PART 2

4.3 – DNA REPLICATION

4.4 – PROTEIN SYNTHESIS
 LESSON LEARNING OUTCOMES
Upon completion of this lecture, students should be able to:
Ò Describe the basic principle and process of MITOSIS and
MEIOSIS.
Ò Differentiate between MITOSIS and MEIOSIS.

Ò Differentiate the three DNA replication models.

Ò Explain the semi conservative of DNA REPLICATION.

Ò Define the function of mRNA, tRNA and ribosome

Ò Describe the process of PROTEIN SYNTHESIS.

 DNA – GENETIC MATERIAL
Ò The structure was discovered in 1953 by James Watson and
Francis Crick
v In 1953, James Watson and
Francis Crick introduced an
elegant double-helical model
for the structure of
deoxyribonucleic acid, or
DNA.
v Hereditary information is
encoded in DNA and
reproduced in all cells of the
body.
Ò DNA –
Deoxyribonucleic Acid
Ò DNA is arranged in an
double-helix
Ò Consist of many
nucleotides
Ò Nucleic acids are polymers of nucleotides
Ò NUCLEOTIDES consist of:
1. Phosphates
2. Pentose sugars
•RNA - ribose
•DNA - deoxyribose
3. Nitrogen bases
 Nitrogenous Bases

Ò PURINES - double carbon-nitrogen

ring
É Adenine

É Guanine

Ò PYRIMIDINES - single carbon-

nitrogen ring
É Uracil- RNA only
É Thymine - DNA only

É Cytosine – DNA only

• DNA is made up of:
– Four nucleotides: Adenine, Thymine,
Guanine and Cytosine
– These follow the rules of base-pairing:
Possible Number Of
Purine Pyrimidines
Base Pairs Hydrogen Bonds
Thymine (T)
Adenine (A) A-T 2
Uracil (U)

Guanine (G) Cytosine (C ) G-C 3

– A sugar-phosphate backbone
• DNA is arranged in an double-helix
4.3 DNA REPLICATION
 When & How Does DNA Replicate?

G1 S
esis
tok
in G2
sis

Cy
ito

MIT
(M) OTIC
M

P HA
SE

DNA replicate during

INTERPHASE –
S phase (“synthesis”).
 Proposed Models of DNA Replication
Ò In the late 1950s, three
different mechanisms Original
double helix
were proposed for the
replication of DNA
1. Conservative model 1st round of
replication
ÐThe two parental strands re-
associate after acting as
templates for new strand, 2nd round of
replication
restoring the parental double
helix
 Proposed Models of DNA Replication

2. Semiconservative model
Original double
ÐThe two strands of the helix
parental molecule
separate and each
function as a template for 1st round of
synthesis of a new, replication

complementary strand

2nd round of
replication
 Proposed Models of DNA Replication

3. Dispersive model Original double

helix
ÐEach strand of both daughter
molecules contains a mixture
of old and newly synthesized 1st round of
DNA replication

2nd round of
replication
 Parent First Second
cell replication replication

Conservative
model
• Watson and Crick’s semiconservative
model of replication predicts that when a
double helix replicates, each daughter
Semiconservative molecule will have one old strand
model (derived or “conserved” from the parent
molecule) and one newly made strand.

• Competing models were the conservative

Dispersive model model (the two parent strands rejoin) and
the dispersive model (each strand is a
mix of old and new)
 Proposed Models by WATSON-CRICK

A T Watson and Crick’s model

C G
suggested a mode of
T A
replication that is semi-
A T
G C
conservative.

(a) Parent molecule

Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model, as
predicted by Watson and Crick.
 Proposed Models by WATSON-CRICK

A T A T
C G C G
T A T A
A T A T
G C G C

(a) Parent molecule (b) Separation of

strands
 Proposed Models by WATSON-CRICK

A T A T A T A T
C G C G C G C G
T A T A T A T A
A T A T A T A T
G C G C G C G C

(a) Parent molecule (b) Separation of (c) “Daughter” DNA molecules,

strands each consisting of one
parental strand and one
new strand

The parental strand now serves as template that determines

the order of nucleotides along a new complementary strand.
DNA REPLICATION

Ò Process of copying a double-stranded DNA strand

Ò Process of replication consists:

STEP 1 – Initiation
STEP 2 – Elongation
STEP 3 – Termination
 Step 1 - INITIATION
Ò The replication of DNA molecule begins at special sites called
origin of replication.
Ò where two DNA strands are separated, opening up a replication “bubble”
Ò A eukaryotic chromosome may have hundreds or even thousands of
origins of replication.
Ò Replication proceeds in both directions from each origin, until the
entire molecule is copied.
Ò At the end of each replication bubble is a replication fork.
É Y-shaped region where the parental strands of DNA are being unwind and
new DNA strands are elongating.
Origins of replication
in a eukaryotic cell
 Step 1 - INITIATION

Ò Helicases are enzymes that untwist the double helix at the

replication forks by breaking the hydrogen bonds between
complementary bases.
Ò Single-strand binding proteins that stabilize single strand DNA
(used to hold the separated DNA strands in place)
Ò Topoisomerase prevent “overwinding” ahead of replication forks
by breaking, swiveling and rejoining DNA strands.
 Step 1 - INITIATION
Single-strand binding proteins Primase

3¢
5¢
Topoisomerase 3¢

RNA primer

5¢
Helicase 5¢
3¢
 Step 2 - ELONGATION
Synthesizing a new DNA strand

Ò Primase enzymes makes ‘RNA primer’ using RNA nucleotides.

(Primer- short RNA sequences of about 10 nucleotides
synthesized by primase)
Ò RNA primers – a short stretch of RNA that need to begin
replication.
Ò Start point for DNA Replication.
 Step 2 - ELONGATION
Synthesizing a new DNA strand

Ò DNA Polymerases
É Require a RNA primer and a DNA template strand.
É Adds a DNA nucleotide to the RNA primer
Ð continues adding DNA nucleotides complementary to the
parental DNA template strand.
É The rate of elongation is about 500 nucleotides per second
in bacteria and 50 per second in human cells.
 Step 2 - ELONGATION
Synthesizing a new DNA strand

Ò The two strands of DNA in a double helix are antiparallel –

they are oriented in opposite directions to each other.
Ò DNA Polymerases can add nucleotides only to the free 3’
end of a primer or growing DNA strand.
Ò Thus, a new DNA strand can elongate only in the 5’ à 3’
direction.
 Step 2 - ELONGATION
Synthesizing a new DNA strand

Ò DNA Polymerase III enzyme catalyzes the elongation of new

DNA strand at the location after the primer by continuously
adds nucleotides to the new complementary strand.
Ò à The new complementary strand growing towards replication
forks – LEADING STRAND.
Ò à The other template strand with direction away from the
replication fork – LAGGING STRAND.
Ò These segment of the lagging strand are called OKAZAKI FRAGMENTS.
LEADING STRANDS
LAGGING STRANDS
 Step 2 - ELONGATION
Synthesizing a new DNA strand

Ò DNA Polymerase I replace the RNA primer DNA

polymerase
with adding complementary DNA nucleotide.
5¢ 3¢
(LEADING and LAGGING strands).
3¢ 5¢
Ò The Okazaki Fragments are still have gap
DNA
between the strand. ligase

Ò DNA ligase facilitates the joining of 5¢ 3¢

Okazaki Fragments together. 3¢ 5¢
 Step 3 - TERMINATION
Proofread sequence
Ò Make sure that nucleotides are paired up correctly.
Ò DNA polymerase double-check the sequence of the new strand to make sure the
nucleotides are paired correctly.
Ò DNA can be damaged by exposure to harmful chemical or physical agents such as
cigarette smoke and X-rays; it can also undergo spontaneous changes
Ò Any mismatched detected will be repaired.
Ò Nuclease enzymes – cuts the damaged DNA strand at 2 points & damaged section
is removed.
Ò DNA polymerase repair by filling in the missing nucleotides
Ò DNA ligases seals the free end of the new DNA to old DNA, making the strand
complete.
Proofreading
&
Repairing
DNA
STEPS OF
DNA REPLICATION 3

1
LEADING STRAND
5’ DNA pol III
3’
Primer Primase LAGGING STRAND
3’
5’ DNA Pol III
3’ DNA Ligase
5’ DNA Pol I
Parental DNA 3’ 5’
4
3 2 1
3¢
4 5¢

5 6 7
STEPS OF DNA REPLICATION

1. Helicase unwinds the parental double strand.

2. Single strand binding protein stabilize the unwound
template strand.
É Topoisomerase help to prevent from over-winding.
3. DNA polymerase III continuously adding new nucleotides in
the 5’ à 3’ direction toward replication fork, producing the
LEADING STRAND.
STEP 3
Origin of replication
1. After RNA primer is made, DNA pol III
starts to synthesize the leading strand
3¢
5¢
5¢ RNA primer

3¢ “Sliding clamp”
5¢ DNA pol III
Parental DNA

3¢
5¢

5¢ 2. The leading strand is elongated

3¢ continuously in the 5’ to 3’
direction as the fork progresses.
5¢
STEPS OF DNA REPLICATION
4. Primase begins synthesis of the RNA primer for the 5th
Okazaki fragment.
5. DNA Polymerase III discontinuously adding new nucleotides of the
4th fragment in the 5’ à 3’ direction away from replication fork,
producing the LAGGING STRAND.
6. DNA Polymerase I remove (delete) RNA primers and replacing the
strand with nucleotides DNA.
7. DNA Ligase bind the 3’ end of the 2nd fragment to the 5’ end of the
1st fragment.
É Joining the lagging strands - Okazaki fragments.
DNA REPLICATION – enzymes
Ò Topoisomerase – untwist the double helix and prevent ‘overwinding’ strain ahead of
replication fork.
Ò Helicase – enzymes that unwind the double strand at the replication fork.
Ò Single-strand binding protein – bind to the unpaired DNA strands, stabilizing single
strand DNA.
Ò Primase – build up the RNA primer
É RNA primers – a short stretch of RNA that need to begin replication.

Ò DNA Polymerase III – Adding new nucleotide at the free 3’ of growing strand (RNA
primer).
É Elongation of new strand from 5’ à 3’

Ò DNA Polymerase I – Remove the primer and replacing new nucleotide (filling the gap).
Ò DNA Ligase – prevent “overwinding” ahead of replication forks.
Q&A
4.4 PROTEIN SYNTHESIS
from Gene to Protein
 PROTEIN SYNTHESIS
From Gene to Protein
Ò The information content of DNA is in the form of specific
sequences of nucleotides.
Ò Proteins are the links between GENOTYPE and PHENOTYPE.
Ò GENE EXPRESSION, the process by which DNA directs protein
synthesis, includes two stages:
É TRANSCRIPTION
É TRANSLATION
DNA mRNA Protein
TRANSCRIPTION TRANSLATION
Overview of Protein Synthesis
 Nuclear
envelope

DNA
TRANSCRIPTION

Pre-mRNA
RNA PROCESSING
Overview of Protein Synthesis
mRNA in Eukaryotic Cell

TRANSLATION Ribosome

Polypeptide

Protein synthesis in eukaryotic cell

DNA vs RNA
Ò There are two types of nucleic acids
É DNA (deoxyribonucleic acid)
É RNA (ribonucleic acid).
DNA RNA
Number of strands Double strand Single strand
Nitrogenous base Adenine – Thymine Adenine – Uracil
pairing Guanine – Cytosine Guanine - Cytosine
Where it is found Inside nucleus Inside nucleus and cytoplasm

Stores genetic information 1. Transfer genetic code out of nucleus

Function
which controls cell functions 2. Translate genetic code into proteins

1. mRNA – messenger RNA

Forms Only 1 form of DNA 2. tRNA – transfer RNA
3. rRNA – ribosomal RNA
RNA STRUCTURE (1 strand polynucleotide)

1. mRNA : Messenger RNA mRNA – linear polynucleotide

É Linear single
polynucleotide
É Carries the genetic code
transcripted from DNA in
the nucleus to the
ribosome in the cytoplasm
for protein synthesis.
RNA STRUCTURE (1 strand polynucleotide)

2. tRNA : Transfer RNA

É Single stranded polynucleotide folded into clover – leaf shape
É Carries amino acid to ribosome during translation.
É 2 sides:
Ð One side of tRNA molecule attaches to amino acids.
Ð Other side has anticodon that complementary to codon on mRNA.
É One unique tRNA for each amino acids.
tRNA
RNA STRUCTURE (1 strand polynucleotide)

Ò rRNA : Ribosomal RNA rRNA - ribosome

É Synthesize in nucleolus - P site (Peptidyl-tRNA Exit tunnel
binding site)
contain RNA & Protein. A site (Aminoacyl-
É Contain large (50s) and E site
tRNA binding site)

small subunit (30s) – (Exit site)

combines form E P A Large

subunit
RIBOSOMES.
mRNA
Ð Organelle involve in binding site Small
translate mRNA to subunit
polypeptide.
(b) Schematic model showing binding sites
 1 TRANSCRIPTION
DNA mRNA
From DNA transcribe into mRNA.
 TRANSCRIPTION

Ò Is the process of making mRNA from a DNA template.

É Very similar to DNA Replication.
Ò Remember!! as in replication, in transcription,
addition of a new nucleotide occurs at the 3’ end!
É Direction will be from 5’ à 3’
Ò The RNA is made so that its sequence is complementary to the DNA:
É A transcribes U

É G transcribes C

É T transcribes A

É C transcribes G
Complementary Coding

Ò Pairing is same like DNA, the Hydrogen bonding:

É A-U : 2 H-bonds
É G-C : 3 H-bonds

If the template DNA is:

A-T-G-C-T-T-A-A-C-C-G-G-T-T

The transcribed mRNA is:

U-A-C-G-A-A-U-U-G-G-C-C-A-A
 STEPS IN TRANSCRIPTION
1. Initiation. RNA Polymerase binds and unwinds
DNA strand at promoter region.
• promoter region – sequence TATAAA (TATA BOX) in DNA.
Transcription begin, RNA polymerase initiates
RNA synthesis at the start point, while promoter is
not transcribed.
2. Elongation. The RNA Polymerase moves
downstream, unwinding the DNA and elongating
the RNA transcript 5’ à 3’ direction, by adding
complementary sequence.
In the wake of transcription, the DNA strands re-
form a double helix.
3. Termination. RNA Polymerase reach at
terminator sequence, all compenents separate:
mRNA is released & RNA polymerase detaches
from the DNA.
 Nuclear
envelope

DNA
Now, DNA is already TRANSCRIPTED into mRNA TRANSCRIPTION

Pre-mRNA
RNA PROCESSING
Next, mRNA will be TRANSLATED into PROTEIN.
mRNA

mRNA move from nucleus

to cytoplasm for TRANSLATION process. TRANSLATION Ribosome
(through pores in nuclear envelope)
Polypeptide

Protein synthesis in eukaryotic cell

THE GENETIC Genetic code - almost universal
Code is non-overlapping groups of 3 mRNA nucleotide, CODON.
CODE NO space separating neighboring CODON.

Ò CODON Ò ANTICODON
É Triplet bases on mRNA strand É A sequence of three nucleotides on tRNA
É The mRNA base which codes for specific that is COMPLEMENTARY with three
nucleotides codon of mRNA.
amino acids
É 64 possible codon, but only 20 amino acid
Ð Not all amino acids have equal number of codon
coding for it
Ð For example; Tryptophan has 1 codon while
Leusine has 6 codons.
É Start codon : AUG corresponding to
amino acid methionine
É Stop codon : UAG, UAA, UGA
 2 TRANSLATION
mRNA Protein
From genetic code translate into
protein code
Codon on mRNA

Ò START CODON
The beginning of
translation
É AUG (methionine)
Ò STOP CODON
The ending of
translation
É UAA, UAG, UGA
The Flow Of Coding Information
Ò Central Dogma: DNA à mRNA à Protein (Proposed by Frances Crick)
Ò The association between DNA, RNA and Protein at the molecular level is
given in this example:
DNA : 3' - ACC AAA CCG AGT - 5’
mRNA: 5' - UGG UUU GGC UCA - 3’ ß codon
(complementary to DNA)
Protein: Trp Phe Gly Ser

Ò The string of amino acids has a direct relationship to nucleotide bases in

RNA and DNA
 TRANSLATION

Ò Is the process of protein production from a mRNA template.

Ò Involves tRNA, codon, anticodon and ribosomes.

Ò The information contained in the nucleotide sequence of the

mRNA is read as 3 letter words (triplets), called CODONS.
É Each codon stands for one AMINO ACID.
Ò Decoded by a ribosome to produce a specific amino acid chain,
or POLYPEPTIDE.
The Roles of Ribosomes

Ò Ribosomes make protein:

É Pair the tRNA with mRNA.
É Catalyze a condensation reaction between the ends of
the amino acids to make the peptide bond.
É The peptidyl transferase activity (the enzymatic activity
that forms the peptide bond) is carried out by rRNA.
É Protein is released either to the cytoplasm or to the
endoplasmic reticulum in eukaryotes.
 The function of tRNA is to
transfer amino acids from the
cytoplasmic pool of amino acids
to a ribosome

Two-dimensional structure of tRNA

Ò 2 subunits of ribosomes, separate in cytoplasm until they join to
begin TRANSLATION
É Large subunit & Small subunit

Ò 3 important site on the ribosome:

É E site (exit site)
É P site (peptidyle tRNA binding site)
– formation of peptide bond
É A site (Aminoacyl-tRNA binding site)
– acceptor site

The ribosome adds each amino acid brought to it by tRNA to the growing
end of a polypeptide chain.
mRNA will attach at the small subunit of ribosome.
 STEPS IN TRANSLATION

1. Initiation
É Formation initiation complex.
Ð mRNA binds to small subunit ribosomes,
Ð aminoacyl-tRNA with complementary anticodon to start codon (AUG) binds
to mRNA.
2. Elongation
É The continued addition of amino acids to the growing polypeptide
chain.
3. Termination
É The end of translation; release of the protein.
Ð When ribosome reach stop codon (UAA, UAG, UGA).
 Step 1 - INITIATION

mRNA binds to small ribosomal The arrival of large ribosomal subunit

subunit. An initiator tRNA, with complete the initiation complex. The
complementary anticodon to start initiator tRNA is in the P site, the A site
codon (AUG) binds to mRNA. is available to the tRNA bearing the
This tRNA carries the amino acid next amino acid.
methionine (Met).
 Step 2 - ELONGATION

1. Codon recognition. The anticodon of an incoming aminoacyl-tRNA

base pairs with the complementary mRNA codon in the A site.
2. Peptide bond formation. The ribosome catalyzes the formation of
a peptide bond between the new amino acid at A site and the
carboxyl end of the growing polypeptide P site.
3. Translocation. The ribosomes moves one codon down mRNA.
- The tRNA in the A site is translocated to the P site.
- Meanwhile, the empty tRNA in the P site moves to the E site,
where it is released from the ribosome.
- A site will be available for the next codon to be translated.
 The Elongation cycle of Translation

1. Codon Recognition
- An incoming aminoacyl tRNA
binds to the codon in the A site

3. Translocation
- The ribosomes moves one codon 2. Peptide bond formation
down mRNA - The ribosome catalyzes the
- The tRNA with growing polypeptide formation of peptide bond between
chain in the A site is translocated the new amino acid and the carboxyl
to the P site. end of the growing polypeptide.
- Meanwhile tRNA in the P site - So the amino acid from P site will
moves to the E site and is released transfer onto aminoacyl-tRNA in
from the ribosomes A site
 Step 3 - TERMINATION

1. When a ribosomal 2. The release factor

3. The two ribosomal
reaches a stop codon hydrolyzes the bond
subunits and the
on mRNA, the A site between the tRNA in
other components
of the ribosome the P site and the last
of the assembly
accepts a ‘release amino acid of the
dissociate.
factor’. polypeptide chain.
Polypeptide from Ribosome to Rough ER
 POLYSOME
Ò Situation occur when cell rapidly make many protein.
Ò Many ribosome will read the same message.

Ò This structure known as POLYSOME.

78
Prokaryote Eukaryote
Q&A