Chapter 4 First part Genes its NERegulation.ppt

“
“Quest to
Quest to
search for
search for
gene
gene
begins……..”
begins……..”

 He injected r strain into mice and mice did
not suffer.
 Griffith was bacteriologist working on
Diplococcus pnuemoniae, S Strain & R Strain.
 Then he injected s strain into mice, mice
suffered & died.
 He then injected heat
killed s strain into mice
and mice did not suffer.
 Then he injected a mixture
of living r strain & heat
killed s strain & to his
surprise the mice suffered
& died.
GRIFFITH’s Experiment

 He concluded that the r
strain have been
transformed to s strain by
some transforming
principle.
 Avery Mac Cleod & McCarty
purified Proteins, DNA, RNA
etc.& infected mice
separately.
 Those injected with DNA showed transformation.
 When Deoxyribonuclease was added which digests DNA
infection was not seen
 Thus DNA was confirmed as transforming principle.
GRIFFITH’s Experiment contd…
 On observing the blood smear, he found colonies of s
strain.

 Hershey & Chase used a
bacteriophage, cultured
them in different mediums
containing radioactive
Sulphur & Phosphorus.
 Both the group were
allowed to infect, E.coli
 Viral coats were removed by
centrifugation.
 Bacteriophage with radioactive DNA were found to be infective
GRIFFITH’s Experiment contd…

 A gene is described as the basic unit of inheritance
influencing specific expressions
 A gene is a CISTRON if it control’s expression.
 A gene is a MUTON if it undergoes mutations.
 A gene is a RECON if it participates in crossing over.
 A gene is found on chromosomes.
.
 DNA / RNA are collectively called as Nucleic acids
Defination & Forms

 James Watson and Francis Crick studied the
structure of DNA with X-ray Crystallography
provided by Maurice Wilkins & Rosalind
Franklin.
 Fredrick Meischer identified cellular
substance from nucleus (nuclein) & studies
showed its acidic properties hence it was
named as nucleic acids
 They received a Nobel prize in 1953
Historical Account
Fredrick
Meischer
James Watson Francis Crick Rosalind Franklin
Maurice Wilkins

 It is a polymer of nucleotide
Chemical Composition of DNA
 A Nucleotide = Nucleoside + Phosphate Group
 A Nucleoside = Pentose Sugar+N2 Base
THEREFORE
THEREFORE
Nucleotide = Pentose Sugar+N2 Base + Phosphate Grp

 It is De-oxyribose C5H10O4 i.e. one
O less than Ribose sugar C5H10O5
I PENTOSE SUGAR
 1st
Carbon atom has N2 base & 5th
carbon is out of the ring and has
Phosphate group.
CH2
O
1
2
3
4
5
H
H
H
OH
H
H
II PHOSPHATE GROUP
 It is represented in the form of Phosphoric acid
 The chemical formula is H3PO4
 The structural formula is
OH P O
OH
OH

III NITROGEN BASES
 Nitrogen containing cyclic
compounds -- Purines &
Pyrimidines.
 Purines are double ring
compounds & hence
larger namely; Adenine &
Guanine.
 Pyrimidines are smaller as
they are single ring
compounds namely;
Cytosine & Thymine.
 Purines pair with
Pyrimidines.

 Phosphate group is
attached to the 5th
Carbon
atom of one de-oxyribose
sugar of a nucleotide & to
the 3rd
Carbon of the de-
oxyribose sugar of
adjacent nucleotide..
 The bond thus formed is
called as Phosphodiester
Phosphodiester
linkage
linkage
O
1
2
3
4
P
5
O
1
2
3
4
P
5
S
S
PHOSPHODIESTER LINKAGES

O
1
2
3
4
P
5
O
1
2
3
4
P
5
NB
NB
S
S
Phosphodi
ester
linkage
O
1
2 3
4
P
5
O
1
2 3
4
P
5
NB
NB
S
S
Phosphodi
ester
linkage

WATSON & CRICK MODEL OF DNA
 Two polynucleotide chains are
held by H2 bonds between N2
bases
 It is double helical, two strands
coil around themselves & an
imaginary axis
 Due to twisting it shows major &
minor grooves
 It appears as a twisted ladder,
strands are formed of sugar &
phosphate, steps are made up of
H2 bonds between N2 bases
 Diameter is 20 AO
(2nm), length of
a spiral is 34AO
(3.4 nm) – pitch of
DNA.
 Each spiral contains 10 pairs of nucleotides hence distance
between two nucleotides is 3.4 AO

Chargaff’s Rue
 At C3 end there is a free OH group
& C5 end there is a free
Phosphate group, it is termed as
polarity.
 Base sequence on one strand
decides the base sequence on the
other strand hence the strands
are called complementary.
 Adenine pairs with Thymine A
T
 Cytosine pairs with Guanine C
G
 Strand shows a C3 & C5 end.
 Number of Purines = Number of Pyrimidines
 It is represented as A+G=T+C or A+G/T+C =1
WATSON & CRICK MODEL OF DNA

 Formation of exact copies is called as Replication or
Duplication.
 It takes place in S phase (synthetic phase)
MECHANISM OF REPLICATON
I.
I. Activation of nucleotides
Activation of nucleotides
 dAMP, dGMP, dTMP & dCMP are found in the nucleoplasm
 All the monophosphates are activated to triphosphotases
by the enzyme Phosphorylase using ATP
REPLICATION OF DNA

II.
II. Origin or
Origin or
Initiation
Initiation
 Thus the two strands
unzip/uncoil.
 Eukaryotes have many
nicks, prokaryotes have
only one nick.
 It begins at a specific place
and involves breakdown of
H2 bonds due to the action
of Endonuclease (initiator
proteins).
REPLICATION OF DNA

 DNA appears like an
inverted “Y” called as
replicating fork.
 DNA unwinding protein
Helicase (rep protein)
separates the strands.
 Separated strands are
stabilized by SSBP (Single
stranded binding protein /
Helix destabilizing
protein)
III.
III. Unwinding of strands
Unwinding of strands
REPLICATION OF DNA

IV.
IV. Synthesis of new
Synthesis of new
strands
strands
 The process is initiated by
RNA primer & in turn
synthesis of primer is
controlled by RNA primase.
 Each strand acts as a
template.
 RNA primer attaches to the
3’ end of template.
REPLICATION OF DNA

 Selected nucleotides are
joined to old strand by H2
bonds, successive
nucleotides by
Phosphodiester linkages
 This process takes place
under the influence of DNA
polymerase.
 RNA Primer helps in
formation of new strand.
IV.
IV. Synthesis of new
Synthesis of new
strands
strands
REPLICATION OF DNA

V.
V. Leading & Lagging
Leading & Lagging
strands
strands
 3’ – 5’ is leading template
& the strand is leading
strand, 5’ – 3’ is lagging
template forming lagging
strand.
 Leading strand is
synthesized continuously
& at a faster speed while
lagging strand is
discontinuous (in fragments)
& at less speed.
REPLICATION OF DNA
 The fragments are called
Okazaki fragments.
 These fragments are finally
joined by DNA ligase.

VI.
VI. Formation of Daughter
Formation of Daughter
DNA
DNA
 After replication each DNA molecule has one old and the
other new strand, hence it is termed as semi-conservative
method of DNA replication.
REPLICATION OF DNA

Replicating fork
SSBP – prevents reunion
Template – model
RNA Primer synthesized by DNA Primase
DNA polymerase – forms new strands
Leading & Lagging strand
Okazaki fragments
DNA Ligase – joins Okazaki fragments
Phosphorylase & ATP – activation of nucleotides
Endonuclease – breakdown of H2 bonds
Helicase – unwinding protein
Semiconservative method

 Usually single stranded, appears double stranded sometimes due
to coiling around itself
 Messenger RNA (m-RNA)
 Ribosomal RNA (r-RNA)
 Transfer RNA (t-RNA)
 Present in nucleus,cytoplasm & of two types;
Genetic (Viruses)
Non genetic (protein synthesis)
 Thymine is replaced by Uracil
 Sugar is ribose C5H10O5
RIBOSE NUCLEIC ACID

 Synthesized in nucleus by transcription.
 Antisense strand of DNA is used in transcription.
 Simple, straight, with triplets/ codons.
Messenger RNA (m-RNA)
 About 5% of total RNA
AUG UGC GAC GGC UAC UAA
UGA
UAG
Start Codon
Initiation
Codon
Stop
Termination
Codon
Codons
 Stop/Termination Codon (UAA Ochre/UGA Opal/UAG Amber) is
present at 3’ end.
 Start /Initiation Codon (AUG) is at 5’ end.

 m-RNA is short lived & is degenerated soon after
protein synthesis
 Function of m-RNA is copy the genetic information
from DNA
 Each codon specifies one amino acid & is called as
RNA language /Genetic code/Cryptogram
AUG UGC GAC GGC UAC UAA
UGA
UAG
Start Codon
Initiation
Codon
Stop
Termination
Codon
Codons
Messenger RNA (m-RNA)

 Functions are not clearly
known.
 Main function is to orient m-
RNA, once it comes in
cytoplasm after transcription.
 Appears double stranded due
to coiling around itself
 About 80 % in quantity.
Ribosomal RNA (r-RNA)

 It shows lump, DHU arm,
TΨC arm, Carrier arm and
anticodon
 Contains anticodon which
corresponds to codon on m-
RNA
 It is in two forms Clover Leaf
& Hair Pin model
 About 15 % in quantity.
Transfer RNA (t-RNA)

Formation of polypeptide chain of amino acids
It is divided into Transcription & Translation
Central Dogma
DNA RNA Proteins
Transcription Translation
In nucleus In cytoplasm
DNA Enzymes
ATP & GTP
Ribosomes
RNA
Amino Acids
Components Involved
PROTEIN SYNTHESIS

 Formation of m-RNA in nucleus is
called transcription
 Promoter is a small DNA sequence
providing binding site for RNA
polymerase.
 Brought about by DNA dependent
RNA polymerase.
 Transcription Unit made up of
Promoter, Structural Gene,
Terminator is required for
transcription.
 The DNA strand used for forming
mRNA is called as antisense
strand/template(3’5’)
PROTEIN SYNTHESIS – Transcription (copy in writing)

 During transcription RNA polymerase binds with promoter site (DNA
segment at 5’ end) and brings about initiation. (initiation factors )
 The two strands separate, complementary RNA nucleotides are
arranged as per the template. (elongation)
 Synthesis continues till the terminator & is called termination
(Termination factors), the RNA formed is called as hnRNA. heterogenous
nuclear RNA (precursor of mRNA )
 hnRNA undergoes splicing, capping & tailing
 A small DNA sequence which terminates the process is called
terminator.
 Structural gene is polycistronic in prokaryotes & monocistronic in
eukaryotes.(cistron -- controls expression)
 The segment of DNA strand is the structural gene.

 SPLICING -- Removal of introns (introns are non coding segments which do
not appear whereas exons – coding segments appear in the processed RNA)
 CAPPING -- addition of methylguanosine triphosphate (unusual
nucleotide) at 5’ end.
 TAILING -- addition of adenylate residues at 3’ end.
 Finally mRNA is synthesized & ready for Translation
 Other enzymes associated with Protein synthesis are
 RNA Polymerase I – forms rRNA
 RNA Polymerase II – forms hnRNA (heterogenous nuclear RNA)
 RNA Polymerase III – forms tRNA & snRNA (small nuclear RNA)

Video
SPLICING
CAPPING & TAILING

 Translation is the process of reading the sequence of codons on
the mRNA strand & form a polypeptide linkage of amino acids
accordingly.
 Following are the steps
1. Activation of Amino acids
2. Formation of polypeptide chain
 ACTIVATION OF AMINO ACIDS
 Aminoacyl synthetase activates the amino acid
 Energy required for activation is provided by ATP.
 Activated Amino acid is attached to 3’ end of tRNA forming
Aminoacyl-tRNA complex
Translation
Translation
 This process is called as charging of tRNA or aminoacylation of
tRNA

 INITIATION
 It begins with initiation complex
 Initiation complex requires
mRNA, larger & smaller
subunits of Ribosome, initial
AA-tRNA complex, ATP & GTP
& also initiation factors
 It starts with binding of mRNA on 30s unit, start codon is
positioned properly, fmet-tRNA complex attaches to start codon
with the help of tRNA (CCA end) with UAC as anticodon.
INITIATION ELONGATION TERMINATION
2. Formation of polypeptide chain involves
Translation contd…..

ELONGATION
Peptide linkages are formed between AA1, AA2,AA3….
Peptidyl transferase catalyzes elongation
Elongation factors are also involved in the process.
Ribosome moves in 5’  3’ direction & is called translocation.
 Ribosome has three sites
namely; Aminoacyl site, Peptidyl
site &Exit site
 Only AA1-tRNA complex binds at P
site while others at A site, then
are shifted to P site. Polypeptide
chain is released from P site

TERMINATION
Stop codon indicates termination, termination factors play an
important role in identifying the stop codon & release.
Smaller & Larger subunits get separated.
Energy required for protein synthesis is given by ATP & GTP.

Ribosome consists of 65% rRNA & 35% protein.
They are not surrounded by membrane, made up of unequal
subunits leaving a cleft when unite together through which the
mRNA passes.
To increase the cellular efficiency many ribosomes may get
attached to a single mRNA forming copies of poly peptide chains,
such a structure is called polyribosomes/polysome.
POLYRIBOSOMES/POLYSOMES

An operon includes structural genes & their control
elements promoters & operators.
Structural genes code for proteins, rRNA’s, tRNA’s.
Gene expression & regulation is brought about by an
operon.
GENE EXPRESSION & REGULATION
Promoters are signal sequences to start Protein
Synthesis, binding sites for RNA polymerases.
Operators are present between Promoters & Structural
genes.
P i O z y a
Promoter Regulator Operator Structural genes.

LAC OPERON
Lac operon has regulatory site, promoter site, and
three structural genes (z,y & a).
z gene  β-galactosidase – hydrolysis of lactose
y gene  Permease – entry of lactose inside the cell
a geneTransacetylase – transfer of acetyl group
from Acetyl Co A to β -galactosidase
If the cell is using normal energy source then i gene
transcribes a repressor mRNA to form a repressor
protein.
Repressor protein binds with the operator site thus
leaving no opportunity for RNA polymerase to bind.
P i O z y a
Promoter Regulator Operator Structural genes.

 Thus the structural gene does not get transcribed and
the enzymes are not formed.
LAC OPERON contd….
 But in the absence of glucose the Permease enzyme
brings lactose inside the cell & also interacts with
the repressor & inactivating the repressor.
 Thus RNA polymerase binds the operator site &
transcribes operon producing lac mRNA.
 This enables expression of the three genes &
formation of the three enzymes assigned to them.

 They form 64 possible combinations of codons (triplets).
GENETIC CODE
In RNA there are 4 types of nitrogen bases (A,U,C,G).
 3 of them serve as termination codons & the rest 61
are called sense codons.
 It is triplet & commaless.
 It is non ambiguous except (AUG – Methionine GUG – Valine
but in absence of AUG GUG as a start codon, codes for Methionine)
CHARACTERISTICS OF GENETIC CODE
 It is degenerate (2 or more Codons may code for a single amino acid
i.e. GGG, GGA, GGC & GGU code for glycine).
 It can be read only in 5’  3’ direction (polarity)
 It is universal. (Same in all organisms exceptions in the mitochondria
of Yeast & Mycoplasma).

 There are 20 amino acids (Amino Acid – Selenocysteine
requires element Selenium)
WOBBLE HYPOTHESIS
 To code for 20 amino acids there are 61 codons
which is more as far as the requirement.
 In 1966 Crick proposed Wobble hypothesis,
Accordingly in codon – anticodon pairing the third
base may not be complementary.
 The third base is called wobble base & the position is
called wobble position.
 The actual pairing occurs at the first two positions
only. Thus though there are 61 codons, tRNA’s are
not of 61 types.

Codon
mRNA
AntiCodon
tRNA
Coded
Amino Acid
Type of
Pairing
GUU CAA Valine Perfect
pairing
GUC CAA Valine Imperfect
pairing
GUA CAA Valine Imperfect
pairing
GUG CAA Valine Imperfect
pairing
WOBBLE HYPOTHESIS

U C A G
U UUU
UUC
Phe UCU
UCC
Ser UAU
UAC
Tyr
y
UGU
UGC
Cys U
C
A
G
UUA
UUG
Leu UCA
UCG
UAA
UAG
Stop UGA
UGG
Stop
Trp
C CUU
CUC
CUA
CUG
Leu CCU
CCC
CCA
CCG
Pro CAU
CAC
His CGU
CGC
Arg U
C
A
G
CAA
CAG
Gln CGA
CGG
A AUU
AUC
AUA
Ile ACU
ACC
ACA
ACG
Thr AAU
AAC
Asn AGU
AGC
Ser U
C
A
G
AUG Met AAA
AAG
Lys AGA
AGG
Arg
G GUU
GUC
Val GCU
GCC
GCA
GCG
Ala GAU
GAC
Asp GGU
GGC
GGA
GGG
Gly U
C
A
G
GUA
GUG
GAA
GAG
Glu

Heterocatalytic function
auto catalytic function

Chapter 4 First part Genes its NERegulation.ppt

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Chapter 4 First part Genes its NERegulation.ppt