2. WHAT IS DNA?
• DNA is deoxyribonucleic acid
• A nucleic acid which is a polymer of a
nucleotide monomers
• DNA present in
Nucleus: nuclear DNA
Mitochondria: mitochondrial DNA
Chloroplast: chloroplast DNA
2
3. DNA AS GENETIC MATERIAL
Frederick Griffith Transformation Experiments with
Streptococcus pneumoniae (1928)
lllS Type Two
strains
llR Type
• Virulent
• smooth colonies
• Cause pneumonia
• Avirulent
• Rough colonies
• No pneumonia 3
6. The Experiments of Avery, MacLeod and McCarty to
identify transforming principle (1944)
Type R
cells
Type R
cells
Type R
cells
Type R
cells
Type R
cells
Type S
DNA
Extract
+
DNase
Type S
DNA
Extract
+
RNase
Type S
DNA
Extract
+
protease
Type S
DNA
extract
Transformed Transformed Transformed
Mix Mix Mix Mix
6
7. Hershey and Chase Experiment (1952)
• Provided further evidence that DNA is the genetic material
• Studied the bacteriophage T2
• This virus infects Escherichia coli bacterial cells and is
therefore known as a bacteriophage
DNA
(inside the
capsid head)
Head
Sheath
Tail fiber
Base plate
Inside the
capsid
Made
up of
protein
7
8. Properties of Genetic Material
• Replication
• Storage of information
• Expression of that information
• Variation by mutation
8
9. DISCOVERING THE STRUCTURE OF DNA
Erwin Chargaff (1952)
James Watson and Francis Crick (1953)
Rosalind Franklin (1952)
9
10. X-Ray Diffraction by Rosalind Franklins
• Used X-ray diffraction to
study wet fibers of DNA
• The diffraction pattern she
obtained suggested several
structural features of DNA
Main Features are:
1. Helical structure containing
one or more strands
2. Proposed “34 Å repeat”
3. Confirmed 3.4 Å
internucleotide distance 10
11. Erwin Chargaff’s Experiment
• Chargaff analyzed the base composition of
DNA, which was isolated from many different
species.
Important conclusions drawn by him are:
1.The sum of pyrimidine bases = sum of purine
bases [C+T (+MC) = A+G]
2.The ratio of adenine to thymine and guanine to
cytosine is one [A/T=1 & G/C(+MC) = 1]
3.Bases with 6-amino groups are equal to bases
with 6-keto groups [A+C(+MC) = G+T]
11
12. 4. The ratio of A + T/G + C(+MC), known as
dissymmetry ratio, varies from one species of
DNA to other
Ratio>1: DNA is called AT type
Ratio<1: DNA is called GC type
Chargaff’s data suggest that A always paired with
T and G always paired with C
12
13. Watson and Crick’s DNA Model
Main features:
1.DNA molecule is a right handed
double helix consists of two
polynucleotide chains
2.Double helix has a major groove
(width 12 Å, depth 8.5 Å) and a minor
groove (width 6 Å, depth 7.5 Å)
3.Two chains of double helix run in
opposite direction and are
complementary to each other
13
14. 4. The diameter of helix is 20 Å
5. Length of pitch is 34 Å
6. Bases are 3.4 Å apart along
the helix axis
7. Each turn of helix contain 10
nucleotide residues
8. The two chains held together
by hydrogen bonds, A pairs with
T by 2 H-bonds and G with C by
3 H-bonds.
14
16. Phosphate
group
Sugars
D-Deoxyribose (in DNA)
Purines
(double ring)
Pyrimidines
(single ring)
Bases
O
O
O–
O–
P
H
H
H
HO
OH
O
HOCH2
H
H
D-Ribose (in RNA)
H
OH
H
HO
OH
O
HOCH2
H
H
Uracil (U) (in RNA)
Thymine (T) (in DNA)
Cytosine (C)
Adenine (A)
Guanine (G)
NH2
N
H
H
H
H
H
O
N
4
3
2
1
5
6
7
8
9
4
3
2
1
5
6
O
CH3 H
4
3
2
1
5
6
7
8
9
5′
O
NH2
H
H
N
N
N
N
NH2
N
N
H
N
N
N
H
H O
N
4
3
2
1
5
6
O
H
H O
4
3
2
1
5
6
N
4′ 1′
2′
3′
5′
4′ 1′
3′ 2′
N
CHEMICAL STRUCTURE OF DIFFERENT
COMPONENTS OF DNA
16
19. Comparison of different forms of DNA
A- DNA B-DNA C-DNA Z-DNA
Conditions 75% RH; Na+,
K+, Cs+ ions
92% RH; Low ion
strength
60% RH; Li+ ions Very high salt
conc.
Helix sense Right-handed Right-handed Right-handed Left-handed
Pitch 25.30 Å 35.36 Å 30.97 Å 45.60 Å
Base pairs per
turn
11 10 9.33 12
Helix Diameter 25.5 Å 23.7 Å 19.0 Å 18.4 Å
Sugar
phosphate
backbone
regular regular regular Zig-zag
Major groove Narrow and
deep
Wide and deep __ No major
groove
Minor groove Wide and
shallow
Narrow and
deep
__ Narrow and
deep
19
24. Helicase unwinds
parental double helix
Single-strand
Binding proteins
stabilize separate
strands
Ligase joins Okazaki
fragments and seals
other nicks in sugar-
phosphate backbone
Primase adds
short primer
to template strand
Enzymes involved in DNA replication
24
25. Binding proteins prevent single strands from rewinding.
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
5’
3’
5’
3’
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
3’
5’
5’
3’
Replication
25
27. DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
5’
5’
Overall direction
of replication
5’
3’
5’
3’
3’
3’
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
27
29. 3’
5’ 5’
5’
3’
5’
3’
3’
5’
3’
Overall direction
of replication
Okazaki fragment
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
29
30. 5’ 5’
5’
3’
5’
3’
3’
5’
3’
Overall direction
of replication
3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Okazaki fragment
30
31. 5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’
3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
31
32. 3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’
3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
32
34. Polymerase activity of DNA polymerase I fills the gaps.
Ligase forms bonds between sugar-phosphate backbone.
3’
5’
3’
5’ 3’
5’
3’
3’
5’
34
35. Proteins involved in DNA replication
Prokaryotes
1.DNA polymerase I
Polymerase activity for
primer extension
Exonuclease activity in
excision of DNA strands
during DNA repair
2. DNA polymerase II
Function in DNA repair
3. DNA polymerase III
Catalyzes DNA
synthesis
Eukaryotes
1.DNA polymerase α:
replication initiation of both
strands and priming
2.DNA polymerase β: DNA
repair
3.DNA polymerase ε:
replication of lagging strand
4.DNA polymerase γ:
replication of mt DNA
5.DNA polymerase δ:
replication elongation of
leading strands
35
36. 1. Direct repair of DNA: simple
removal of damage to the
DNA e.g., removal of
thymine dimers in E. coli by
gene phr
2. In mismatch repair of DNA,
repair enzymes correct
errors in base pairing
3. In nucleotide excision DNA
repair nucleases cut out and
replace damaged stretches
of DNA
DNA Repair
36
38. TRANSCRIPTION
• Production of RNA from nucleotide sequence
of DNA
THREE STEPS:
1.Initiation
2.Elongation
3.Termination
38
39. Transcription in prokaryotes
1. INITIATION
Promoter sequence:
• Present upstream of site of transcription
• Short sequence elements are particularly
critical for promoter recognition that is
The sequence in the top DNA strand at the -35
region is 5ʹ–TTGACA–3ʹ,
and the one at the -10 region is 5ʹ–TATAAT3ʹ.
39
40. • The enzyme that catalyzes the synthesis of
RNA is RNA polymerase and complete
molecule of this is called Holoenzyme required
to initiate transcription
Holoenzyme composed of:
Core enzymes: composed of five subunits,
α2ββʹϖ.
Sigma factor: role of σ factor is to recognize the
promoter.
40
43. 3. TERMINATION
The end of RNA synthesis is referred to as
termination.
1)Rho-dependent: Two components
rut site: acts as a recognition site for the binding
of the ρ protein
Termination site: DNA encodes an RNA
sequence containing several GC base pairs that
form a stem-loop
43
45. 2. Rho independent:
a process that does not require the ρ protein
Termination depend on two adjacent nucleotide
sequences
One is a uracil-rich sequence located at the 3ʹ
end of the RNA.
The second sequence is adjacent to the uracil-
rich sequence and promotes the formation of a
stem-loop.
45
47. TRANSCRIPTION IN EUKARYOTES
• Three different RNA polymerases transcribe the
nuclear DNA of eukaryotes. Each synthesizes one or
more classes of RNA.
Types of RNA polymerase:
1. RNA polymerase I : resides in nucleolus and
responsible for synthesizing rRNA molecules.
2. RNA polymerase II : is found in nucleoplasm and
synthesizes mRNA that codes for protein.
3. RNA polymerase III : is also a nucleoplasmic enzyme
and synthesizes tRNA
47
49. INITIATION
• RNA polymerase II and the general
transcription factors assemble at the core
promoter
Different transcription factors:
TFIIA, TFIIB,TFIID, TFIIE, TFIIF, and TFIIH
A first step in initiation is the binding of TFIID
to the TATA box on the DNA template.
Other transcription factors bind to core
promoter and to RNA polymerase and
position it over the transcription start site.
49
52. Characteristic Prokaryotes Eukaryotes
RNA polymerase • one in each species • Three, RNA polymerase
I, II and III
Promoter • A simpler and relatively
smaller sequence
• A relatively larger
sequence
Transcription
initiation
• Holoenzyme binds to
promoter and initiates
transcription
• Transcription factors
first bind to promoter,
then RNA polymerase
associates with them
and initiates
Transcription
complex
Composition
Separation of
components
Core polymerase + sigma
factor
Sigma factor dissociates
from core enzyme after
initiation
RNA polymerase +
transcription factors
Transcription factors
dissociate when
transcription initiated
End product • Polycistronic RNA
transcripts
• Monocistronic RNA
transcripts
A COMPARISON OF TRANSCRIPTION PROCESS
52
53. RNA PROCESSING
An RNA molecule newly produced by
transcription called a primary transcript,
frequently must undergo changes before it
can function in the cell.
Changes are made to the 5′ end, the 3′ end,
and the protein coding section of the RNA
molecule by addition of
1.5’ cap
2.3’ poly-A tail
3.RNA splicing
53
54. • A 5’ cap is simply a guanosine
nucleotide that has been
methylated at position 7 of the
purine ring.
• Enzyme involved is guanyl
transferase
Functions:
Protect the molecules from
degradation by nucleases.
Positioning of mRNA on the
ribosome for the initiation of
translation.
CAPPING
54
56. SPLICING
• In eukaryotic cells, the precursors for most mRNAs contain
introns, which are sequences within the primary transcript
that do not appear in the mature/functional RNA.
• To produce a functional mRNA molecule, the entire
process of removing introns and rejoining the exons is
termed RNA splicing.
• The process of intron removal is catalyzed by an RNA-
protein complex called Spliceosome
56
57. TRANSLATION
For translating mRNA in to polypeptides involves five
major components :-
1.Ribosomes
2.tRNA
3.Aminoacyl-tRNA synthatase
4.mRNA
5.Protein factors
57
65. Characteristic Prokaryotes Eukaryotes
tRNA • tRNAf
met carries
formylmethionine to
initiation site of mRNA
• tRNAi
met carries
methionine to initiation
site of mRNA
rRNA and
ribosomes
• 70S dissociates into
30S and 50S subunits
• Free in cytoplasm
• 80S dissociates into 40S
and 60S subunits
Translation Simultaneously with
transcription
Translation initiation
involves base pairing
between 16S rRNA and
mRNA in Shine-
Dalgarno consensus
sequence
Formylmethionine is
incorporated by
initiation codon AUG
Not
It is based on recognition
of 5’-cap of mRNA by 40S
subunit and some
proteins
Methionine is
incorporated at initiation
point by codon AUG
A COMPARISON OF TRANSLATION PROCESS
65
