WELCOME

MOLECULAR BASIS OF
INHERITANCE

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Semiconservative model proposed by
Watson and Crick
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 SEMICONSERVATIVE DNA
REPLICATION: AN EXPERIMENTAL
PROOF (MESSELSON AND STAHL’S
EXPERIMENT)
2 preparations of E. coli
E. coli in a medium containing E. Coli in N salt medium labeled
15
NH4Cl (15N: heavy isotope of N) with 14N.
15
N was incorporated in both DNA 14
N was incorporated in both DNA
strands and became heavier. strands and became lighter.

Transferred E. coli cells from 15N medium to 14N

medium.
After one generation, DNA was isolated and
centrifuged using CsCl density gradient.
Its density was intermediate between 15N DNA and 14N DNA.
This shows that the newly formed DNA one strand is old (15N
type) and one strand is new (14N type). 4
MESSELSON AND STAHL’S
EXPERIMENT

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 THE MACHINERY AND
ENZYMES FOR REPLICATION
 Origin of replication: DNA replication starts at a point
called origin.
 A unit of replication with one origin is called a replicon.
 During replication, the 2 strands unwind and separate by
breaking the H-bonds in presence of an enzyme,
Helicase.

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THE MACHINERY AND ENZYMES
FOR REPLICATION

 The separated strands act as

templates for the synthesis
of new strands.
 DNA replicates in the 5’-3’
direction.
 Firstly, a small RNA primer is
synthesized. An enzyme,
primase produces the primer.

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THE MACHINERY AND ENZYMES
FOR REPLICATION

 In the presence of DNA dependent DNA polymerase, many

nucleotides join with one another to the primer strand and form a
polynucleotide chain (a new strand).
 Unwinding of the DNA molecule at a point forms a ‘Y’-shaped
structure called replication fork.
 The DNA polymerase forms one new strand (leading strand) in a
continuous stretch in the 5’-3’ direction. 8
THE MACHINERY AND ENZYMES
FOR REPLICATION

 The other new strand is formed in small stretches (Okazaki

fragments) in the 5’-3’ direction.
 The Okazaki fragments are then joined together to form a new
strand (lagging strand) by an enzyme, DNA ligase.
 If a wrong base is introduced in the new strand, DNA polymerase
can do proof reading.
 E. coli completes replication within 38 minutes. i.e. 2000 bp/s. 9
THE MACHINERY AND ENZYMES
FOR REPLICATION

 The other new strand is formed in small stretches (Okazaki

fragments) in the 5’-3’ direction.
 The Okazaki fragments are then joined together to form a new
strand (lagging strand) by an enzyme, DNA ligase.
 If a wrong base is introduced in the new strand, DNA polymerase
can do proof reading.
 E. coli completes replication within 38 minutes. i.e. 2000 bp/s. 10
THE MACHINERY AND ENZYMES
FOR REPLICATION

 Energetically, replication is a very expensive

process. Deoxyribonucleoside triphosphates act as
substrate and also provide energy for
polymerization.
 In eukaryotes, the replication of DNA takes place
at S-phase of the cell cycle.
 A failure in cell division after DNA replication
results into polyploidy.
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DNA REPLICATION

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 CENTRAL DOGMA OF
MOLECULAR BIOLOGY

Replication Transcription Translation

DNA DNA mRNA
Protein

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TRANSCRIPTI
ON
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 TRANSCRIPTION

 It is the process of copying genetic information from one

strand of the DNA into RNA.
 Here, adenine forms base pair with uracil instead of

thymine.
 Both strands are not copied during transcription, because

◦ The code for proteins could be different in both strands. This would
complicate the genetic information transfer machinery.
◦ If 2 RNA molecules are produced simultaneously this would be
complimentary to each other, hence form a double stranded RNA.
This would prevent translation process.

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 TRANSCRIPTION UNIT
 Consists of 3 regions:
◦ A promoter: Binding site for RNA polymerase.
◦ The structural gene:
◦ A terminator: End of the process of transcription.
 Since the 2 strands have opposite polarity and the DNA-

dependent RNA polymerase also catalyze the

polymerization in only one direction, i.e. 5’→3’.
 3’ →5’ acts as template strand. 5’→3’ acts as
coding strand.
 3’-ATGCATGCATGCATGCATGCATGC-5’ template strand

5’-TACGTACGTACGTACGTACGTACG-3’ coding strand

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SCHEMATIC STRUCTURE OF
A TRANSCRIPTION UNIT
Transcription
start site

Promoter Structural Template Terminator

gene strand
3’ 5’

5’ 3’
Coding strand

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 TRANSCRIPTION UNIT AND GENE
 Gene: Functional unit of inheritance.
 The DNA sequence coding for tRNA or rRNA molecule is
considered as gene.
 A segment of DNA coding for a polypeptide is called a Cistron.
 The structural gene in a transcription unit is said as
monocistronic (mostly in eukaryotes) or polycistronic (mostly
in prokaryotes).
 In eukaryotes, the monocistronic structural genes have
interrupted coding sequences-the genes in eukaryotes are
split.
 The coding sequences (expressed sequences) are defined as
exons. The exons are interrupted by introns (intervening
sequences).
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TRANSCRIPTION IN
BACTERIA

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TRANSCRIPTION

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STEPS OF TRANSCRIPTION IN
PROKARYOTES
 Initiation: Here, the enzyme RNA polymerase binds at the
promoter site of DNA. This causes the local unwinding of
the DNA double helix. An initiation factor (σ) present in RNA
polymerase initiates the RNA synthesis.
 Elongation: The RNA chain is synthesized in the 5’-3’

direction. In this process, activated ribonucleoside

triphosphates (ATP, GTP, UTP and CTP) are added. This is
complementary to the base sequence in the DNA template.
 Termination: A termination factor (ρ) binds to the RNA

polymerase and terminates the transcription.

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In eukaryotes, there are 2 additional complexities:
◦There are at least 3 RNA polymerases in the
nucleus.
 RNA polymerase I: Transcribes rRNAs (28S, 18S & 5.8S)
 RNA polymerase II: Transcribes precursor of mRNA,
the heterogeneous nuclear RNA (hnRNA).
 RNA polymerase III: Transcribes tRNA, 5S rRNA and
snRNAs (small nuclear RNAs).

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24
◦ The primary transcripts (hnRNA ) contain both the
exons and introns. Introns are non-functional and
have to be removed. For this it undergoes the
following processes:
 Splicing: Introns are removed and exons are spliced
(joined) together.
 Capping: Here, an unusual nucleotide (methyl guanosine
triphosphate) is added to the 5’ end of hnRNA.
 Poly A-Tailing (Polyadenylation): Here, adenylate
residues (200-300) are added at 3’-end. It is the fully
processed hnRNA, now called mRNA.

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GENETIC CODE
It is the sequence of nucleotides (nitrogen
bases) in the mRNA which contains the
information for protein synthesis.

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 SALIENT FEATURES OF
GENETIC CODE
 Triplet code (three-letter code)
 Genetic code is universal
 No punctuations are present between the adjacent codons
(comma less code)
 Non-overlapping
 A single amino acid is represented by many codons. Such
codons are called degenerate codons.
 The genetic code is non-ambiguous. i.e. one codon specify
only one amino acid.
 AUG is the initiator codon. In eukaryotes, methionine is the
first amino acid and formyl methionine in prokaryotes.
 Termination codons are UAA, UAG and UGA (non-sense
codons) 27
 THE GENETIC CODE DICTIONARY
(20 AMINO ACIDS)
1. Alanine (ala) 11. Leucine (leu)
2. Arginine (arg) 12. Lysine (lys)
3. Asparagine (asn) 13. Methionine (met)
4. Aspartic acid (asp) 14. Phenyl alanine (phe)
5. Cystein (cys) 15. Proline (pro)
6. Glutamine (gln) 16. Serine (ser)
7. Glutamic acid (glu) 17. Threonine (thr)
8. Glycine (gly) 18. Tryptophan (trp)
9. Histidine (his) 19. Tyrosine (tyr)
10. Isoleucine (iso) 20. Valine (val)

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 U C A G
UUU Phe UCU Ser UAU Tyr UGU Cys U
UUC phe UCC Ser UAC Tyr UGC Cys C
U
UUA Leu UCA Ser UAA Stop UGA Stop A
UUG Leu UCG Ser UAG Stop UGG Trp G
CUU Leu CCU Pro CAU His CGU Arg U
CUC Leu CCC Pro CAC His CGC Arg C

THE GENETIC CODE

C
CUA Leu CCA Pro CAA Gln CGA Arg A
CUG Leu CCG Pro CAG Gln CGG Arg G
AUU Ile ACU Thr AAU Asn AGU Ser U
A
AUC
AUA DICTIONARY
Ile
Ile
ACC
ACA
Thr
Thr
AAC Asn
AAA Lys
AGC Ser
AGA Arg
C
A
AUG Met ACG Thr AAG Lys AGG Arg G
GUU Val GCU Ala GAU Asp GGU Gly U
GUC Val GCC Ala GAC Asp GGC Gly C
G
GUA Val GCA Ala GAA Glu GGA Gly A
GUG Val GCG Ala GAG Glu GGG Gly G
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TRANSLATION

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 TYPES OF RNA
 mRNA (messenger RNA): Provides template for the
protein synthesis.
 tRNA (transfer RNA or sRNA or soluble RNA): Brings

amino acids for the protein synthesis and reads the

genetic code.
 rRNA (ribosomal RNA): Play structural and catalytic role

during translation.

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 tRNA- THE ADAPTER MOLECULE
Clover leaf model of tRNA

Ser Tyr

UCA Anticodon AUG

5’ AGU Codon UAC 3’

 DNA

Gene
Protein

Trait
 TRANSLATION
(PROTEIN SYNTHESIS)
 It takes place in ribosomes. Includes 4 steps
◦CHARGING OF tRNA
◦INITIATION
◦ELONGATION
◦TERMINATION

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 1. CHARGING OF tRNA
(AMINOACYLATION OF tRNA)

◦ Formation of peptide bond requires energy

obtained from ATP.
◦ For this, amino acids are activated and linked to
their cognate tRNA in the presence of aminoacyl
tRNA synthetase. This is called charging.

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 2. INITIATION
◦ It begins at the 5’-end of mRNA in
the presence of an initiation factor.
◦ mRNA is transcribed from DNA to
the ribosomes for protein synthesis.
◦ The mRNA binds to the small
subunit of ribosome. Now the large
subunit binds to the small subunit
to complete the initiation complex.
◦ Large subunit has 2 binding sites for
tRNA- aminoacyl tRNA binding site
(A site) and peptidyl site (P site).
◦ Initiation codon for methionine is
AUG. So methionyl tRNA complex
would have UAC at the Anticodon
site.
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 3. ELONGATION

◦ At the P site the first codon of

mRNA binds with Anticodon of
methionyl tRNA complex.
◦ Then another aminoacyl tRNA
complex with an appropriate
amino acid enters the ribosome
and attaches to the A site. Its
Anticodon binds to the second
codon on the mRNA and a peptide
bond is formed between first and
second amino acids. This is
catalyzed by an enzyme, peptidyl
transferase.

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 3. ELONGATION

◦ The first amino acid and its tRNA

are broken. This tRNA is removed
from the P site and second tRNA at
the A site is pulled to the P site
along with mRNA. This is called
translocation.
◦ Then the third codon comes into
the A site and an appropriate tRNA
with third amino acid binds at the
A site. This process is repeated.
◦ A group of ribosomes associated
with a single mRNA for translation
is called a polyribosome
(polysomes).
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 4. TERMINATION

◦ When the aminoacyl tRNA reaches

the termination codon like UAA,
UAG and UGA, the termination of
translation occurs. The
polypeptide and tRNA are released
from the ribosomes.
◦ The ribosome dissociates into
large and small subunits at the end
of protein synthesis.

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TRANSLATION

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TRANSLATION

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 42

SEE PART 3
Prepared by
MUHAMMED ALI. K.C
DEPARTMENT OF ZOOLOGY
Ph: 9544187632
Email: [email protected]
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