The information encoded in the DNA is transferred to messenger

RNA and then decoded by the ribosome to produce proteins.

DNA REPLICATION DNA replication is the biological process of
producing two identical replicas of DNA from
one original DNA molecule.
WHEN DOES IT OCCUR?
DNA replication occurring at S Phase (DNA
synthesis phase)

WHERE DOES IT OCCUR? Inside the

nucleus

WHY DOES IT OCCUR?

DNA replication needs to occur because
existing cells divide to produce new cells.
Each cell needs a full instruction manual to
operate properly. So the DNA needs to be
copied before cell division so that each new
cell receives a full set of instructions!

WHAT KIND OF CELLS WILL BE ABLE TO COPY DNA?

ALL CELLS
The Chemistry of DNA
Replication:

THE POLYMERIZATION OF
NUCLEOTIDE MONOMER

NUCLEOTIDE MONOMER
• Double helix unwinds

• Each strand acts as

template

• Complementary base
pairing

• Two daughter helices

produced after replication
Basic Rules of DNA Replication

A) Semi-conservative

B) Starts at the ‘origin’

C) Semi-discontinuous

D) Involves DNA templating

E) RNA primers required

A) DNA Replication – three possible models

• Semiconservative
replication Watson and
Crick model

• Conservative
replication: The
parental double helix
remains intact; both
strands of the daughter
double helix are newly
synthesized

• Dispersive replication:
At completion, both
strands of both double
helices contain both
original and newly
synthesized material.
 Meselson-Stahl experiments confirm
semiconservative replication
Semi-conservative
replication:

An experiment by
Meselson and Stahl
showed that one
strand of each DNA
strand is passed on
unchanged to each of
the daughter cells.
This 'conserved'
strand acts as a
template for the
synthesis of a new,
complementary
strand by the enzyme
DNA polymerase
B) DNA Replication starts at origin of replication
Initiator proteins identify specific base sequences on
DNA
Prokaryotes – single ori site E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating sequences)

Prokaryotes
 C) Semi-discontinuous replication: DNA replication is
continuous on the leading strand and semidiscontinuous on
the lagging strand
Problem: The two DNA strands are of opposite polarity and DNA polymerases only
synthesize DNA 5’ to 3’.
Solution: DNA is made in opposite directions on each template.

Leading strand : synthesized 5’ to 3’ in the direction of the replication fork

movement. Continuous => requires a single RNA primer

Lagging strand synthesized 5’ to 3’ in the opposite direction. semidiscontinuous

(i.e., not continuous); requires many RNA primers , DNA is synthesized in short
ragments
1955: Arthur Kornberg

Worked with E. coli. Discovered the mechanisms of DNA synthesis.

Steps of DNA Replication:

INITIATION
• Proteins bind to DNA and open
up double helix
• Prepare DNA for
complementary base pairing
ELONGATION
Proteins connect the correct
sequences of nucleotides into a
continuous new strand of DNA
TERMINATION
Proteins release the replication
complex
E) RNA primers required to start DNA
Replication
WHAT DO YOU THINK REPLICATION NEEDS?

NUCLEOTIDES [dNTPs: dATP, dTTP, dGTP, Dctp

(deoxyribonucleoside 5’-triphosphates which is sugar-
base + 3 phosphates)
DNA TEMPLATE
DNA POLYMERASE ((Kornberg enzyme): can only add
nucleotides to the 3’ end of the DNA. This causes the
NEW strand to be built in a 5’ to 3’ direction.
Mg 2+ (optimizes DNA polymerase activity)
PRIMER
PRIMASE
LIGASE
TOPOISOMERASE II
Core proteins at the replication fork

Topoisomerases - Prevents torsion by DNA breaks

Helicases - separates 2 strands
Single strand - prevent reannealing
binding proteins of single strands
Primase - RNA primer synthesis
DNA polymerase - synthesis of new strand
Tethering protein - stabilises polymerase
DNA ligase - seals nick via phosphodiester linkage
 DNA
REPLICATION
INITIATION
 DNA
REPLICATION
ELONGATION
Fidelity of DNA Replication
• DNA polymerase initially makes
about 1 in 10,000 base pairing
errors
• Enzymes proofread and correct
these mistakes
• The new error rate for DNA that
has been proofread is 1 in 1
billion base pairing errors

16
 DNA REPLICATION TERMINATION

• At the end, two Replication fork of E.coli

reached at the termination sequences (Ter)
• The Terminal Utilization (Tus) Protein will
bind to Ter and form Tus-Ter complex and
stop the rep fork completing two interlinked
circular chromosome (catenated)
• DNA Topoisomerase catalyses the
decatenation. A break is made in one
daughter molecule and the second one pass
through the break. Thus separates the
chromosomes.
 Basic Principles of Gene Expression

DNA encodes hereditary information (genotype)->decoded into RNA -> protein (phenotype)

DNA

Transcription

RNA

Translation

Protein
 Transcription
Transcription is the synthesis of RNA
molecules using DNA strands as the
templates so that the genetic information can
be transferred from DNA to RNA.
• The whole genome of DNA needs to be
replicated, but only small portion of genome
is transcribed in response to the
development requirement, physiological
need and environmental changes.
• DNA regions that can be transcribed into
RNA are called structural genes.
 Similarity between
replication and transcription

• Both processes use DNA as the

template.
• Phosphodiester bonds are formed in
both cases.
• Both synthesis directions are from 5´
to 3´.
 Differences between
replication and transcription

replication transcription

template double strands single strand

substrate dNTP NTP

primer yes no

Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pair A-T, G-C A-U, T-A, G-C

 Template strand and coding strand
The template strand is the strand from which the RNA is actually
transcribed. It is also termed as antisense strand. The coding
strand is the strand whose base sequence specifies the amino
acid sequence of the encoded protein. Therefore, it is also
called as sense strand.

5' GCAGTACATGTC 3' coding

strand
3' CGTCATGTACAG 5' template
strand

transcription

5' GCAGUACAUGUC 3' RNA

 Asymmetric transcription
• Only the template strand is used for the
transcription, but the coding strand is
not.
• Both strands can be used as the
templates.
• The transcription direction on different
strands is opposite.
• This feature is referred to as the
asymmetric transcription.
5' 3'
3' 5'
§1.2 RNA Polymerase
• The enzyme responsible for the RNA
synthesis is DNA-dependent RNA
polymerase.
– The prokaryotic RNA polymerase is a
multiple-subunit protein of ~480kD.
– Eukaryotic systems have three kinds of
RNA polymerases, each of which is a
multiple-subunit protein and responsible
for transcription of different RNAs.
 Holoenzyme

The holoenzyme of RNA-pol in E.coli

consists of 5 different subunits: 2  
.


 
 

 RNA-pol of E. Coli

subunit MW function
Determine the DNA to be
 36512
transcribed

 150618 Catalyze polymerization

 155613 Bind & open DNA template

Recognize the promoter
 70263
for synthesis initiation
• Rifampicin, a therapeutic drug for
tuberculosis treatment, can bind
specifically to the  subunit of RNA-
pol, and inhibit the RNA synthesis.
• RNA-pol of other prokaryotic
systems is similar to that of E. coli in
structure and functions.
§1.3 Recognition of Origins
• Each transcriptable region is called
operon.
• One operon includes several structural
genes and upstream regulatory
sequences (or regulatory regions).
• The promoter is the DNA sequence that
RNA-pol can bind. It is the key point
for the transcription control.
 General concepts

• Three phases: initiation, elongation,

and termination.
• The prokaryotic RNA-pol can bind to
the DNA template directly in the
transcription process.
• The eukaryotic RNA-pol requires co-
factors to bind to the DNA template
together in the transcription process.
§2.1 Transcription of Prokaryotes

• Initiation phase: RNA-pol recognizes

the promoter and starts the
transcription.
• Elongation phase: the RNA strand is
continuously growing.
• Termination phase: the RNA-pol stops
synthesis and the nascent RNA is
separated from the DNA template.
a. Initiation
• RNA-pol recognizes the TTGACA region, and slides to the
TATAAT region, then opens the DNA duplex.
• The unwound region is about 171 bp.

• No primer is needed for RNA synthesis.

• The  subunit falls off from the RNA-pol once the first 3,5
phosphodiester bond is formed.
• The core enzyme moves along the DNA template to enter the
elongation phase.
b. Elongation

• The release of the  subunit causes the

conformational change of the core
enzyme. The core enzyme slides on the
DNA template toward the 3 end.
• Free NTPs are added sequentially to the
3 -OH of the nascent RNA strand.

(NMP)n + NTP (NMP)n+1 + PPi

elongated
RNA strand substrate RNA strand
• RNA-pol, DNA segment of ~40nt and the nascent RNA form a
complex called the transcription bubble.
• The 3 segment of the nascent RNA hybridizes with the DNA
template, and its 5 end extends out the transcription bubble as the
synthesis is processing.
 c. Termination
• The RNA-pol stops moving on the DNA template. The RNA
transcript falls off from the transcription complex.
• The termination occurs in either  -dependent or  -independent
manner.

The termination function with  factor

The  factor, a
hexamer, is a
ATPase and a
helicase.
-independent termination
• The termination signal is a stretch
of 30-40 nucleotides on the RNA
transcript, consisting of many GC
followed by a series of U.
• The sequence specificity of this
nascent RNA transcript will form
particular stem-loop structures to
terminate the transcription.
• The stem-loop structure alters the
conformation of RNA-pol, leading to
the pause of the RNA-pol moving.
• Then the competition of the RNA-
RNA hybrid and the DNA-DNA
hybrid reduces the DNA-RNA hybrid
stability, and causes the
transcription complex dissociated.
• Among all the base pairings, the
most unstable one is rU:dA.