Review

Evidence supports
the semi-conservative mode

One strand of the parental DNA helix is conserved in each new double helix,
paired with a newly synthesized complementary strand. This is called
semiconservative replication.
 Replication is bi-directional

During DNA replication, the two forks arise at a fixed starting point-the origin
of replication and move in opposite direction away from the origin.
Replication origin in prokarytoes

Replication bubble also known as

replication eye. Each replication
bubble found to have two replication
forks, each at the corner of an eye.
Multiple origins of replication in eukaryotic cells

Local opening
of double helix

The space between two adjacent origins is called the

replicon, a functional unit of replication.
Enzymes involved in DNA replication
DNA polymerase: responsible for replicating genome
Helicase: An enzyme that separate the strands of DNA
SSB: Single-strand binding protein
Primase is an RNA polymerase that is used only to synthesize
short stretches of RNA
DNA topoisomerasereleasing tension created by the supercoil
ahead of replication forks
Enzymes responsible for primer removal: removing the RNA
primers
DNA ligase: connecting two adjacent ssDNA
Telomerase: maintain the integrity of DNA telomere
Always 5 to 3, but why?
DNA polymerase reacts on the 3 end
only, thus, new DNA elongates from 5 to
3 direction.

DNA polymerase can only add a

nucleotide onto a pre-existing 3-OH
group, and therefore needs a primer at
which it can add the first nucleotide.

In DNA replication, the first few bases

are always RNA, and are synthesized by
another enzyme called primase.

When nucleotide is added to chain, the

phosphodiester bond is formed between
the two adjacent nucleotides coupling
with the release of diphophate.
 nucleotide 5
PO4
PO4

base
N base 5 CH
2
O
4 1
C
5 CH2 3 2
O O
O P O

4 ribose 1 O base
5 CH
2
O
3 2 4 1
OH 3 2
OH
3
 3
5

5 3
3 5

3
5

DNA replication initiates from special DNA sequence called origin. Because of the
antiparallel orientation of the two DNA strands in the DNA double helix, it requires
one daughter strand to polymerize in the 5 to 3 direction and the other in the 3 to 5
direction. However, DNA polymerase can only catalyze polymerization of DNA in 5 to
3 direction.

Oops, paradox? answer ?

 Okazaki fragments

In 1968, Japanese scientist Reiji

Okazaki revealed the transient
existence of DNA fragment later
called Okazaki fragments and
proposed that semi-discontinuous
replication is a mode in which one
new strand is synthesized
continuously while the other is
synthesized discontinuously.
RNA primer, leading and lagging strands,
Okazaki fragment
The continuous strand, or leading
3 strand, is the one in which 5 to 3
5 synthesis proceeds in the same
direction as replication fork
movement.
ing
ad
le The other strand, the lagging
strand, is synthesized
discontinuously in short pieces
(Okazaki fragments) in a direction
opposite to that in which the
lag replication fork moves.
gin
g
5 Synthesis of each Okazaki
3 fragments required a RNA primer
to provide free 3 hydroxyl group.

5
 Okazaki fragments

Okazaki fragments are short DNA fragments produced on the lagging

strand during DNA replication. They are polymerized only in the 5-to 3
direction and rapidly joined by DNA ligase to form a continuous DNA
strand.

Although the lagging strand is synthesized discontinuously in short

pieces (Okazaki fragments) in a direction opposite to that in which the
 Leading and lagging strands

The leading strand of DNA is synthesized continuously in the 5-3

direction.
The lagging strand of DNA must grow overall in the -3 to-5
direction and is synthesized discontinuously in the form of short
fragments (5-3) that are called Okazaki Fragments and later
connected covalently by DNA ligase.
Enzymes involved in DNA replication
DNA polymerase: responsible for replicating genome
Helicase: An enzyme that separate the strands of DNA
SSB: Single-strand binding protein
Primase is an RNA polymerase that is used only to synthesize
short stretches of RNA
DNA topoisomerasereleasing tension created by the supercoil
ahead of replication forks
Enzymes responsible for primer removal: removing the RNA
primers
DNA ligase: connecting two adjacent ssDNA
Telomerase: maintain the integrity of DNA telomere
 helicase

Also referred to as DnaB.

An enzyme that separate the strands

of DNA, usually using the hydrolysis
of ATP to provide the necessary
energy.

A helicase is generally multimeric. A

common form of helicase is a
hexamer.
Single-strand binding protein (SSB)

SSB protein binds to the single-stranded

DNA, maintaining the DNA template in the
single strand form in order to
1. Prevent it from reforming the duplex state;
2. Protect the vulnerable ssDNA from
nucleases

The SSB proteins bind tightly and

cooperatively to exposed single-stranded
DNA without covering the bases, which
therefore remain available for templating.

The E.coli SSB is a tetramer of 74KD that

binds cooperatively to single-stranded DNA.
 During replication, DNA ahead of the transcription fork becomes overwound,
or positively supercoiled, thus DNA topoisomerase is needed to solve
topological problems.
 Each Okazaki fragment is synthesized
individually
1primase synthesizes RNA primer

2DNA polymerase (III for prokaryotes and

for eukaryotes) extends RNA primer into
Okazaki fragment;

3next Okazaki fragment is

synthesized;

4RNA primer is removed and the

resulting gap is replaced by DNA.

5DNA ligase seals the nick between

the two Okazaki fragment
What happens after
removing the last RNA
primer in bacterial DNA ?

Perfect !
End-loss problem in replication

Because DNA cannot be

extended in the 3 to 5 direction,
and no 3 end is upstream.

Note: The very 5 of the

leading strand, Although not
drawn here, also may have the
same end-loss Problem
because it is primed by an
RNA primer as well

?
 telomere
A telomere is the natural end of a chromosome.
A typical telomere has a simple repeating structure with a G-rich strand
that extends beyond the C-rich strand, generating a G-tail.

repetitive nucleotide sequences of telomere: TTGGGG(T2G4)

5 TTGGGGTTGGGGTTGGGGTTGGGG 3 (G-rich strand)

3 AACCCCAACCCCAACCCC 5 C-rich strand

Telomerase also called telomere terminal transferase is a reverse transcriptase

enzyme that adds the nucleotide TTGGGG to the 3 end of telomeres.
 Telomerase

The eukaryotic cells use telomerase to maintain the integrity of DNA telomere

The telomerase is composed of

telomerase RNA
telomerase association protein
telomerase reverse transcriptase

It is able to synthesize DNA using RNA as the template

Synthesis of telomeric DNA by telomerase
(a)The starting point is the chromosome
end with 5 gap left after primer removal.

(b) Binding of telomerase to the

overhanging telomere repeat at the end of
the chromosome.

(c) Synthesis of three-nucleotide DNA

segment at chromosome end, using the
RNA template of telomerase.

(d) The telomerase translocates to the

new 3end of the telomere so that the RNA
template can bind to the newly synthesized
TTG in a different way.
Synthesis of telomeric DNA by telomerase

(e) Telomerase catalyzes the synthesis of a

new telomere repeat, using the RNA
template. The process recurs, to add more
telomere repeats.

(f) New DNA is made on the template,

starting with an RNA primer.

(g) After the primer is removed, the result is

a longer chromosome than at the start, with
a new 5 gap.

This mechanism ensures that chromosome ends can be rebuilt and therefore
do not suffer shortening with each round of replication.
 oriC region

Consensus sequence of bacterial replication origin.

Has two types of repetitive sequences: four 9bp and three 13bp repeats
locate in 245bp oriC region
Initiation of replication at oriC starts with formation of a complex that
requires six proteins: DnaA, DnaB, DnaC, HU, Gyrase and SSB.
 a) The four 9bp consensus sequences
on the oriC provide the initial
binding sites for DnaA which is in
ATP
ATP-bound state.
HU

b) DnaA binds cooperatively to form a

central core around which oriC DNA
is wrapped.

ATP

C) DnaA acts at three AT-rich 13bp

tandem repeats and melts the DNA
strands at each of these sites to form
an open complex.
 The open complex

Helicase
Open
complex

(d) By forming the DnaC-DnaB

comlex, DnaB was recruited to the
oriC to form pre-priming complex.

Prepriming complex
 2. primer synthesis
(e) The DnaB in pre-priming complex recruits primase
DnaG to form a complex called primosome.

Primase starts the synthesis of primers on the ssDNA

template using NTP as the substrates in the 5 -3 direction
at the expense of ATP.

The short RNA fragments provide free 3-OH groups for

DNA elongation.
Formation of primosome

+ DnaG

pro
primosome
 DNA repair systems
1.

2.

3.

4.

5. Error-prone repair
6. Recombinational DNA repair
 2.2 Few definitions of transcription
The two complementary DNA
strands have different roles in
transcription.
mRNA
Template strandThe strand that
serves as template for RNA
synthesis is called the template
strand.
The opposite strand is called the Coding strand or the sense strand
which is complementary to the template strand
The base sequence is identical to the RNA transcript
Except for the substitution of uracil in RNA for thymine in DNA
 Termination of Bacterial Transcription
Termination is the end of RNA synthesis
When the polymerase reaches a terminator at the

end of a gene it falls off the template, releasing

the RNA. This releases the newly made RNA as well as
the RNA polymerase

E. coli has two different mechanisms for termination

1. rho-dependent termination
Requires a protein known as (rho)
2. rho-independent termination
Does not require
Most -independent terminators have two
important features

(1) A region produces an RNA transcript with self-

complementary sequences which is GC-rich, allowing a
hairpin to form at the end of the transcript to destabilize
the RNA-DNA hybrid.

(2) A highly conserved string of A residues in the

template strand that are transcribed into U residues
near the 3 end of the hairpin.

Although -dependent terminators consist of an inverted repeat,

the number of C-G base pairs is less than the -independent
terminators. In addition there is no string of A residues in the
template strand.
 Operator is the regulatory region in DNA that interacts with a repressor
protein which localizes between the promoter and structural genes.
Whether the RNA polymerase can arrive the structural genes is
determined by the operator.

Without repressor, operator open, RNA polymerase can pass operator

and transcribe the downstream structural genes.

In the presence of repressor, operator off, blocking the way of RNA

polymerase to the downstream structural genes. Transcription off.

I P O Z Y A
Lac operon
 Lac Operon
Lac operon
CAP binding site
I
(activator binding site) P O Z Y A

Under double regulation:

1. negative induction: (1)In the absence of the lactose, repressor binds to the
operator and blocks transcription of the lac operon.

(2)In the presence of the lactose, repressor undergoes conformational change and
becomes inactive. Thus the repressor dissociates from the operator, allowing lac
operon to be transcribed and leading to higher levels of the encoded proteins.
2. Positive regulation by CAP: CAP-cAMP binds to DNA most avidly when cAMP
concentrations are high.
In the presence of glucose, the synthesis of cAMP is inhibited.

When glucose is absent, CAP-cAMP binds to a CAP-binding site and activates

RNA transcription. CAP-cAMP is therefore a positive regulatory element
responsive to glucose levels.
Four regulatory states of the lac operon

Operon off,
CAP not bound

Operon off,
Lac repressor bound
CAP not bound

Operon off
Lac repressor bound

Operon on
Organization of controlling sites and the structural genes of
the E.coli Trp operon

Regulatory regiontrpP, trpO, trpL

Negative repression of the trp operon

Tryptophan acts as a co-repressor

Low Tryptophan levels, High Tryptophan levels, tryptophan
no repressor binding, Trp operon on, binds to repressor, then repressor
structural genes are expressed. can bind to operator, Trp operon off.
Attenuation can be controlled by
translation
In trytophan starvation or at low
levels of trytophan:
When ribosome translates two
successive Trp codons in the
sequence 1, the ribosome pauses
at one of the Trp codons due to the
short supply of the Tryptophan.

transcript stalls at Trp codons.

Sequence 2 bases pair with
sequence 3, preventing formation
of 3-4 hairpin. Transcription
continues.
 When tryptophan levels are high,
Now the dual Trp codons present
no barrier to translation, so the
ribosome continues through
sequence 1 until it reaches the
stop signal (UGA) between
sequence 1 and 2 and falls off.
With no ribosome to interfere,
the two hairpins can form,
resulting in the formation of
transcriptional termination
structure and transcription of the
Trp operon ceases.

Which types of structures can be formed in the attenuator is

determined by the position of the ribosome on mRNA.
The cis-acting elements of eukaryotic gene includes:

1. Promoter

2. Enhancer

3. Silencer
 The composition of eukaryotic
promoter

or upstream
activating
sequence Core promoter
(UAS)

DPEdownstream promoter element

Inr initiator
A single-base change in TATA box will weaken transcription.
Some genes without TATA boxes are generally transcribed at low rates.
Transcription of these genes begins at any one of multiple possible site over
an extended region, producing different version of mRNAs with multiple
alternative 5 ends.
2. Enhancer
enhancerDNA sequences that facilitate the expression of a
given gene; may be located a few hundred, or even thousand,
base pairs away from the gene.

The first enhancer was discovered in a polyomavirus called

SV40 (Simian virus 40). It contains a conspicuous
duplication of a 72 bp sequence.

72bp repeat still simulates transcription even if they were

inverted or moved over 2Kb away from the promoter.
 General characteristics of enhancers
1.They increase the rate of transcription of nearby gene.

2. Their enhancing activity is independent of orientation with respect to

the gene.

3. Their location is not fixed. They may be found hundreds or even

thousands of base pairs upstream from the transcription start site, or may
even be downstream, within the gene itself.

4. Enhancer is additional cis-acting DNA sequence.

4.4 Trans-acting factors of eukaryotic genes

Definition A term that describes a genetic element, such as

a repressor gene or transcription factor gene, that can be on a

separate chromosome and still influence another gene.

Trans-acting factors exert their functions via the interaction

with other proteins or with DNA.
Eukaryotic transcription factors include:
1.General transcription factorsGTF: bind to promoters of all
protein-encoding genes.

2.Special transcriptional factors: activator and repressor.

GTFs

UAS TATA Initiator

Activators Basal Transcription

machinery
 General transcription factors of
eukaryotes
Transcription
functions
proteins

TBP The subunit of TFIID, specifically recognizes the TATA box

TFIIA Stabilizes binding of TFIIB and TBP to the promoter

TFIIB Binds to TBP; recruits poly II-TFIIF complex

TFIIE Recruits TFIIH; has ATPase and helicase activities

Binds tightly to Pol II; binds to TFIIB and prevents binding of Pol
TFIIF
II to nonspecific DNA sequence

Unwinds DNA at promoter(helicase activity); phosphorylates Pol

TFIIH
II(within CTD); recruits nucleotide-excision repair proteins
Assembly of pre-initiation complex

TFIID

TFIIB

RNA pol II/ TFIIF

TFIIE

TFIIH

Forming a closed complex

 Initiation of transcription

Closed complex

TFIIH

Form the transcription initiation

complex
Low but measurable
level transcription =basal transcription

TFIIH has several activities, including an ATPase, a helicase, and a kinase

activity that can phosphorylate the CTD tail of RNA polymerase II