Gene: Structure and Functions

A gene is the basic physical and functional unit of

heredity.
Genes are made up of DNA.
Each chromosome contains many genes
Prokaryotic Gene Structure:
 Promoter, Terminator and coding region

A promoter is a region of DNA that initiates transcription of a

particular gene. Promoters are located near the transcription start
sites of genes, on the same strand and upstream on the DNA.

Transcription terminator is a section of nucleic acid sequence that

marks the end of a gene during transcription. This sequence
mediates transcriptional termination by providing signals in the newly
synthesized mRNA.

Open Reading Frame/RNA coding sequence/Coding region: The

region which is read by RNA polymerase for mRNA transcription
Fig. Basic structure of eukaryotic gene

Fig. Promoter region

Fig. Schematic representation of eukaryotic DNA to mRNA
 Gene Expression
The central dogma of biology describes the flow of information from gene sequence
to protein product. This whole process is called gene expression.

Gene expression is the process by which information from a gene is used in the
synthesis of a functional gene product. These products are often proteins, but in
non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA
(tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
 Trnscription and translation
Transcription: It is the first step of gene expression, in which a
particular segment of DNA is copied into mRNA by the enzyme RNA
polymerase.

Translation: Messenger RNA (mRNA) is produced

by transcription from DNA—is decoded by a ribosome to produce a
specific amino acid chain, or polypeptide.
 Promoter, Terminator and codon

A promoter is a region of DNA that initiates transcription of a

particular gene. Promoters are located near the transcription start sites of genes, on
the same strand and upstream on the DNA.

Transcription terminator is a section of nucleic acid sequence that marks the end of
a gene during transcription. This sequence mediates transcriptional termination by
providing signals in the newly synthesized mRNA.

A codon is a series of three nucleotides (a triplet) that encodes a specific amino acid
residue in a polypeptide chain. There are 64 different codons (61 codons encoding
for amino acids plus 3 stop codons) but only 20 different translated amino acids. The
most common start codon is AUG. The three stop codons are UAG, UGA and UAA.
Transcription occurs in three stages:
1. Initiation:
a. RNA polymerase binds to DNA at a specific sequence of nucleotides
called the promoter.
b. The promoter contains an initiation site where transcription of the
gene begins.
c. RNA polymerase than unwinds DNA to initiate transcription process.
2. Elongation:
a. Only one of the DNA strands acts as a template for the RNA synthesis.
b. RNA polymerase can only add nucleotides to the 3' end of the strand so
like DNA, RNA must be synthesized in the 5' to 3' direction.
c. Free ribonucleotides from the cytoplasm are paired up with their
complementary base on the exposed DNA template.
d. RNA polymerase joins the ribonucleotides to form an mRNA strand.
e. As RNA polymerase advances, the process continues.
f. The DNA that has been transcribed, re-winds to form a double helix.
Termination:
a. RNA polymerase continues to elongate until it reaches the terminator, a
specific sequence of nucleotides that signals the end of transcription.
b. Transcription stops and RNA polymerase and the new mRNA transcript
are released from DNA.
c. The DNA double helix reforms.
d. The termination sequence usually consists of a series of adjancent
adenines preceded by a nucleotide palindrome.
e. This gives an RNA molecule that assumes a stem-and loop configuration.
f. This configuration stops RNA polymerase from transcribing any further.
There are two ways that bacterial RNA polymerase ‘knows’ when it has
reached the end of a transcription unit.
In one case, as the RNA polymerase nears the 3’ end of the nascent
transcript, it transcribes a 72 base, C-rich region. At this point, a termination
factor called the rho protein binds to the nascent RNA strand. Rho is an ATP-
dependent helicase that breaks the H-bonds between the RNA and the
template DNA strand, thereby preventing further transcription.
In the other mechanism of termination, the polymerase transcribes RNA
whose termination signal assumes a secondary hairpin loop structure that causes
the dissociation of the RNA polymerase, template DNA and the new RNA transcript.
The role of the hairpin loop in rho-independent termination is illustrated below.
 Processing of mRNA:
In prokaryotes, no RNA processing is necessary, the nascent RNA
is usually the mRNA. But, In eukaryotes, the nascent RNA is called
primary transcript and it needs to be processed.

The processing steps are:

Capping: Addition of a 5’ 7-methyl guanosine cap (capping) which
prevent pre mRNA from degradation by exonucleases, regulates
nuclear export and promotes translation.

Splicing: It is the process by which pre-mRNA is modified to remove

certain stretches of non-coding sequences called introns; the
stretches that remain include protein-coding sequences and are
called exons.
Polyadenylation:
Addition of a poly-A tail at the 3’end (polyadenylation) of
mRNA. Polyadenylation is important for transcription
termination, export of the mRNA from the nucleus, and
translation. The poly(A) tail and the protein bound to it
aid in protecting mRNA from degradation by
exonucleases
 Translation

Translation: The process by which mRNA is decoded to produce a

specific amino acid chain, or polypeptide or protein is termed as
translation.
 Translation: The Process of Protein Synthesis
• Ribosomes translate the genetic message of mRNA into
proteins. Proteins are composed of one or more
polypeptides. Polypeptides are linear chains of amino
acids.
• The mRNA is translated 5’3’, producing a
corresponding N-terminal  C-terminal polypeptide.

• Amino acids bound to tRNAs are inserted in the proper

sequence due to:
a. Specific binding of each amino acid to its tRNA.
b. Specific base pairing between the mRNA codon and
tRNA anticodon.
 RNA Used in Protein Synthesis

A copy of the gene that is being

expressed. Groups of 3 bases in mRNA, called “codons/ Genetic
code” code for each individual amino acid in the protein
synthesized by that respective gene.

Four different RNA molecules that make

up part of the structure of the ribosome. They perform the actual
catalysis of adding an amino acid to a growing peptide chain.

Small RNA molecules that act as adapters

between the codons of messenger RNA and the amino acids they
code for.
Usually the genetic code is almost universal for both

prokaryotes and eukaryotes.

 Ribosome
Each ribosome has:
• a binding site for mRNA
• three binding sites for tRNA

• A-site: aminoacyl-tRNA
• P-site: peptidyl-tRNA
• E-site: exit
 tRNA

•Transfer RNA molecules are short

RNAs that fold into a characteristic
clove leaf pattern.

•Each tRNA has 3 bases that make up

the anticodon. These bases pair with
the 3 bases of the codon on mRNA
during translation.

•Each tRNA has its corresponding

amino acid attached to the 3’ end.
 Initiation of Translation
Protein synthesis is similar in prokaryotes and eukaryotes. Some
significant differences do occur, and are noted below.
2. In both it is divided into three stages:
a. Initiation.
b. Elongation.
c. Termination.
3. Initiation of translation requires:
a. An mRNA.
b. A ribosome.
c. A specific initiator tRNA.
d. Initiation factors.
e. Mg2+ (magnesium ions).
 Initiation of Prokaryotic Translation
Ribosome binding to mRNA requires more than the AUG:
An additional sequence 8–12 nucleotides upstream from the AUG
is commonly involved. Discovered by Shine and Dalgarno, these
purine-rich sequences (e.g., AGGAGG) are complementary to the
3’ End of the 16S rRNA appears to be important in ribosome
binding to the mRNA
• The 30S ribosomal subunit requires the following components to
initiate translation:
a. All three initiation factors, IF1, IF2 and IF3.
b. GTP.

c. Mg2+ : Regulate the association and dissociation of the

ribosomal subunit.
 Initiation factors IF-1, IF-2, & IF-3

 IF-3 binds to the 30S ribosomal subunit, freeing it from its

complex with the 50S subunit.

 IF-1 assists binding of IF-3 to the 30S ribosomal subunit.

IF-1 also occludes the A site of the small ribosomal subunit,
helping insure that the initiation aa-tRNA fMet-tRNAfMet can bind
only in the P site & that no other aa-tRNA can bind in the A site
during initiation.

 IF-2 is a small GTP-binding protein. IF-2-GTP binds the initiator

fMet-tRNAfMet & helps it to dock with the small ribosome
subunit.
Initiation of protein synthesis in prokaryotes
 Initiation of protein synthesis in prokaryotes

The initiation process

involves first joining the
mRNA, the initiator
methionine-tRNA, and the
small ribosomal subunit.
Several “initiation factors”--
additional proteins--are also
involved. The large
ribosomal subunit then
joins the complex.
 Elongation of protein synthesis in prokaryotes
• The ribosome has 2 sites for tRNAs, called P and A.
• The initial tRNA with attached amino acid is in the P site.
• A new tRNA, corresponding to the next codon on the
mRNA, binds to the A site.
• The ribosome catalyzes a transfer of the amino acid from
the P site onto the amino acid at the A site, forming a new
peptide bond.
• The ribosome then moves down one codon.
• The now-empty tRNA at the P site is displaced off the
ribosome, and the tRNA that has the growing peptide
chain on it is moved from the A site to the P site.
Elongation of protein synthesis in prokaryotes
Termination of protein synthesis in prokaryotes

• Three codons are called “stop

codons”. They code for no amino acid,
and all protein-coding regions end in a
stop codon.

• When the ribosome reaches a stop

codon, there is no tRNA that binds to it.
Instead, proteins called “release
factors” bind, and cause the ribosome,
the mRNA, and the new polypeptide to
separate. The new polypeptide is
 Polysomes
•Each mRNA transcript is read simultaneously by more
than one ribosome.

•A second, third, fourth, etc. ribosome starts to read the

mRNA transcript before the first ribosome has completed
the synthesis of one polypeptide chain.

•Multiple ribosomes on a single mRNA transcript are

called polyribosomes or polysomes. Multiple ribosomes
can not be positioned closer than 80 nt.
 Polysomes

Fig. Diagram of a polysome, a number of ribosomes each translating the

same mRNA sequentially
 Polysomes

Electron micrographs of ribosomes actively engaged in

protein synthesis revealed by "beads on a string"
appearance.
 Polyprotein

• A single translation
product that is cleaved
to generate two or more
separate proteins is
called a polyprotein.
Many viruses produce
polyprotein.
 Post Translational Modification
The chemical modifications that take place at certain amino acid
residues after the protein is synthesized by translation are known
as post-translational modifications. These are essential for
normal functioning of the protein. PTMS occur mostly in E.R and
golgi apparatus. Simply,
Addition of groups or deletion of parts to make a finished and
active protein

Why PTM is necessary???

 Stability of protein
 Biochemical activity (activity regulation)
 Protein targeting (protein localization)
 Protein signaling (protein-protein interaction,cascade
amplification)
Protein Modification
 Protein translocation
Protein translocation or targeting or protein sorting is the biological mechanism by
which proteins are transported to the appropriate destinations in the cell or outside
of it. Proteins can be targeted to the inner space of an organelle, different
intracellular membranes, plasma membrane, or to exterior of the cell via secretion

ATP !!!
 Recombinant DNA Technology

 Recombinant DNA (rDNA) is a form of DNA that is created by combining

two or more DNA sequences from different sources and that would not normally

occur together.

 Recombinant DNA technology: Joining together of DNA molecules from

two different species that are inserted into a host organism to produce new

genetic combinations that are of value to science, medicine, agriculture, and

industry.

The procedure has been used to change genomic constitution of living organisms

and may have even more practical uses in the future.

 How recombinant technology works
These steps include isolating of the target gene and the vector, specific

cutting of DNA at defined sites, joining or splicing of DNA fragments,

transforming of replicon to host cell, cloning, selecting of the positive cells

containing recombinant DNA, and either express or not in the end. Six

basic steps are:

1. Isolating (vector and target gene)
2. Cutting (Cleavage)
3. Joining (Ligation)
4. Transforming
5. Cloning
6. Selecting (Screening)
Restriction enzyme, also called restriction endonuclease, a protein produced by bacteria
that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes
cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated
from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those
that contain genes; for this reason they are indispensable tools of recombinant
DNA technology.
 Nomenclature of Restriction enzymes

 Restriction enzymes are named after the bacterium from which they

are isolated

For example, Eco RI is from Escherichia coli, and Bam HI is

from Bacillus amyloliquefaciens. The first three letters in the

restriction enzyme name consist of the first letter of the genus (E)

and the first two letters of the species (co). These may be

followed by a strain designation (R) and a roman numeral (I) to

indicate the order of discovery (eg, EcoRI, EcoRII).

Named for bacterial genus, species, strain, and type

Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order of discovery: 1
Restriction Endonuclease scan the length of the DNA , binds to the DNA molecule

when it recognizes a specific sequence and makes one cut in each of the sugar

phosphate backbones of the double helix – by hydrolyzing the phoshphodiester

bond. Specifically, the bond between the 3’ O atom and the P atom is broken.

3’OH and 5’ PO4 is produced.

•Most bacteria use Restriction Enzymes as a defence against bacteriophages.

•Restriction enzymes prevent the replication of the phage by cleaving its DNA at

specific sites.

•The host DNA is protected by Methylases which add methyl groups to adenine or

cytosine bases within the recognition site thereby modifying the site and

protecting the DNA.

 Why don’t bacteria destroy their own DNA
with their restriction enzymes?
Methylation
DNA ligase is a specific type of enzyme, a ligase, (EC 6.5.1.1) that facilitates the joining
of DNA strands together by catalyzing the formation of a phosphodiester bond.

•DNA ligase has applications in both DNA repair and DNA replication
•DNA ligase has extensive use in molecular biology laboratories for recombinant
DNA experiments
•Purified DNA ligase is used in gene cloning to join DNA molecules together to
form recombinant DNA.
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3'

hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another

("donor"). ATP is required for the ligase reaction, which proceeds in three steps:

1. Adenylation (addition of AMP) of a lysine residue in the active center of the

enzyme, pyrophosphate is released

2. Transfer of the AMP to the 5' phosphate of the so-called donor, formation of a

pyrophosphate bond;

3. Formation of a phosphodiester bond between the 5' phosphate of the donor and the 3'

hydroxyl of the acceptor.

 Vector
A vector is a DNA molecule used as a vehicle to artificially carry foreign
genetic material into another cell, where it can be replicated and/or
expressed. A vector containing foreign DNA is termed recombinant DNA.

A good vector must have the following properties:

1. It should be able to replicate autonomously.

2. It should be easy to isolate and purify.
3. It should be easily introduced into the host cells, i.e. transformation of the
host with the vector should be easy.
 Properties of a good Vector
4. The vector should have suitable marker genes that allow easy detection
and/or selection of the transformed host cells.

5. When the objective is gene transfer, it should have the ability to integrate
either itself or the DNA insert it carries into the genome of the host cell.

6. A vector should contain unique target sites for as may restriction enzymes as
possible into which the DNA insert can be integrated without disrupting an
essential function.
7. When the expression of the DNA insert is desired, the vector should contain
regulatory elements, e.g. promoter, operator and ribosome binding sites;
several other features also be important.
 Properties of a good host
A good host should have the following features:

(1) Is easy to transform

(2) supports the replication of recombinant DNA

(3) is free from elements that interfere with replication of recombinant DNA

(4) lacks active restriction enzymes, e.g. E. coli K12 substrain HB 101

(5) does not have methylases since these enzymes would methylate the

replicated recombinant DNA which, as a result, would become resistant

to useful restriction enzymes

 Cloning Vector
A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or
the cell of a higher organism, that can be stably maintained in an
organism, and into which a foreign DNA fragment can be inserted
for cloning purposes.

The vector therefore contains features that allow for the convenient
insertion or removal of DNA fragment in or out of the vector.
Cloning Vector
Cloning Vector
 Expression Vector
An expression vector is usually a plasmid or bacteriophage designed
for protein expression in cells i.e; expression of the DNA insert.

The vector is used to introduce a specific gene into a target cell, and can
commandeer the cell's mechanism for protein synthesis to produce
the protein encoded by the gene.

The plasmid is engineered to contain regulatory sequences such as

promoter, operator, ribosome binding site that can lead to efficient
expression of the gene carried on the expression vector.
Expression Vector
 Different Types of Vector

Vector system Host cell Insert capacity

(kb)
Plasmid E. coli 0.1-10

Bacteriophage lambda E. coli 10-20

Cosmid E. coli 35-45

Bacteriophage P1 E. coli 80-100

BAC (Bacterial Artificial E. coli 50-300

Chromosome)

Yeast Artificial Chromosome Yeast 100-2000

 Plasmids
A plasmid is a small DNA molecule within a cell that is physically separated
from a chromosomal DNA and can replicate independently. They are most
commonly found in bacteria as small, circular, double-stranded DNA molecules.
They are not essential for host cell except under specific environmental
condition.
 Example of plasmid vector: pBR322
pBR322 is a widely used E. coli cloning vectors. Constructed in 1977 in the
laboratory of Herbert Boyer at the University of California, San Francisco. It was
named after the two scientists who constructed it. The p stands for "plasmid," and
BR for "Bolivar" and "Rodriguez."

pBR322 is 4361 base pairs in length and harbors

• The origin of replication.
• The ampR gene, encoding the ampicillin resistance protein and
• The tetR gene, encoding the tetracycline resistance protein.
• The plasmid has unique restriction sites for more than 40 restriction enzymes.
11 of these lie within the tetR gene and 6 restriction sites inside the ampR gene.
• The circular sequence is numbered such that 0 is the middle of the unique EcoRI
site and the count increases through the tetR gene.
Schematic representation of pBR322
 Limitations of plasmids as vector

• Cannot accept large fragments

• Sizes range from 0- 10 kb

• Standard methods of transformation are not always successful

 Bacteriophage lambda as vector

• Phage lambda is a bacteriophage or phage, i.e. bacterial virus,

that uses E. coli as host.

• Its structure is that of a typical phage: head, tail, tail fibres.

• Lambda viral genome: 48.5 kb linear DNA with a 12 base ssDNA

"sticky end" at both ends; these ends are complementary in
sequence and can hybridize to each other (this is the cos site:
cohesive ends).

• Infection: lambda tail fibres adsorb to a cell surface receptor, the

tail contracts, and the DNA is injected.

• The DNA circularizes at the cos site, and lambda begins its life
cycle in the E. coli host.
Bacteriophage lambda as vector
 Marker Genes

A marker gene is used in molecular biology to determine if a piece of

DNA has been successfully inserted into the host organism. There are
two types of marker genes: selectable markers and markers for
screening.

A selectable marker will protect the organism from a selective agent

that would normally kill it or prevent its growth. In most
applications, only one in a several million or billion cells will take up
DNA. Rather than checking every single cell, scientists use a selective
agent to kill all cells that do not contain the foreign DNA, leaving
only the desired ones.
• Antibiotics are the most common selective agents. There are about 50
selectable Marker genes. Most commonly used antibiotic resistance genes
are Ampr, Tetr, Kanr, Cmr etc.

Ampicillin resistance gene (ampr) : It codes for an enzyme (b-lactamase) that

is secreted into the periplasmic space of the bacterium where it catalyzes
hydrolysis of the b-lactam ring of the ampicillin. Thus, the gene product of the
ampr gene destroys the antibiotic.

Tetracycline resistance gene (tetr) : It encodes an outer membrane

associated protein of gram negative cells that prevents the antibiotic from
entering the cell. Thus, tetr containg bacterial cell survive in media with
tetracyclin antibiotic.

Chloramphenicol resistance gene (Cmr): It codes for a protein known as

the cat protein which destroys the antibiotic.
 —Kanamycin resistance gene (NPT II)
• NPT II = neomycin phosphotransferase II
• Normally, plant cells are sensitive to Km.
• Km inhibits protein synthesis and protein translocation
across membranes.
• Expression of the NPTII in plant cells results in synthesis of
NPTII enzyme
• The enzyme detoxifies Km by phosphorylation
 Markers for screening

A marker for screening will make cells containing the gene look
different. There are three types of screening commonly used:
• Green fluorescent protein (GFP) makes cells glow green under
UV light. A specialized microscope is required to see individual
cells. Yellow and red versions are also available, so scientists can
look at multiple genes at once. It is commonly used to measure
gene expression.
• GUS assay (using β-glucuronidase) is an excellent method for detecting a
single cell by staining it blue without using any complicated equipment.
The drawback is that the cells are killed in the process. It is particularly
common in plant science.
• Blue/white screening is used in bacteria. The lacZ gene makes cells
turn blue in special media (e.g. X-gal). A colony of cells with the gene
can be seen with the naked eye. The foreign DNA/gene destroy the lacZ
gene and the transformed cell appeared white in the selection media.
 Schematic representation of pUC19

Insertion of foreign DNA into the MCS located within the lac Z gene causes
insertional inactivation of this gene. Thus bacteria carrying recombinant plasmids
in the MCS, giving rise to white colonies, which can be distinguished on culture
media from non-recombinant cells, which are blue.