MOLECULAR BASIS OF INHERITENCE 1-Transforming principle/

Griffith effect (1928)

 This experiment is conducted by Frederick
Introduction Griffith in 1928.
 There are 2 types of nucleic acids  He conducted experiment on Streptococcus
 DNA (Dexoy ribonucleic acid ) pneumoniae (Pneumococcus-It is a coccus
 RNA (Ribo nucleic acid ) bacteria that cause pneumonia ).
 DNA is the genetic material in most of the  During the course of his experiment, a living
organism including human being organism (bacteria) had changed in physical
 But there are some viruses (Retrovirus) in form.
which genetic material are RNA.  There are 2 strains of pneumococcus bacteria
Examples
 S-Strain
 HIV,
 R-Strain
 TMV,
 QB Bacteriophage,
 Corona Virus
 DNA is acidic substance present in the nucleus
was first identified by Friedrich Meischer
(1869). He called it as Nuclein.
 Due to technical limitation he could not isolate
such a long polymer  S- strain bacteria:
The Search for the Genetic The bacteria produce smooth and shiny colony
in culture plate. This is because the S strain
Material bacteria has mucous (Polysacharide ) Coat.
 The discovery of nuclein by Meischer (1869) This strain of bacteria is virulent and cause
and the proposition for principles of pneumonia
inheritance by Mendel (1865) were almost at  R strain :
the same time, but that the DNA acts as a It produce rough colonies when grown on
genetic material took long to be discovered culture plate. The rough appearance is due to
and proven. the lack of mucous coat. This type of bacteria
 By 1926, the quest to determine the is non virulent and do not cause pneumonia
mechanism for genetic inheritance had Experiment
reached the molecular level.  When Griffith injected S strains (Virulent) into
 Previous discoveries by Gregor Mendel, Walter mice, they killed them by causing pneumonia
Sutton, Thomas Hunt Morgan and numerous  When mice were injected with R strain (Non
other scientists had narrowed the search to virulent ) there was no effect
the chromosomes located in the nucleus of  Then, mice were injected with heat killed S strain.
There was no effect.
most cells. But the question of what molecule
was actually the genetic material, had not
been answered.
 He then injected a mixture of Heat killed S strain cells. They discovered that DNA alone from S
and Live R strain bacteria, the mice died due to bacteria caused R bacteria to become
pneumonia, moreover , he recovered living S transformed.
bacteria from died mice  They also discovered that protein-digesting

enzymes (proteases) and RNA-digesting
enzymes (RNases) did not affect
transformation, so the transforming substance
was not a protein or RNA.
 Digestion with DNase did inhibit
transformation, suggesting that the DNA
caused the transformation.
 They concluded that DNA is the hereditary
material, but not all biologists were
convinced.
2-Hershey-Chase experiment (1952)
 The unequivocal proof that DNA is the genetic
material came from the experiments of Alfred
Hershey and Martha Chase (1952).
 They worked with viruses that infect bacteria
called bacteriophages.
 He concluded that the R strain bacteria had
somehow been transformed by the heat- Life cycle of Bacteriophage
killed S strain bacteria. Some ‘transforming  The bacteriophage attaches to the bacteria
 Bacteriophage introduce its genetic material
principle’, transferred from the heat-killed S into the bacterial cell.
strain, had enabled the R strain to synthesise  The bacterial cell treats the viral genetic
a smooth polysaccharide coat and become material as if it was its own and subsequently
manufactures more virus particles.
virulent.
Ie. R strain were converted into S strain
 This must be due to the transfer of the genetic
material. However, the biochemical nature of
genetic material was not defined from his
experiments.
Biochemical Characterisation of
Transforming Principle
 Prior to the work of Oswald Avery, Colin
MacLeod and Maclyn McCarty (1933-44), the
genetic material was thought to be a protein.
 They worked to determine the biochemical Hershey and Chase worked to discover
nature of ‘transforming principle’ in Griffith's whether it was protein or DNA from the viruses
that entered the bacteria
experiment.
 They purified biochemicals (proteins, DNA, Experiment
RNA, etc.) from the heat-killed S cells to see  Hershey and chase grew some viruses in a
which ones could transform live R cells into S medium containing radioactive phosphorus
 (32P). Viruses grown in the presence of  Bacteria which was infected with viruses that
radioactive Phosphorus (32P) contain had radioactive DNA were radioactive,
radioactive DNA but not radioactive protein, indicating that DNA was the material that
because DNA contains phosphorus but passed from the virus to the bacteria.
protein does not  Bacteria that were infected with viruses that
 They also grew some viruses (bacteriophages) had radioactive proteins were not radioactive.
on a medium containing radioactive Sulphur This indicates that proteins did not enter the
(35S). Viruses grown in the presence of bacteria from the viruses. DNA is therefore the
radioactive sulphur (35S) contains radioactive genetic material that is passed from virus to
protein but not radioactive DNA because bacteria
protein contains sulphur but DNA does not. DNA (Deoxy Ribonucleic acid)
 These Radioactive phages were allowed to  It is an acidic substance present in the nucleus
attach to E. coli bacteria. This step is called  It was first identified by Friedrich meischer in
Infection 1869
 Then, as the infection proceeded, the viral  He named it as Nuclein. However due to
coats were removed from the bacteria by technical limitation in isolating such a long
agitating them in a blender. This process is polymer, the elucidation of structure of DNA
called Blending remains elusive
 The virus particles were separated from the  In 1953, James Watson and Francis Crick
bacteria by spinning them in a centrifuge. This proposed a very simple but famous Double
process is called centrifugation Helical Structure of DNA. They proposed this
model based on X ray diffraction data
proposed by Maurice Wilkins and Rosalind
Frankiln
 One of the hallmarks of their proposition was
base pairing between the two strands of
polynucleotide chains.
 The length of DNA is usually defined as the
number of nucleotides (Or Base pairs: a pair of
nucleotides)
Eg: Man = 6.6×109 BP
E.Coli =4.6×106 BP
Lambda phage =48502 BP
Ø X 174 phage =5386 nucleotides
(Single stranded DNA)

Structure of DNA
 The salient features of the Double-
helix structure of DNA
01- It is made of two polynucleotide chains, where the
backbone is constituted by sugar -phosphate, and
the bases project inside.
02- The two chains have anti-parallel polarity. It
means, if one chain has the polarity 5'3', the other
has 3'5'.
03- The bases in two strands are paired through
hydrogen bond (H-bonds) forming base pairs (bp).
Adenine forms two hydrogen bonds with Thymine
from opposite strand and vice-versa. Similarly,
Guanine is bonded with Cytosine with three H-
bonds. As a result, always a purine comes
opposite to a pyrimidine. This generates
approximately uniform distance between the two
strands of the helix (20AO)
04- The two chains are coiled in a right -handed
fashion. The pitch of the helix is 3.4 nm (a nanometre
is one billionth of a metre, that is 10-9 m) and there
are roughly 10 bp in each turn. Consequently, the
distance between a bp in a helix is approximately
equal to 0.34 nm.
05- The plane of one base pair stacks over the other in
double helix. This, in addition to H-bonds, confer
stability of the helical structure

Chargaff Rule
 Proposed y Erwin Chargaff
 For a double stranded DNA, the ratios between
Adenine and Thymine, and Guanine and Cytosine
are constant and equal one.
Ie: A+G=T+C
(or)
A+G/T+C=1
 Packaging of DNA
Helix
a)Packaging of DNA in Eukaryote
Length of DNA =Total number of Base pair × distance
between adjacent base pair
= 6.6 × 109BP × 0.34 ×10-9  A typical nucleosome contains 200 bp of DNA
= 2.2m helix.
There for total number of nucleosome in
 This length that is far greater than the human :
dimension of a typical nucleus (approximately
10–6 m).
 The negatively charged DNA is wrapped  The beads-on-string structure in chromatin is
around the positively charged histone octamer packaged to form chromatin fibers that are
further coiled and condensed at metaphase
to form a structure called nucleosome.
stage of cell division to form chromosomes.
 The packaging of chromatin at higher level
requires additional set of proteins that
collectively are referred to as Non-histone
Chromosomal (NHC) proteins.

 In Eukaryotes there is a set of +vely charged

basic proteins called Histones.
 Histones are rich in +vely charged basic amino
acids (Amino acids are Lysine and arginine )
 The +ve charge is due to the presence of +ve
charge on both the amino acid residues.
 Histones are organised to form a unit of eight
molecules called as histone octamer.
 Nucleosomes constitute the repeating unit of
a structure in nucleus called chromatin,
thread-like stained (coloured) bodies seen in
nucleus.
 The nucleosomes in chromatin are seen as
‘beads-on-string’ structure when viewed
under electron microscope (EM)
 iii. It can perform dynamic functions of a
messenger.
iv. It can functions as adapter ,structure molecule

 RNA being a catalyst was reactive and hence

unstable. Therefore, DNA has evolved from
RNA with chemical modifications that make it
more stable.

b)Packaging of DNA in Prokaryote

Eg : E.coli
 In prokaryotes, such as, E. coli, though they do
Properties of Genetic Material
not have a defined nucleus, the DNA is not
(i) It should be able to generate its
scattered throughout the cell. replica (Replication).
 -vely charged DNA is held with +vely charged  Both the nucleic acids (DNA and RNA) have the
proteins in a region termed as ‘nucleoid’. ability to direct their duplications/Replication
 The DNA in nucleoid is organised in large loops
 But protein do not replicate
held by proteins.

(ii) It should chemically and

Structurally be stable.
 The genetic material should be stable enough
not to change with different stages of life
cycle, age or with change in physiology of the
organism.
 Stability as one of the properties of genetic
material was very evident in Griffith’s
RNA (Ribo nucleic acid) ‘transforming principle’ itself that heat, which
 It is formed of a single polynucleotide chain killed the bacteria, at least did not destroy
 It acts as the genetic material in some viruses. some of the properties of genetic material.
Such viruses are called Retroviruses  But in RNA, Present of 2’ OH group make it
Eg: HIV, TMV, QB Bacteriophage. catalytic and reactive hence RNA is unstable.
 RNA was the first genetic material.
(iii) It should provide the scope for
Functions of RNA slow changes (mutation) that are
i. RNA used to act as a genetic material (eg.in required for evolution.
Retro virus )  Both DNA and RNA are able to mutate.
ii. It can act a catalyst (there are some important  RNA being unstable, mutate at a faster rate.
biochemical reactions in living systems that are Consequently, viruses having RNA genome
catalyzed by RNA catalysts (RIBOZYME) and not and having shorter life span mutate and
by protein enzymes). evolve faster
 (iv) It should be able to express itself
in the form of 'Mendelian Characters’.
 For the storage of genetic information, DNA is
better due to its stability.
 But for the transformation of genetic
information RNA is better. Because RNA can
directly code for the protein synthesis. Hence
can easily express the character. But DNA
depend on RNA for protein synthesis.

Qn- Among two nucleic acid, DNA is better

genetic material. Explain
Ans : DNA is stable and less reactive.
For the storage of genetic information,
DNA is better due to its stability.
Q- What is the reason for the stability of DNA?
Ans: -DNA is double stranded
-Presence of thymine
-Absence of 2’ OH in sugar
Q- Why retrovirus mutates and Evolve faster?
Ans : Retroviruses are viruses in which
genetic material is RNA. RNA is
unstable, due to the presence of 2’OH in
the sugar (Ribose). It make RNA unstable
and catalytic. Hence retrovirus mutate
faster rate.

Q- Difference between DNA and RNA ?

DNA RNA
-It is formed of 2 poly -It is formed of single
nucleotide chain poly nucleotide chain
-The sugar in DNA is -The Sugar in RNA is
dexoy ribose Ribose
-The nitrogen base in -The nitrogen base in
DNA is A,T,G,C RNA is A,U,G,C
-Stable -Unstable and catalytic

Qn. If the length of E. coli DNA is 1.36 mm, can

you calculate the number of base pairs in
E.coli?
 CENTRAL DOGMA IN MOLECULAR Pairing we have postulated immediately suggests
BIOLOGY a possible copying mechanism for the genetic
material’’ (Watson and Crick, 1953).
 Proposed by Francis Crick
 It is the unidirectional flow of information
 DNA replication takes place in the S phase of cell
from DNA-RNA-Protein
division cycle.
 Failure of cytokinesis after DNA replication results
Polyploidy (Increase in the wholes set of
chromosome). Polyploidy is common in plant cells

Experimental verification of semiconcervative

 In some viruses the flow of information is in method of DNA replication
reverse direction  It was first shown in E.coli and later in higher
organisms such as plants and Human cell.
1) Meselson-Stahl experiment (1958)
 It is an exception to central dogma of molecular  Proposed by Matthew Meselson and Franklin
biology.
Stahl performed the following experiment in 1958
 They grew E. coli in a medium containing 15NH4Cl
DNA REPLICATION (15N is the heavy isotope of nitrogen). 15N was
 DNA replication is the copying for DNA from
incorporated in to both strand of bacterial DNA
parent DNA and the DNA become heavier. This DNA is called
 Watson and crick proposed Semiconcervative Heavy DNA.
method of DNA replication
 They also made medium containing 14NH4Cl (14N
 According to this 2 daughter DNA are produced
is the normal isotope of nitrogen).
from parent DNA. Each daughter DNA consists of 2
strands, one strand is newly synthesised and other  They took E.coli from 15N medium (Heavy DNA)
strand belongs to parent. and transferred into 14N medium. After one
Ie: Parent’s strand is conserved generation (20 minutes), they isolated and
centrifuged the DNA. Its density was
intermediate (hybrid). This shows that , in the
newly formed DNA, one strand is newly formed
(14N) and other strand belongs to parent
(15N).This confirms semi conservative method
DNA replication.

 After second generation (40 minutes), there was

equal amount of hybrid DNA and light DNA.

Heavy DNA can be distinguished from light DNA y

centrifugation in CSCl (cesium chloride) density gradient.

Watson-Crick model for

semiconservative DNA
replication
 While proposing the double helical structure for
DNA, Watson and Crick had immediately proposed
a scheme for replication of DNA.
To quote their original statement that is as follows:

‘‘It has not escaped our notice that the specific

  It has 2 functions,
i)It act as substrate.
ii)It provide energy for polymerisation

The 2 terminal phosphate in a dexoyribo nucleoside

triphosphates are high energy phosphate. It provides
energy for polymerisation.

Replication Origin
DNA replication starts at a point called Replication
Origin (Ori).
Replication Fork
2) Taylor’s Experiment (1958) Unwinding of DNA molecule at a point forms Y shaped
 Conducted by Taylor and Colleague structure called Replication Fork
 He used Vicia faba (Faba beans) as Template
experimentall material . During DNA replication, the 2 strands separate and act
 He used Radioactive thymidine to detect as a template for the synthesis of new strand. New
presence of newly synthesised DNA
strands are synthesised based on template sequence.

Enzymes in DNA Replication Leading strand and Lagging strand

(Continous and discontinuous strand)
a) Helicase
 In the presence of DNA dependent DNA
 During replication, the 2 strands unwind and
polymerase, many nucleotides are joined to one
separated by breaking the H bond in the presence
another to form poly nucleotide (new strand). The
of Helicase enzyme
DNA polymerase forms one new strand (leading
 DNA Replication is energetically expensive that is
strand) in continuous stretch in 5’-3’ direction
why during replication 2 strands of DNA cannot be
(Continous synthesis )
separated in its entire length
 The other new strand is formed in small fragment.
b) RNA Primase
This fragment of DNA is called Okazaki fragment.
 It synthesise RNA Primer (Small stretch of RNA)
Okazaki fragment is seen in discontinues strand.
c) DNA Dependent DNA polymerase
d) DNA Ligase
 It is the main enzyme in DNA replication
 Okazaki fragments are joined together to form new
 It uses a DNA template to catalyse polymerisation
strand by DNA ligase.
of dexoy nucleotides
 In E.Coli, it polymerise the nucleotides in faster
rate (2000BP/second).
 In E.coli DNA replication completes in 18 minutes.
 This enzyme has high accuracy (Any mistake
during replication would result into
mutations)
 It polymerise the nucleotides in 5’ 3 direction
Deoxy Ribonucleoside Triphsophate
DNA transcription a) A promoter
 The process of copying genetic information from  It is the site where DNA dependent RNA
one strand of DNA (Template) into RNA is called polymerase bind
transcription.  Transcription starts from promoter site.
 The enzyme involved in transcription is DNA  Promoter is located towards the 5’ end (Upstream)
dependent RNA polymerase. of the structural gene (The reference is made with
 In DNA transcription only a segment of DNA (Gene, respect to the polarity of Coding strand )
A gene is defined as the functional unit of  it is the presence of a promoter in a transcription
inheritance) and only one of the strand unit that defines the template and coding strands
(Template strand, 3’—5’) is copied to RNA b) The structural gene
 The promoter and Terminator flank the structural
gene in transcription unit.
 RNA is produced from the structural gene
c) Terminator
 It is the site at which transcription stops.
 The strand with polarity 3’—5’ act as a template  It is located towards the 3’ end (Down stream )of
(For mRNA synthesis), the other strand with coding strand
polarity 5’—3’ is called coding strand (It do not
code for anything). Steps in DNA Transcription
 It consist of 3 steps
Q-Why Both strands of DNA are not copied into i)Initiation
RNA during transcription? ii)Elongation
Ans : If both strand act as a template iii)Termination
i)Two RNA molecule will be produced with
different sequences . It results in the i)Initiation
formation of 2 different proteins.  The sigma factor (σ factor/initiation factor) bind
ii)Two RNA molecule will be produced, they transiently with RNA polymerase.
are complementary to each other, hence it  This RNA polymerase bind to the promoter of
may for double stranded DNA. This RNA will Transcription unit and initiate transcription
not translate into protein. ii) Elongation
 The DNA dependent RNA polymerase uses It uses
nucleoside triphosphates as substrate, and
Transcription Unit
polymerase in a template dependent manner in
 A transcription unit in DNA is defined by 3 regions
5’-3’ direction.
a) A promoter
 RNA polymerase somehow also facilitates opening
b) The structural gene
of the helix
c) A terminator
iii) Termination
 Terminator is located towards the 3’ end of coding
strand, it usually defines the end process of
transcription.
 The Rho factor (termination factor) terminates the
process of transcription.(by binding transiently
with RNA polymerase )
 Once the RNA polymerases reaches the terminator
region, the nascent RNA falls off, so also the RNA
polymerase. This results in termination of
transcription.
 The RNA Produces as a result of transcription in
prokaryote is called mRNA (Messenger RNA).
 The RNA Produces as a result of transcription in
Eukaryote is called hnRNA (Heterogeneous
nuclear RNA).
 Both transcription and translation are coupled in
bacteria. Many times translation can begin much
before the m RNA is fully transcribed

Transcription in Eukaryotes
 The process of transcription is same in both
prokaryotes and eukaryotes.
 In Eukaryotes, there are 2 additional complexities
i) There are at least 3 RNA polymerease in the
nucleus
RNA Pol I : it transcribe r RNA
(28S,18S,5.8S)
RNA Pol II : It transcribe hnRNA
(Heterogenous nuclear RNA/
Precursor of mRNA )
RNA Pol III : It transcribe tRNA ,
5srRNA, and snRNAs (small nuclear
RNAs)
ii) The hnRNA Produced as a result of
transcription contains both Exons (Coding
sequences) and Introns (non coding
sequences ),such RNA are non functional.
This hnRNA is subjected to processing
(Splicing, capping, tailing ) to become mRNA .
Hence hnRNA is called precursor of mRNA .

Splicing : It is the process by which Introns

(Non coding sequences) are removed and
Exons are join together in a defined order

Capping : Here an unusual nucleotides mGPPP

(methyl guanosine triphosphate) is added to
the 5'-end of hnRNA.

Tailing: In tailing, adenylate residues (200-

300) are added at 3'-end in a template
independent manner.
 The fully processed hnRNA, now called mRNA, It
is then transported out of the nucleus for
translation.

 Difference between DNA replication and  Introns or intervening sequences do not appear in
Transcription mature or processed RNA.
DNA REPLICATION DNA TRANSCRIPTION
DNADNA DNARNA
Enzyme in DNA Enzyme in DNA
replication is DNA transcription is DNA
dependent DNA dependent RNA
polymerase polymerase
The entire DNA is Only one of the
duplicated strand (Template)
and a segment (Gene)
DNA is copied into
RNA

Difference between Prokaryotic and Eukaryotic

transcription
Prokaryotic Eukaryotic transcription
transcription
Both transcription and They are separate
translation are couple in
prokaryotes
The RNA contains only The RNA contains exons
exons, this RNA is called and Introns ,hence this
mRNA RNA is called hnRNA
No processing required HnRNA Undergo
for mRNA processing
(Splicing,capping, tailing)
to become mRNA
A single DNA dependent At least 3 types of RNA
RNA polymerase is polymerase is involved
involved in synthesizing
all types of RNA

What is a gene?
 A gene is defined as the functional unit of
inheritance.
 The DNA sequence coding for tRNA or rRNA
molecule also define a gene.
 a cistron is a segment of DNA coding for a
polypeptide.
 The structural gene in a transcription unit could be
said as monocistronic (mostly in eukaryotes) or
polycistronic (mostly in bacteria or prokaryotes).
 In eukaryotes, the monocistronic structural genes
have interrupted coding sequences – the genes in
eukaryotes are split.
 The coding sequences or expressed sequences are
defined as exons. Exons are said to be those
sequence that appear in mature or processed RNA.
The exons are interrupted by introns.
 Genetic Code The salient features of genetic code are as follows:
i) The codon is triplet. 61 codons code for
 It is the sequence of nucleotides (Nitrogen base) in
aminoacids and 3 codons do not code for any
the mRNA that containing information for Protein
amino acids, hence they function as stop
synthesis (Translation )
codons.
ii) One codon codes for only one amino acid,
hence, it is unambiguous and specific.
iii) Some amino acids are coded by more than one
codon, hence the code is degenerate.
Nondegenerate codon
AUG :Methionine
UGG: tryptophan

iv) The codon is read in mRNA in a contiguous

Scientists involved in cracking/Deciphering
fashion. There are no punctuations.
the Genetic code
v) The code is nearly universal: for example, from
bacteria to human UUU would code for
Several scientists belongs to several
Phenylalanine (phe). Some exceptions to this
branches of science involved in cracking the genetic
code such as it physicists, organic chemists, rule have been found in mitochondrial codons,
biochemists and geneticists. Some of the scientists and in some protozoans.
are given below vi) AUG has dual functions.
-It codes for Methionine (met) ,
1-George Gamow (Physicist ) : he argued that since - it also act as initiator/start codon
there are only 4 bases and if they have to code for 20
amino acids, the code should constitute a combination Genetic code Amino acids
of bases. He suggested that in order to code for all the AAA Lysine
20 amino acids, the code should be made up of three CCC Proline
nucleotides (Triplet codon) GGG Glycine
UUU Phenyl alanine
2-Har Gobind Khorana : The chemical method GAG Glutamic acid
developed by Har Gobind Khorana was instrumental in GUG Valine
synthesising RNA molecules with defined combinations AUG Methionine
of bases (homopolymers and copolymers). AGU Serine
3-Marshall Nirenberg : He developed cell free system UAC Tyrosine
for protein synthesis UAA STOP
4-Severo Ochoa :Severo Ochoa enzyme UGA (do not code for
(polynucleotide phosphorylase) was also helpful in UAG any amino acid)
polymerising RNA with defined sequences in a
template independent manner (enzymatic synthesis
of RNA). MUTATION
 Finally a checker-board for genetic code was (Refer principles of inheritance notes-Mutation and Types)
prepared which is given below .
tRNA (Transfer RNA)/Adapter molecule
 Proposed by Francis crick
 From the very beginning of the proposition of code,
it was clear to Francis Crick that there has to be a
mechanism to read the code and also to link it to
the amino acids, because amino acids have no
structural specialities to read the code uniquely
Ie: Presence of tRNA was known before the genetic
code was postulated. However, its role as an adapter
molecule was assigned much later.
 tRNA has
 Anticodon loop : That has bases (Anticodon)
DNA Translation
complementary to the triplet codon on the  The process of polymerisation of amino acids to
mRNA form Polypetide chain (Protein) is called
Translation.
 Aminoacid acceptor end to which it bind to
specific amino acids.  The order and sequence of amino acids in a
protein is defined by the sequence of triplet
codon in the mRNA
 In a Protein, amino acids are joined by Peptide
bond. Formation of a peptide bond requires
energy
 A translational unit in mRNA is the sequence of
RNA that is flanked by the start codon (AUG) and
the stop codon and codes for a polypeptide.

tRNA-The Adapter Molecule  The process of translation consists of 4 steps

1-Charging Of Trna/ Aminoacylation of tRNA
 tRNA has specific for each amino acids  Formation of peptide bond required
 there are no tRNA for STOP codon energy. In the first steps amino acids are
 For initiation of translation process, there involved activated in the presence of ATP
initiator tRNA Ie: Aminoacid+ATP= Activated amino acids
 The secondary structure of tRNA looks like Clover  Such charged Amino acids are linked to
Leaf structure specific tRNA (Bind to amino acid binding
 3D structure of tRNA looks like Inverted ‘L’ site of tRNA ). This process is called
charging of tRNA / Aminoacylation of
Ribosome tRNA

 Ribosome is the cellular factory for protein 2-Inititation

synthesis.  Small sub unit of ribosome bind to the mRNA
 The ribosome consists of structural RNA and 80  The tRNA with methionine (Inititator tRNA) enter
different proteins. to the P site , then another Amino acids with
t RNA Enter to the A site
If 2 such charged tRNA are brought close enough, the
formation of peptide bond between them would be
favoured energetically. The Presence of a catalyst
would enhance the rate of peptide bond formation.

3-Elongation
 During this linkage 1st amino acid and 2nd amino
acid, 1st amino acid’s tRNA linkage is broken. This
tRNA is removed from the P site. And the 2nd tRNA
 In its inactive state, it exists as two subunits; a at the A site is pulled to the P site along with mRNA
large subunit and a small subunit . this process is known as translocation
 The large sub units has 2 sites (P and A site ).  It result the 3rd codon coming into the A site and
 The ribosome also acts as catalyst (23srRNA) in appropriate tRNA with amino acid bind to the A
Bacteria is the enzyme Ribozyme. It helps in for the site. This process is repeated result in the
formation of peptide bond. elongation of Protein chain

Q-Why tRNA is called adapter molecule? 4-Termination

Ans : A tRNA can read triplet codon on the mRNA with  When the ‘A site’ reaches on to the stop codon
one hand and can carry a specific amino acids with termination of translation occur because there is ni
other hand hence tRNA is called Adapter molecule. tRNA for stop codon (UAA,UGA,UAG)
Q-mRNA : Triplet codon  Release factor binds to Stop codon also helps in
tRNA : Anticodon termination
 coordinated regulation of expression of several
sets of genes.

OPERON
 Operons are cluster of genes responsible for
controlling metabolic reaction within a living
system.
OR
 All the genes regulating a metabolic reaction
constitute an Operon.
Eg: Lac operon
Trp Operon
Ara Operon
His Operon
UTR (Untranslated region) Val Operon
 An mRNA also has some additional sequences that
are not translated and are referred as untranslated LAC OPERON
regions (UTR).  Proposed by a geneticist, Francois Jacob and a
 The UTRs are present at both 5' -end (before start biochemist, Jacque Monod
codon) and at 3' -end (after stop codon).  They were the first to elucidate a
transcriptionally regulated system
 They are required for efficient translation
 The operon controlling lactose metabolism is
process.
called Lac Operon. It consists of
1-A regulator gene (i gene/ inhibitor gene) :
It code for repressor protein
2-three structural gene
a)Lac Z gene :It code for Beta galactosidase (-
gal . It hydrolyze lactose to glucose and
galactose)
REGULATION OF GENE EXPRESSION b)Lac y gene : It code for Permease (Increase
permeability of cell to-galactosides/ Lactose)
A. Regulation of gene expression in c)Lac a gene : it code for Transacetylase
Eukaryotes
 In eukaryotes, the regulation could be exerted at
i) Transcriptional level (formation of
primary transcript),
ii) Processing level (regulation of splicing),
In the Absence of Lactose (Inducer)
iii) Transport of mRNA from nucleus to the  If there is no inducer (Lactose ), lac operon remains
cytoplasm, switched off. The regulator gene synthesizes mRNA
iv) Translational level. to produce the repressor protein. This protein
binds to the operator gene and blocks RNA
polymerase movement. So structural genes are
B. Regulation of gene expression in
not expressed.
Prokaryotes
 The metabolic, physiological and environmental
conditions regulate expression of genes in
Prokaryotes
 Eg: In E coli the enzyme, beta galactosidase
hydrolyses lactose into glucose and galactose. In
the absence of lactose, the synthesis of beta
galactosidase stops.
 The development and differentiation of embryo
into adult organisms are also a result of the
In the presence of Lactose (Inducer) Human Genome Project
 If lactose is provided in the growth medium, the  It is the Finding out the complete DNA sequence of
lactose is transported into E.coli cells by the action human genome.
of permease.  It is launched in 1990
 Lactose (Inducer) binds with repressor protein. So  Completed in 2003
Repressor protein cannot bind to operator gene.  It was a 13 year project
The operator gene become free and induces the  But the sequence of chromosome 1 was
RNA polymerase to bind with promoter gene. Then completed only in May 2006 (this was the last of
RNA polymerase transcribe the structural RNA the human chromosomes – 22 Autosomes and X
result in lac mRNA formation. The lac mRNA and Y – to be sequenced).
translated to produce beta galactosidase,  The project was coordinated by
permease and trans acetylase  the U.S. Department of Energy and
 the National Institute of Health
 During the early years of the HGP, the Wellcome
Trust (U.K.) became a major partner;
 Additional contributions came from Japan, France,
Germany, China and others.

Why HGP is called a Mega project...?

  Human genome is said to have approximately 3 x

 109 bp, and if the cost of sequencing required is US
 Regulation of lac operon by repressor is referred to $ 3 per bp (the estimated cost in the beginning),
as negative regulation. the total estimated cost of the project would be
approximately 9 billion US dollars.
 Each operon has its specific operator and  Further, if these sequences were to be stored in
specific repressor. typed form in books, and if each page of the book
 For example, lac operator is present only contained 1000 letters and each book contained
in the lac operon and it interacts 1000 pages, then 3300 such books would be
specifically with lac repressor only. required to store the information of DNA sequence
from a single human cell.
Qn. Why lactose is called as an Inducer in Lac Operon
? Goals of HGP
Ans. Lactose is the substrate for the enzyme beta-
galactosidase and it regulates switching on and off of (i) Identify all the approximately 20,000-25,000
the operon. Hence, it is termed as inducer genes in human DNA;
Qn. how long the lac operon would be expressed (ii) Determine the sequences of the 3 billion
in the presence of lactose? chemical base pairs that make up human DNA;
Ans.The Lactose operon expresses as long as
the Lactose is present. When all lactose is converted
(iiii) Store this information in databases;
into glucose and galactose, the reaction stops
(iv) Improve tools for data analysis;
Qn. Why glucose or galactose cannot act as inducers
for lac operon ?
(v) Transfer related technologies to other sectors,
Ans. Glucose structures are not sufficient to bind with
such as industries;
repressor (a protein sensor) so they cannot acts as
inducer for lac operon.where as lactose acts as an
(vi) Address the ethical, legal, and social issues
inducer because it binds with the repressor
(ELSI) that may arise from the project.
Methodologies in Human genome project Salient Features of Human Genome
The methods involved two major approaches. I. The human genome contains 3164.7 million
a)Expressed sequence tags(ESTs). nucleotide bases.
Here we focused on identifying all the genes that are II. The average gene consists of 3000 bases, but
expressed as RNA sizes vary greatly, with the largest known
human gene being dystrophin at 2.4 million
b)Sequence Annotation bases.
This is a blind approach of simply sequencing the whole III. The total number of genes is estimated at
set of genome that contained all the coding and non- 30,000–much lower than previous estimates
coding sequence of 80,000 to 1,40,000 genes. Almost all (99.9
Steps in HGP per cent) nucleotide bases are exactly the
same in all people.
For sequencing, IV. The functions are unknown for over 50 per
1. The total DNA from a cell is isolated cent of the discovered genes.
2. Convert DNA into random fragments of relatively V. Less than 2 per cent of the genome codes for
smaller sizes (because DNA is a very long polymer, proteins.
and there are technical limitations in sequencing VI. Repeated sequences make up very large
very long pieces of DNA) portion of the human genome.
3. Clone each piece of DNA in suitable host using VII. Repetitive sequences are stretches of DNA
specialised vectors. The cloning resulted into sequences that are repeated many times,
amplification of each piece of DNA fragment so sometimes hundred to thousand times. They
that it subsequently could be sequenced with ease. are thought to have no direct coding
The commonly used hosts were bacteria and yeast, functions, but they shed light on chromosome
and the vectors were called as BAC (bacterial structure, dynamics and evolution.
artificial chromosomes), and YAC (yeast artificial VIII. Chromosome 1 has most genes (2968), and
chromosomes). the Y has the fewest (231).
4. The fragments were sequenced using automated IX. Scientists have identified about 1.4 million
DNA sequencers that worked on the principle of a locations where singlebase DNA differences
method developed by Frederick Sanger. (Sanger is (SNPs – single nucleotide polymorphism,
also credited for developing method for pronounced as ‘snips’) occur in humans. This
determination of amino acid sequences in information promises to revolutionise the
proteins). processes of finding chromosomal locations
5. These sequences were then arranged based on for disease-associated sequences and tracing
some overlapping regions present in them. This human history.
required generation of overlapping fragments for Application
sequencing. 1-One of the greatest impacts of having the HG
6. Alignment of these sequences was humanly not sequence may well be enabling a radically new
possible. Therefore, specialized computer based approach to biological research.
programs were developed (bioinformatics—It is 2- it can study all the genes in a genome, for example,
the application of computer science and all the transcripts in a particular tissue or organ or
information technology to the field of biology and tumor, or how tens of thousands of genes and proteins
medicine). work together in interconnected networks to
7. These sequences were subsequently annotated orchestrate the chemistry of life.
and were assigned to each chromosome.
Genome sequencing in other organisms
Genome Sequencing also done in
 Bacteria,
 Yeast,
 Caenorhabditis elegans (a free living non-
pathogenic nematode),
 Drosophila (the fruit fly),
 Plants (rice and Arabidopsis),
A representative diagram of human
genome project
Repetitive DNA  Alec Jeffrey used satellite DNA as the basis of
These are sequences, a small stretch of DNA is DNA fingerprinting that shows very high
repeated many times. These repetitive DNA can be degree of polymorphism. It was called as
separated from bulk genomic DNA as different peaks Variable Number Tandem Repeats.(VNTR)
during density gradient centrifugation. The bulk
DNA forms a major peak an the other small peaks are
referred to as satellite DNA.
Depending on • Different steps of DNA fingerprinting are:-
 base composition (A : T rich or G:C rich),  Isolation of DNA.
 Length of segment, and  Digestion of DNA by restriction
 Number of repetitive units (Copy number) endonucleases.
the satellite DNA is classified into many  Separation of DNA fragments by gel
categories, electrophoresis.
such as  Transferring (blotting) of separated DNA
a)micro-satellites, fragment to synthetic membranes, such as
b)mini-satellites nitrocellulose or nylon.
 Double stranded DNA made single stranded.
 The VNTR belongs to a class of satellite DNA
 Hybridization using labeled VNTR probe.
referred to as mini-satellite. A small DNA
sequence is arranged tandemly in many copy  Detection of hybridized DNA fragments by
numbers. The copy number varies from autoradiography
chromosome to chromosome in an individual(this  After hybridization with VNTR probe the
number varies from person to person ). autoradiogram gives many bands of different
 The size of VNTR varies in size from 0.1 to 20 kb sizes
 These bands give a characteristic pattern for
an individual DNA. It differs from individual to
Functions of repetitive DNA individual.
 It normally do not code for any proteins, Applications:
 These sequence show high degree of • Test of paternity.
polymorphism (Variation at genetic levels). • Identify the criminals.
 DNA Polymorphism means any difference • Population diversity determination.
in the nucleotide sequence observed in a
• Determination of genetic diversity.
population.( Eg SNPs)
 It is inheritale and arises due to mutation
and form the basis of DNA fingerprinting.
 Since DNA from every tissue (such as blood,
hair-follicle, skin, bone, saliva, sperm etc.),
from an individual show the same degree of
polymorphism, they become very useful
identification tool in forensic applications

Polymorphism in DNA sequence is the basis of

genetic mapping of human genome as well as of
DNA fingerprinting,

DNA FINGERPRINTING
 DNA fingerprinting was initially developed by
Alec Jeffreys.
 DNA finger printing is a very quick way to
compare the DNA sequences of any two
individual.
 The DNA from a single cell is enough to
perform DNA fingerprinting.
 DNA fingerprinting involves identifying
differences in some specific regions in DNA
called repetitive DNA, because in these
sequences, a small stretch of DNA is repeated
many times