enzymes to take in the vector that has
been modified with the desired DNA.
 
These recombinant DNAs are injected into the host in a variety of ways, including
microinjection, biolistics or gene gun, alternate cooling and heating, calcium ion usage, and
soon.
 
Goals of rDNA Technology
Some of the goals of this technology  are mentioned below: 

 Isolation and characterization of genes.

 Desired modification in isolated genes.
 Artificial synthesis of new genes.
 Modification of organisms’ genome.
 Interpretation of hereditary diseases and related cures.
 Enhancement of the human genome.
 
Steps of Recombinant DNA Technology
1. DNA Isolation 

 DNA is isolated in its pure form, which means they are devoid of other
macromolecules.
 In rDNA technology, the initial step is to extract the desired DNA in its purest form,
that is, free of extraneous macromolecules.
 Because DNA coexists with other macromolecules such as RNA, polysaccharides,
proteins, and lipids within the cell membrane, it must be separated and purified using
enzymes such as lysozymes, cellulase, chitinase, ribonuclease, and proteases.
 Other enzymes or treatments can remove other macromolecules. The DNA eventually
precipitates out as fine threads as a result of the presence of ethanol. After that, the
pure DNA is spooled out.
 
2. Cutting of DNA/Restriction Enzyme Digestion

 For this step, the restriction enzymes are quite vital. It helps to identify the location
wherein a designated gene is introduced into a vector genome. The said reaction is
known as restriction enzyme digestions. 
 They entail incubating pure DNA with a restriction enzyme of choice at conditions
that are appropriate for that enzyme.
 The 'Agarose Gel Electrophoresis' technology displays the restriction enzyme
digestion's progress.
 This method entails passing the DNA across an agarose gel. When current is applied,
negatively charged DNA flows to the positive electrode and is divided into different
sizes. This permits the digested DNA fragments to be separated and snipped out.
 The same method is used to process the vector DNA.
 
3. Amplifying of DNA

 Copies of genes are amplified through PCR or polymerase chain reaction. It is

essentially a process to increase a single DNA copy into several copies after the
desired gene of interest is cut with restriction enzymes. 
 It allows a single copy or a few copies of DNA to be amplified into thousands or
millions of copies.
 The following components are used in PCR reactions that are conducted on 'thermal
cyclers':
 1. Template:DNA that has to be amplified.
2. Primers: oligonucleotides are tiny, chemically produced oligohnucleotides
that are complementary to a DNA region.
3. Enzyme: DNA polymerase.
4. Nucleotides: The enzyme is required to lengthen the primers.

 PCR can be used to amplify the cut DNA fragments, which can subsequently be
ligated with the cut vector.
 
(Image will be uploaded soon)
 
4. Joining DNA

 The vector and a section of DNA are joined in this step. It is achieved with the help of
the enzyme DNA ligase. 
 With the same restriction enzyme, the pure DNA and the vector of interest are cut.
 This yields the cut DNA fragment and the cut vector, both of which are now open.
 Ligation is the process of putting these two parts together with the enzyme 'DNA
ligase.'
 The resulting DNA molecule is a hybrid of the interest molecule and the vector DNA
molecules. Recombination is the term used in genetics to describe the merging of
different DNA strands.
 As a result, this new hybrid DNA molecule is known as a recombinant DNA
molecule, and the process is known as recombinant DNA technology.
 
5. Insertion of rDNA into a Host

 Here rDNA is added to the recipient host cell, and the entire process is called
transformation. Post insertion, the recombinant DNA multiplies and manifests as
manufactured protein under favorable conditions.
 The recombinant DNA is then transferred into a recipient host cell, most commonly a
bacterial cell, in this stage. The term for this procedure is 'Transformation.'
 Bacterial cells have a hard time accepting foreign DNA. As a result, they are given
treatments to make them 'capable' of accepting new DNA. Thermal shock, Ca++ ion
therapy, electroporation, and other procedures may be applied.
 
6. Recombinant Cell Isolation

 A mixed population of converted and non-transformed host cells results from the
transformation process.
 Only the transformed host cells are filtered during the selection procedure.
 The marker gene of the plasmid vector is used to distinguish recombinant cells from
non-recombinant cells.
 PBR322 plasmid vector, for example, comprises two marker genes (Ampicillin
resistant gene and Tetracycline resistant gene). When pst1 RE is utilised, it eliminates
the Ampicillin resistance gene from the plasmid, causing the recombinant cell to
become Ampicillin sensitive.
 
(Image will be uploaded soon)
 
Application of Recombinant DNA Technology
Recombinant DNA technology has been widely used in medical science, industries, animal
husbandry, and agriculture.
The following highlights the application of r DNA technology in brief -

 To produce recombinant HB vaccines.

 For producing human insulin.
 To facilitate better crop production.
 For producing growth hormones in humans to treat dwarfism.
 For better gene therapy. 
 To acquire DNA fingerprinting.
 To diagnose several types of diseases. 
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2. What is DNA Cloning?
Typically, a clone is defined as a cluster of individual cells that come from a progenitor. A
clone is genetically similar to its parent cell from which it replicates. 
 
DNA cloning is initiated when DNA fragments are inserted into DNA molecules. The said
replicating molecule is the carrier of DNA vectors. A clone is a group of individuals or cells
that derive from a single progenitor. Clones are genetically identical because each time a cell
replicates, it produces identical daughter cells. Scientists have discovered a way to make
many copies of a single DNA fragment, a gene that can be used to make identical copies of a
DNA clone. DNA cloning is accomplished by inserting DNA pieces into a small DNA
molecule. This molecule is designed to multiply inside a living cell, such as a bacteria. The
carrier of the DNA vector is a small replicating molecule. Plasmids, yeast cells, and viruses
are among the prominent vectors of rDNA technology examples. Plasmids are circular DNA
molecules that bacteria inject into the human body. They aren't a part of the cell's core
genome. It carries genes that confer favourable traits on the host cell, such as the ability to
mate and drug resistance. They can be easily controlled since they are small enough and can
carry extra DNA that has been woven into them.
 
Importance of DNA Cloning:

 Agriculture is one of the fields where gene cloning is used. Nitrogen fixation is
carried out by cyanobacteria, and desired genes can be used to boost crop output and
improve health. As a result of this method, the consumption of fertilisers is reduced,
and chemical-free products are produced.
 In the world of medicine, gene cloning is extremely significant. Hormones, vitamins,
and medicines are all made with it.
 It can be used to identify and detect a clone that contains a specific gene that can be
modified by growing in a controlled environment. 
 It is utilised in gene therapy, in which a damaged gene is replaced with a healthy one.
This technique can be used to cure diseases like leukaemia and sickle cell anaemia.

An Introduction to Eukaryotic and Prokaryotic Cells

Eukaryotic and prokaryotic cells are present in organisms, depending on whether they include
membrane-bound organelles. Whether a nuclear membrane protects its genetic components is
a matter of debate. In this article, we will be looking at the differences between eukaryotic
and prokaryotic cells. Let us start by understanding what is DNA, in the coming section.
 
What is DNA?
Deoxyribonucleic acid (DNA) is the hereditary material in most living organisms. It is a
molecule comprising two helices that remain coiled around each other and has the genetic
information to be transmitted to the RNA or ribonucleic acid through a process called
transcription. 
Human DNA is located in the nucleus as a chromatin reticulum where DNA remains
associated with histone and non-histone proteins. In prokaryotes like in bacteria, the DNA
remains in the nucleoid region and does not remain associated with proteins. The DNA
contains the code for protein synthesis as nucleotides with four types of nitrogen bases,
Adenine, Guanine, Cytosine and Thymine. The double-helical structure of DNA is typical for
both eukaryotic and prokaryotic cells. However, DNA coiling is different. 
 
3. What Are Prokaryotic and Eukaryotic Cells? And write the differences between
prokaryotic cells and eukaryotic cells?
Prokaryotic cells were formed before eukaryotic cells and have a much simpler structure than
the latter. Both eukaryotic and prokaryotic cells have diverse DNA structures and chemical
compositions. There is no nucleus, no organelles, and just a minimal amount of DNA in
prokaryotic cells. Eukaryotic cells, on the other hand, have a nucleus and cell organelles, as
well as a large amount of DNA. The cytoplasm contains prokaryotic DNA, which is smaller
and circular. Inside the nucleus, eukaryotic DNA is large and linear.
 
The Differences Between Prokaryotic and Eukaryotic Cells

Prokaryotic Cell Eukaryotic Cells

They do not possess a well-defined nucleus. They possess a well-defined nucleus with
The genetic material remains diffused in a nuclear membrane, nucleolus, nucleoplasm
region of the cytoplasm called the nucleoid. and nuclear reticulum.

The cell is much smaller in size, usually These cells are larger, usually ranging from
ranging between 1 to 5 micrometres.  10 to 100 micrometres. 

Membrane-bound cell organelles like

Membrane-bound cell organelles are
mitochondria, endoplasmic reticulum, Golgi
present. 
bodies are absent 

Ribosomes are smaller in size with a Ribosomes are larger with a sedimentation
sedimentation coefficient 70S coefficient of the 80S
 The cell wall is simple and is made up of The cell membrane is a complex structure
peptidoglycan, muramic acid, etc.  made up of cellulose. 

The process of DNA replication is highly

During the process of DNA replication, the regulated and has selected origins of
entire genome is replicated at once.  replication for replicating portions of the
DNA. 

Organisms with eukaryotic cells are usually

Organisms which have prokaryotic cells are
multicellular but may be unicellular as
usually unicellular
well. 

Examples: Bacteria and Archaebacteria Examples: Animals, Plants, Fungi, Protists

 
4. Write the differences Between Eukaryotic and Prokaryotic DNA
Prokaryotic DNA: Prokaryotic DNA is double-stranded circular DNA that remains diffused
in a dense region of cytoplasm called the nucleoid. There is no nuclear membrane
surrounding the DNA in prokaryotes. The single circular DNA represents a single
chromosome. This DNA is not a supercoiled structure as in eukaryotes as they do not have
histone proteins in their structure. They form loops with the help of nucleoid-associated
proteins. Due to the absence of the histone scaffolding, the prokaryotic DNA is often referred
to as “naked DNA”. 
The size of the DNA is around 160000 to 12.2 million base pairs. The genes present in this
kind of DNA is less in number and is present in the form of operons. Operons are a group of
genes with a common promoter and a common terminator sequence as a result of which these
genes are expressed simultaneously. Since the number of base pairs is less, it does not keep
much scope for “junk DNA”. 
This means that most of the genes are without introns or non-functional DNA. There is a
single origin of replication as a result of which the entire genome is replicated at once.
During this type of replication, a single replication fork is formed. The rate of replication is
close to 2000 nucleotides per second. Transposons or mobile genetic elements are small
segments of DNA that can jump from one place to another in the genome. Such elements are
present in prokaryotes and help in bringing in genetic variation. During transcription, the
RNA formed is polycistronic, which contains information for more than one protein. When
ribosomes attach to each of these cistrons, it forms a beaded structure called polyribosomes. 
Apart from the DNA present in the nucleoid, prokaryotes also have extranuclear, double-
stranded, circular DNA in the cytoplasm. These are known as plasmids. The genes present in
the plasmids help in the survival of the bacteria and contains antibiotic-resistant genes. They
replicate autonomously and are transferred from one bacterium to another during
conjugation. 
Eukaryotic DNA: The DNA in eukaryotes is found in the nucleus enclosed in the nuclear
membrane. It is linear in shape. This DNA is present in the form of chromatin reticulum
when the cell is not dividing and condenses to form rod-shaped structures called
chromosomes during cell division. The DNA molecule remains tightly coiled and supercoiled
against basic proteins called histone proteins. Non-histone proteins are also present in this
DNA. The DNA forms a coiled structure called nucleosome which supercoils many folds to
form the condensed structure of the eukaryotic DNA. This coiling allows the large bulk of
DNA to be incorporated into the nucleus. 
The number of chromosomes varies from species to species and is unique for each one of
them. The human genome consists of 23 pairs of chromosomes, consisting of 2.9 billion base
pairs. There is a large amount of non-coding or junk DNA present in eukaryotes. Hence
introns need to be cut and removed from the RNA during gene expression. This process is
known as RNA splicing. There is more than one origin of replication allowing different genes
to be transcribed separately. The rate of replication is 100 nucleotides per second and is hence
much slower than that in prokaryotes. Transposons are inactive in eukaryotes. The mRNA
formed during transcription aremonocistronic, that is codes for only one protein.
Polyribosomes are absent in eukaryotes.  
 
Comparison of Eukaryotic DNA with Prokaryotic DNA 
Prokaryotic cells are much more complicated than eukaryotic cells because eukaryotic cells
are considered to exist in the later stages of evolution. It is probable that eukaryotic cells
developed from prokaryotic cells. At the cellular level, the difference in complexity can be
seen. The necessary and sufficient feature to define an organism as a eukaryote is that a
nuclear membrane surrounds the nucleus with nuclear pores. 

All existing eukaryotes have cells with a nucleus; most of the genetic material of eukaryotic
cells is contained in the nucleus. In contrast, prokaryotic DNA is not contained in the nucleus,
but attached to the plasma membrane and contained in the form of a nucleoid. The nucleoid is
an irregularly shaped area that is not surrounded by the nuclear membrane. Eukaryotic DNA
is packaged into a bunch of chromosomes, and each chromosome is composed of a linear
DNA molecule that is wound around basic proteins called histones, which coils the DNA into
a more compact structure.

Circular, non-chromosomal DNA is found in prokaryotic cells. Prokaryotes also have

plasmids, which are tiny circular DNA fragments that can replicate independently of
prokaryotic genomic DNA. Because eukaryotic DNA is linear, telomeres, or repeating non-
coding DNA sequences, are present on both ends of chromosomes to preserve them from
deterioration.

Mitosis, a nuclear division process wherein replicated chromosomes are divided and
separated via cytoskeleton components, is present in all eukaryotes. Actin microfilaments and
microtubules are structural and motility components of the cytoskeleton. These cytoskeletal
elements are found in all existing eukaryotes. Prokaryotes, on the other hand, go through a
binary fission process in which the DNA is copied, splits into two poles of the cell, and
ultimately divides completely.

The existence of mitochondrial DNA is a major difference between eukaryotes and

prokaryotes. As eukaryotes have mitochondria and prokaryotes do not have.  As a result, we
may split cells into prokaryotes and eukaryotes based on genetic materials enclosed by a
nuclear envelope. Prokaryotes do not have membrane-bound organelles, but eukaryotes do.

5. Write a note on the DNA replication?

DNA is the genetic material which has to be distributed to the daughter cells. Hence the
entire DNA molecule should be replicated before the cell division.

DNA replication is an important process which takes place in every organisms, be it

prokaryotic or eukaryotic. The DNA replication process produces two identical copies of
daughter DNA molecules using the existing DNA molecule as template. Each daughter DNA
molecule inherits one strand from the parent cell and the other strand is newly synthesized.
This is known as semiconservative mode of replication, demonstrated by Meselson and Stahl
(see the original paper here).
Replication process in eukaryotic and prokaryotic organisms share a lot similarity with few
differences. In this post we discuss about the DNA replication in the prokaryotes.

The Stages in Replication:

The DNA replication in prokaryotes (and eukaryotes) are divided into three
stages: initiation, elongation, and termination.

Initiation:

During initiation the different proteins bind and form complex called orisome at origin which
unwinds the DNA. The different components and their functions are as follows;

• The origin:

The replication of DNA initiates at a particular sequence of nucleotides in the genome,

known as origin. The prokaryotic cell usually has one origin as the genome is small and
circular. The initial loading of the replication machinery known as orisome, a nuclear-protein
complex, and the initial unwinding takes place at origin.

In E. coli chromosomal DNA replication initiates at origin of chromosomal DNA, oriC. The

oriC is a 245-bp region containing four 9-bp and are three repeats of a 13-bp A/T-rich
sequence.

(Just for info: Read this paper if want to know more about orisomes.)

• DnaA protein: It is also known as the initiator protein. This protein with 4 different domains
is the first one to recognize and bind to the origin of replication at the 9 bp regions. It is the
binding of the initiator protein that actually causes the DNA to stretch and leads to
the separation of the strands at the AT-rich region. As the two strands separate more DnaA
proteins bind the unwound DNA.

• Dna C helicase loader: These proteins interact with the ssDNA-bound DnaA proteins. Dna
C helicase loaders help load the Dna B helicase onto the unwound DNA.

• Dna B helicase: Helicase continues the unwinding of the DNA.

• Single-stranded DNA binding protein (SSB): These proteins bind and stabilize the unwound
DNA. Hence the unwound DNA do not form base pairs again.
• DNA gyrase: DNA gyrase is a topoisomerase that removes the twist resulting from the
unwinding of the DNA, by cleaving a strand of the DNA helix, untwisting it and then
resealing the broken strand again.

As the DNA is unwound, the area appears like a bubble (fig 1) and is known as a replication
bubble.

Fig 1: Circular prokaryotic DNA and the replication bubble.

The replication bubble has two Y shaped ends known as replication forks, (fig 2 shows one of
the two replication fork) which is the junction between the wound ds DNA and ss unwound
DNA.

Fig 2: Replication fork.

The two unwound strands are complementary and each can serve as the templates for the
synthesis of the new strands of DNA.

Elongation:

During elongation, the DNA pol III extends the daughter strands as the replication complex
known as replisome travels along the length of the chromosome. The replisome includes a
helicase, double-stranded DNA, a DNA polymerase(s) and a clamp loader.

• DNA Polymerase

The first DNA polymerase enzyme to be characterized, DNA polymerase I (pol I) from the
bacterium Escherichia coli. DNA pol I is not the main DNA replicating enzyme but is
involved in processing of RNA primers during lagging-strand synthesis and DNA repair
mechanisms.

The major polymerase involved in the replication is DNA polymerase III, which is a dimer,
with two similar multisubunit complexes.

Fig 3: DNA pol III (Fijalkowska et al, 2012).

In DNA pol III monomer is made up of core enzyme, γ complex and the β clamp.

is The DNA pol III core is made up of the αεθ complex, wherein α is the Polymerase subunit,
ε is the exonuclease (proofreading) subunit, and θ is involved with the stabilizing.
The γ complex (γδδ′χψ) is required for the loading and unloading of the β clamps, the ring-
shaped protein complexes that holds and slides along duplex DNA. β clamps allow the
polymerase to remain associated with DNA and still slide along the DNA.

Each pol III monomer of the complex catalyzes the replication of either of the two DNA
strands at the replication fork at the rate of around 1000 bp per second.

DNA polymerase enzymes join the phosphate group at the 5′ carbon of a new nucleotide to
the hydroxyl (OH) group of the 3′ carbon of a nucleotide already in the chain. Because of this
there are two needs that arise;

~ First the DNA pol needs an existing 3’OH end.

This problem is solved by creating RNA primer

• Dna G primase: As the replication bubble is formed, the Dna G primase initiates the

synthesis of RNA primer, a sequence of about 10 RNA nucleotides complementary to the
parent DNA template. RNA primer provides a free 3-OH end for the addition of the base
pairs by the DNA polymerase. The RNA nucleotides in the primers are then replaced by
DNA nucleotides.

~ Second the replication can only proceed in 5′→3′ direction.

Now as DNA polymerase III can add nucleotides only to the 3′ end of a DNA strand having
OH group, the replication always proceeds only in the 5′ → 3′ direction on a growing DNA
strand.

However, as the two parent strands of a DNA molecule are antiparallel, one strand is
unwound from the 5′ end and other from the 3′ end at the replication fork (fig 4a).

In the strand which is unwound from the 5′ end (Fig 4b), the primer is synthesized, such that
it’s 3′ end faces towards the unwinding replication fork. Hence the DNA pol III
can continuously add nucleotides to the 3′ end (Fig 4c) and extend the DNA daughter strand
(fig 4d). This strand which is synthesized continuously is known as the leading strand, in
which the direction of daughter strand elongation is same as the direction of unwinding.
Fig 4: The replication in the leading strand.

The other strand, known as lagging strand, experiences the unwinding from the 3′ end. The
primase synthesizes the primer with 5′ end towards the unwinding end and the and the 3′ end
is on the opposite side. Now the DNA pol III extends the daughter DNA strand at the 3′ end.
Hence the unwinding of the parent duplex and extension of daughter DNA strand takes place
in the opposite direction.

In this lagging strand the replication cannot be continuous as in the leading strand. The
primase synthesizes the first primer (say P1 in fig 5b) and moves towards the 3′ end along the
direction of replication fork. The DNA pol III adds nucleotides to the 3′ end of the primer as
the replication fork moves in opposite direction. As the DNA is unwound, the region to the 5′
end of the primer (P1) remains unreplicated (please see fig 5c).

To replicate this region primase synthesizes another primer (P2) at the point closer to the
replication fork (see fig 5c). Hence the new RNA primer is constructed and the DNA pol III
jumps ahead to it and begin synthesis of the next DNA fragment (from the 3′ end of the
primer P2 to the 5’end of the primer P1, as seen in fig 5d).
Fig 5: Replication in the lagging strand.

Hence the replication of the lagging strand takes place in discontinuously as a series of short
fragments of DNA. These fragments are called Okazaki fragments, and are about 1000 to
2000 nucleotides long in prokaryotes.
Fig 6: The Okazaki fragments in the lagging strand.

As the Okazaki fragments are completely synthesized, the enzyme DNA polymerase

I removes the RNA primer and fills in the gap. DNA pol I also fills in any gaps between
Okazaki fragments following which the enzyme DNA ligase joins the adjacent Okazaki
fragments to the lagging strand.

“Hence one of the important feature of Replication is that the synthesis of the leading strand
is continuous, while that of the lagging strand is discontinuous.”

Termination:

Replication of the circular chromosome initiates at a origin, with two replication forks
proceeding in opposite directions. The two forks move in the opposite direction around the
circular chromosome and finally move towards the site opposite to the initiation site. This
region is known as the replication terminus.

Fig 7: The Termination sites (Beattie & Reyes-

Lamothe, 2015)

The replication terminus contains series of sites called Termination or ‘Ter’ sites. Ter sites
sequences are recognized and bound by the Tus protein monomers.
 Fig 8: Termination of replication
in E.coli (Kaplan, 2006).
The Tus–Ter complex is asymmetric and has one face that blocks replisome progression, the
‘nonpermissive’ face, and a second face that allows replication to continue, the ‘permissive’
face (Figure 1A) Thenon permissive face of Tus protein blocks DnaB helicase passage while
permissive face permits the passage of the replisome complex.

When a replisome proceeds towards the nonpermissive face, a cytosine at position 6 of Ter

site binds to the Tus protein. This strengthened association between Tus and Ter site, causes
the replisome to disassemble from the DNA and single-stranded binding protein (SSB) binds
and stabilises the ssDNA.

Later as the second replisome approaches the permissive face of the Tus–Ter complex,
affinity of Tus for Ter site decreases and the Tus protein dissociates.

6. Explain the process of transcription?

Transcription is the first step in gene expression, in which information from a gene is used to
construct a functional product such as a protein. The goal of transcription is to make a RNA
copy of a gene's DNA sequence. For a protein-coding gene, the RNA copy, or transcript,
carries the information needed to build a polypeptide (protein or protein subunit). Eukaryotic
transcripts need to go through some processing steps before translation into proteins.

In transcription, a region of DNA opens up. One strand, the template strand, serves as a
template for synthesis of a complementary RNA transcript. The other strand, the coding
strand, is identical to the RNA transcript in sequence, except that it has uracil (U) bases in
place of thymine (T) bases.

Example:

Coding strand: 5'-ATGATCTCGTAA-3' Template strand: 3'-TACTAGAGCATT-5' RNA

transcript: 5'-AUGAUCUCGUAA-3'

For a protein-coding gene, the RNA transcript contains the information needed to synthesize a
polypeptide (protein or protein subunit) with a particular amino acid sequence. In this case:

RNA transcript (acting as messenger RNA): 5'-AUGAUCUCGUAA-3' Polypeptide: Met-Ile-

Ser-STOP

RNA polymerase

The main enzyme involved in transcription is RNA polymerase, which uses a single-
stranded DNA template to synthesize a complementary strand of RNA. Specifically, RNA
polymerase builds an RNA strand in the 5' to 3' direction, adding each new nucleotide to the
3' end of the strand.

RNA polymerase synthesizes an RNA strand complementary to a template DNA strand. It

synthesizes the RNA strand in the 5' to 3' direction, while reading the template DNA strand in
the 3' to 5' direction. The template DNA strand and RNA strand are antiparallel.

RNA transcript: 5'-UGGUAGU...-3' (dots indicate where nucleotides are still being added at
3' end) DNA template: 3'-ACCATCAGTC-5'
 Stages of transcription

Transcription of a gene takes place in three stages: initiation, elongation, and termination.
Here, we will briefly see how these steps happen in bacteria. You can learn more about the
details of each stage (and about how eukaryotic transcription is different) in the stages of
transcription article.

1. Initiation. RNA polymerase binds to a sequence of DNA called the promoter, found near

the beginning of a gene. Each gene (or group of co-transcribed genes, in bacteria) has its own
promoter. Once bound, RNA polymerase separates the DNA strands, providing the single-
stranded template needed for transcription.

The promoter region comes before (and slightly overlaps with) the transcribed region whose
transcription it specifies. It contains recognition sites for RNA polymerase or its helper
proteins to bind to. The DNA opens up in the promoter region so that RNA polymerase can
begin transcription.

2. Elongation. One strand of DNA, the template strand, acts as a template for RNA

polymerase. As it "reads" this template one base at a time, the polymerase builds an RNA
molecule out of complementary nucleotides, making a chain that grows from 5' to 3'. The
RNA transcript carries the same information as the non-template (coding) strand of DNA, but
it contains the base uracil (U) instead of thymine (T). 
 RNA polymerase synthesizes an RNA transcript complementary to the DNA template strand
in the 5' to 3' direction. It moves forward along the template strand in the 3' to 5' direction,
opening the DNA double helix as it goes. The synthesized RNA only remains bound to the
template strand for a short while, then exits the polymerase as a dangling string, allowing the
DNA to close back up and form a double helix.

In this example, the sequences of the coding strand, template strand, and RNA transcript are:

Coding strand: 5' - ATGATCTCGTAA-3'

Template strand: 3'-TACTAGAGCATT-5'

RNA: 5'-AUGAUC...-3' (the dots indicate where nucleotides are still being added to the RNA
strand at its 3' end)

3. Termination. Sequences called terminators signal that the RNA transcript is complete.

Once they are transcribed, they cause the transcript to be released from the RNA polymerase.
An example of a termination mechanism involving formation of a hairpin in the RNA is
shown below.
 The terminator DNA encodes a region of RNA that forms a hairpin structure followed by a
string of U nucleotides. The hairpin structure in the transcript causes the RNA polymerase to
stall. The U nucleotides that come after the hairpin form weak bonds with the A nucleotides
of the DNA template, allowing the transcript to separate from the template and ending
transcription.

Eukaryotic RNA modifications

In bacteria, RNA transcripts can act as messenger RNAs (mRNAs) right away. In

eukaryotes, the transcript of a protein-coding gene is called a pre-mRNA and must go
through extra processing before it can direct translation.

 Eukaryotic pre-mRNAs must have their ends modified, by addition of a 5' cap (at the
beginning) and 3' poly-A tail (at the end).

 Many eukaryotic pre-mRNAs undergo splicing. In this process, parts of the pre-mRNA

(called introns) are chopped out, and the remaining pieces (called exons) are stuck back
together.
Top of image: Diagram of a pre-mRNA with a 5' cap and 3' poly-A tail. The 5' cap is on the 5'
end of the pre-mRNA and is a modified G nucleotide. The poly-A tail is on the 3' end of the
pre-mRNA and consists of a long string of A nucleotides (only a few of which are shown).

The pre-mRNA still contains both exons and introns. Along the length of the mRNA, there is
an alternating pattern of exons and introns: Exon 1 - Intron 1 - Exon 2 - Intron 2 - Exon 3.
Each consists of a stretch of RNA nucleotides.

During splicing, the introns are removed from the pre-mRNA, and the exons are stuck
together to form a mature mRNA.

Bottom of image: Mature mRNA that does not contain the intron sequences (Exon 1 - Exon 2
- Exon 3 only).

End modifications increase the stability of the mRNA, while splicing gives the mRNA its
correct sequence. (If the introns are not removed, they'll be translated along with the exons,
producing a "gibberish" polypeptide.)

To learn more about pre-mRNA modifications in eukaryotes, check out the article on pre-
mRNA processing.

Transcription happens for individual genes

Not all genes are transcribed all the time. Instead, transcription is controlled individually for
each gene (or, in bacteria, for small groups of genes that are transcribed together). Cells
carefully regulate transcription, transcribing just the genes whose products are needed at a
particular moment.

For example, the diagram below shows a "snapshot" of an imaginary cell's RNAs at a given
moment in time. In this cell, genes 1, 2 and 3, are transcribed, while gene 4 is not. Also,
genes 1, 2, and 3 are transcribed at different levels, meaning that different numbers of RNA
molecules are made for each.

Diagram showing that individual genes are transcribed in different amounts.

A region of DNA containing four genes is shown, with the transcribed region of each gene
highlighted in dark blue. The number of transcripts of each gene is indicated above the DNA
(on a Y- axis). There are six transcripts of gene 1, one transcript of gene 2, twelve transcripts
of gene 3, and no transcripts of gene 4.

This is not an illustration of any actual set of genes and their transcription levels, but rather,
illustrates that transcription is controlled individually for genes and other transcription units.

In the following articles, we'll take a more in-depth look at RNA polymerase, the stages of
transcription, and the process of RNA modification in eukaryotes. We'll also consider some
important differences between bacterial and eukaryotic transcription.