MOLECULAR BIOLOGY – DNA replication, transcription
DNA REPLICATION
(semi-conservative method)
MOLECULAR BIOLOGY – DNA replication, transcription
Meselson-Stahl experiment
REPLICATION FORK
1000 nt / sec !
The enzyme DNA polymerase uses the two parental strands as a template to
faithfully synthesise new daughter strands according to the specific Watson & Crick
base-pairing system (A-T and G-C)
Figure 5-2 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Semi-conservative replication by
DNA polymerase requires that the
two anti-parallel parental DNA
strands are unwound to give a
single stranded templates
Unwinding and strand separation
achieved by DNA helicase using
the enegy released from ATP
hydrolysis
Figure 5-14 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Secondary structure in the single stranded template can hinder DNApol
Single-strand binding
proteins facilitate
DNApol’s progress
Figure 5-16 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Newly synthesised strand
replicated in a 5’ - 3’
direction
New dNTPs are added to the free 3’OH
group of preceding nucleotides in the
DNA strand by means of a
condensation reaction thus forming a
new phosphodiester bond
Pyrophosphate and water are byproducts
Incoming deoxyribonucleotide trisphosphates or dNTPs
(i.e. dATP, dCTP, dTTP or dGTP depending on base pairing with
TEMPLATE STRAND)
Figure 5-3 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Methotrexate
Synthesis of dNTP
substrates required
for DNA replication
anti-cancer drug
Folate cofactor only
obtainable as a dietary
supplement
(extra supplements for children
and pregnant women)
dTDP
UDP
ADP
GDP
CDP
deoxyUDP
deoxyADP
deoxyGDP
deoxyCDP
ribonucleotide
reductase
dTTP
dATP
dGTP
dCTP
kinase
MOLECULAR BIOLOGY – DNA replication, transcription
Chromosomal DNA synthesis
catalysed by DNA polymerase III
(DNApol III)
DNApol III requires the help of
‘sliding clamp’ in order to bind DNA
and start replication
The sliding clamp however requires
a complex of proteins (i.e. the
‘clamp loading complex’) plus the
energy released from ATP hydrolysis
to be loaded onto the DNA
DNA synthesis can now occur in 5’ 3’ direction although any basepairing mistakes can be corrected by
removing the incorrect base via the
‘3’-exonuclease activity’ of the
DNApol III complex
DNApol III
Figure 5-18c Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
DNA polymerase synthesizes in 5’ 3’ direction therefore
the two newly synthesized daughter strands are made
differently
replication direction
3’
5’
leading strand
lagging strand
3’
5’
3’ 3’
5’ 5’
5’
3’
Okazaki fragmentsare eventually joined (ligated)
together to form a complete strand
MOLECULAR BIOLOGY – DNA replication, transcription
How does DNA synthesis get
started?
DNA polymerase III can not simply
start synthesizing a new strand
It can only elongate from existing
one (i.e. a free 3’OH group is
required)
A specialized RNA polymerase called
‘DNA primase’ can simply start the
synthesis of a new strand using the
template DNA strand as a guide
These 11-12 nucleotide RNA primers
then provide the free 3’OH required
by DNApol III to replicate the rest of
the DNA
Figure 5-11 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
What happens to the RNA
primers and Okazaki fragments
on the lagging strand?
DNApol III completes the synthesis
of the DNA Okazaki fragment up until
the previous RNA primer without
joining the two molecules together
The ‘gap’ between the Okazaki DNA
fragment and the RNA primer is
recognised by ‘DNA polymerase I’
that then removes the RNA primer
and fills in the space with template
directed DNA
DNApol I
Lastly the two adjacent DNA Okazaki
fragments are joined by the enzyme
‘DNA ligase’
Figure 5-12 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
DNA Replication
Summary
N.B. the lagging strand template is bent round so
both DNApol’s proceed in the same direction!
Figure 5-19a Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Detailed electronmicrograph of a bacterial DNA
replication fork
Figure 5-19b,c Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Electronmicrograph of two replication forks
progressing around a circular bacterial DNA genome
Figure 5-6 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Summary of
bidirectional
replication forks
with leading and
lagging strand
synthesis
Figure 5-25 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
The action of helicase at the
replication fork places great strain
on the DNA double helix ahead of
it because the two ends of the
helix cannot freely rotate with
respect to each other
Figure 5-21 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
An enzyme called ‘DNA topisomerase I’ relieves this strain by
catalysing a break in the phosphodiester backbone of one DNA strand
allowing the two ends of the helix to rotate relative to each other
Figure 5-22 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
DNA replication summary
http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter11/animation_quiz_2.html
MOLECULAR BIOLOGY – DNA replication, transcription
Known as OriC in
E-coli.
N.B. bi-directional
replication from a
single replication
origin.
Relatively small
bacterial genomes
are circular and
are usually
replicated from a
single ‘replication
origin’ that
consists of
tandem repeat
rich DNA
sequences
Figure 5-26 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Large eukaryotic genomes (multiple linear chromosomes) are
replicated from many different ‘origins of replication’
• Multiple origins of replication comprising
many different sequence variations (approx
100 000 in human genome)
• Not all activated at the same time
• Mechanism involves the assembly of the
‘pre-replication complex (pre-RC)’ of proteins
prior to their activation and initiation of DNA
synthesis
Figure 5-34 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
During DNA replication there is aProblem with the
end of the lagging strand:
... progressive shortening of chromosomal ends and
genetic instability
MOLECULAR BIOLOGY – DNA replication, transcription
Chromosome ends comprised of TELOMERES
Telomeres made of many repeats
(TTAGGG)20-HUNDREDS
TTAGGGTTAGGGTTAGGGTTAGGG
Species
Repeat Sequence
Arabidopsis
TTTAGGG
Human
TTAGGG
Oxytricha
TTTTGGGG
Slime Mold
TAGGG
Tetrahymena
TTGGGG
Trypanosome
TAGGG
Yeast
(TG)1-3TG2-3
Telomeres provide a kind of ‘buffer’ for the chromosomal ends
that protect genes located in the ‘sub telomeric’ regions
Therefore only telomeric sequence is lost during DNA
replication
HOW ARE TELOMERES MAINTAINED?
MOLECULAR BIOLOGY – DNA replication, transcription
TELOMERES ARE ELONGATED BY ACTION OF TELOMERASE
Conventional DNA synthesis is achieved by RNA priming
of the elongated parental strand thus maintaining
telomere length and chromosome integrity
Figure 5-41 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
• ~ 150 genes control telomere length in yeast and shortening
telomeres is associated with cell senescence and death
• telomerase highly active in >90% of tumors i.e. cell
immortalization and uncontrolled proliferation (drug target)
• many adult cell types have detectable telomerase activity,
but it is a highly regulated, fine tuned activity
MOLECULAR BIOLOGY – DNA replication, transcription
Correlation between levels of perceived stress and
telomere length and action of telomerase
RELAX AND KEEP YOUR TELOMERES LONG!
MOLECULAR BIOLOGY – DNA replication, transcription
Telomere and Telomerase video/ tutorial
http://www.youtube.com/watch?v=AJNoTmWsE0s
MOLECULAR BIOLOGY – DNA structure, genetic code
TRANSCRIPTION
double stranded DNA
5’
3’
ATG GCT CCT TCT TCC AGA GGT GGC . . . . . . TAA
TAC CGA GGA AGA AGG TCT CCA CCG . . . . . . ATT
3’
5’
TRANSCRIPTION
single stranded mRNA
TRANSLATION
AUG GCU CCU UCU UCC AGA GGU GGC . . . . . . UAA
AUG
UAA
protein coding sequence
or
open reading frame
MAPSSRGG…..
Functional Protein
Focus on how the genetic information
contained within DNA is copied into
RNA and it’s consequences
MOLECULAR BIOLOGY – DNA replication, transcription
Eukaryotic mRNAs are
not synthesised in a form
that can be immediately
translated to protein
RNA
processing
i.e. there are processing
and translocation steps
Figure 6-21 Molecular Biology of the Cell (© Garland Science 2008)
translocation
MOLECULAR BIOLOGY – DNA replication, transcription
Polycistronic mRNA
(e.g related gene operons)
Monocistronic mRNA
Figure 6-22a Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Table 6-1 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
TRANSCRIPTION INITIATION
Single stranded mRNA
RNA polymerse complex
Doubel stranded DNA
How does the RNA polymerase recognise the the correct
place within DNA to start transcribing mRNA for a gene?
MOLECULAR BIOLOGY – DNA replication, transcription
TRANSCRIPTION START IN PROKARYOTES
The core ‘RNA polymerase complex’ (consisting of
 & ’catalytic and 2x  regulatory subunits) alone
is unable to recognise and bind DNA
Molecular interaction with the ‘sigma () factor’ permits RNA polymerase
(designated ‘holoenzyme’) to bind DNA at specific regions called ‘promoters’
Figure 6-11 (part 1 of 7) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
The sigma factor recognises ‘consensus’ sequences in
the promoter DNA
There are x2 consensus
sequences at the -35 and -10
positions of the DNA
The sigma factor recognises
-35 & -10 and positions the
RNA polymerase in the
correct position to start
synthesis of mRNA starting
from the +1 position
-35
-10
+1
-35
-10
PROMOTER
1st ribonucleotide to
be incorporated into
mRNA
+1
GENE
Variations in the -35 & -10 DNA sequences of different gene promoters affect how often mRNA
synthesis can be initiated. Additionally bacteria have multiple sigma factors each with subtle
differences in binding affinity for -35 & -10 sequence variants.
Figure 6-12a Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
After initiation, sigma factor disassociates and synthesis of
the new mRNA molecule occurs in a 5’ - 3’ direction i.e.
‘elongation phase’
3’
5’
5’
The mRNA sequence is specified by base-pair complementarities with the
‘template/ non-coding/ antisense’ DNA strand and therefore is a copy/
‘transcript’ of the ‘non-template/ coding/ sense’ strand wit U replacing T
MOLECULAR BIOLOGY – DNA replication, transcription
How does RNA polymerase know when to stop?
Just as promoter sequences in DNA instruct where transcription should begin, other DNA
sequences called ‘terminators’ specify where it should stop.
‘terminators’ sequences are
inverted DNA repeats followed
by a run of A nucleotides
The repeats are copied into the
transcribed mRNA and form a ‘hairpin
loop’ that is sensed by the RNA
polymerase causing it to pause/ stutter.
Weak hydrogen bonding between the
run of A nucleotides in the DNA template
strand and the U ribonucleotides of the
mRNA cause the disassociation of the
whole RNA polymerase complex i.e.
‘intrinsic termination’
In other cases the binding of special
helicases called ‘Rho ()proteins’
facilitate the disassociation i.e. ‘rhodependent termination’
MOLECULAR BIOLOGY – DNA replication, transcription
Prokaryotic transcription cycle video/ tutorial
http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_1.html
MOLECULAR BIOLOGY – DNA replication, transcription
TRANSCRIPTION IN EUKARYOTES
Additionally transcription occurs in the
nucleus and not in the cytoplasm
Table 6-2 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Recognition of the promoter and the initiation of transcription
in eukaryotes is more complex
In addition to RNA polymerase a host of other proteins known as the ‘General
Transcription Factors (GTFs)’ are required
i.e. PROTEIN CODING GENE
TRANSCRIPTION
GTF’s help RNA polymerase recognise a T/A rich DNA sequence motif in the promoter
called the ‘TATA box’, correctly position it on the ‘chromatin template’ with respect to the
+1 position and convert it to form capable of RNA synthesis
i.e. help form a ‘pre-initiation complex’
Table 6-3 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Binding of ‘specific transcription factors’ positively or
negatively affect frequency of transcription initiation
• specific transcription factors bind particular DNA sequences only found in the
locality of certain genes
• therefore can help regulate which genes mRNAs are synthesised within a cell (e.g.
confine the production of antibodies in white blood cells but not in neurones!)
e.g. ‘short-range upstream regulator
elements’
e.g. ‘long range enhancer sequences’
Specific transcription factor binding
Pre-initiation complex of RNA polymerase II and GTFs at TATA box
MOLECULAR BIOLOGY – DNA replication, transcription
CORE PROMOTER
Figure 6-19 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Initiation of eukaryotic transcription video/
tutorial
http://bcs.whfreeman.com/thelifewire/content/chp14/1402002.html
MOLECULAR BIOLOGY – DNA replication, transcription
RNA PROCESSING in Eukaryotes
Unlike prokaryotes, the DNA sequence encoding eukaryotic
protein is not often organised in one continuous length that
when transcribed is a fully functional mRNA that can be
translated into protein. The mRNA first needs to be ‘processed’
Eukaryotic genes typically consist of segments of protein coding DNA sequence called ‘exons’ that are
interspersed with non-protein coding sequences called ‘introns’ (that are often very long).
• both the exons and introns are
transcribed by RNA polymerase II to yield
long RNA molecules called ‘Pre-mRNAs’
Pre-mRNA
CAP
AAA(n)
SPLICING
Mature mRNA
CAP
AAA(n)
• Pre-mRNAs are modified (cotranscriptionally) by addition of a 5’-cap
and 3’ polyadenylation motifs (important
for stability and translation).
• the sequence corresponding to the
introns is removed from the pre-mRNA in a
process called ‘SPLICING’ to yield a
‘mature mRNA’
• the ‘spliced’ together protein coding mRNA sequence corresponding to the exons in the DNA can
now be translated into function protein (except for ‘untranslated regions/ UTRs’ at the 5’ & 3’ ends
MOLECULAR BIOLOGY – DNA replication, transcription
Capping Eukaryotic Pre-mRNA
RNA terminal
phosphatse
Leads to the covalent attachment
of a ‘7-methylguanosine cap (m7G
cap)’ at the 5’ end of the premRNA via an unusual 5’ - 5’
triphosphate bond
‘Enzyme capping
complex (CEC)’
Bound to RNApolII complex
and caps the pre-mRNA cotranscriptionally
The m7G cap ensures:
• the (pre)mRNA is protected from degradation
• mRNA export from nucleus
• mRNA is translated to give functional protein
Figure 6-22b Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Polyadenylation of Eukaryotic Pre-mRNA
Cleavage and polyadenylation
specificity factor (CPSF) &
cleavage stimulation factor
(CstF)
PAP extends the 3’ end of the RNA
by untemplated addition of up to 200
adenosine (A) nucleotides that are
then bound by PABPs
CPSF & CstF recognise the
cleavage and polyadeylation
signals once they have been
transcribed into the pre-mRNA
CPSF & CstF binding recuits
other factors that cleave the
pre-mRNA chain free of the
RNA polymerase II complex
Additionally polymerase (PAP)
and poly-A-binding proteins
(PABPs) are also recruited to
this polyadenylation complex
Polyadenylation importance:
• participates in transcriptional termination
• protects mRNA from degradation
• mRNA export from nucleus
Figure 6-38 Molecular Biology of the Cell (© Garland Science 2008)
• mRNA translation to give functional protein
MOLECULAR BIOLOGY – DNA replication, transcription
mRNA capping and polyadenylation video/
tutorial
http://www.youtube.com/watch?v=YjWuVrzvZYA
MOLECULAR BIOLOGY – DNA replication, transcription
SPLICING of Eukaryotic Pre-mRNA
The very precise removal of non protein coding intron derived sequences from pre-mRNAs (N.B.
genetic code) is catalysed by the multiple subunit containing complex called the ‘SPLICEOSOME’
Spliceosome
Pre-mRNA
The subunit composition of the spliceosome changes as the
splicing of introns progresses
The main class of subunit are the ‘small nuclear
ribonucleoproteins/ snRNPs (“snurps”)’
snRNA
snRNPs are complexes of
proteins and ‘small nuclear
RNA (snRNA)’
The snRNAs in snRNPs
permit spliceosome
assembly
&
There are 5 different
snRNPs designated U1,
U2, U4, U5 & U6
Protein
Recoginse ‘specific splice
signals’ within the premRNA by complementary
base-paring
MOLECULAR BIOLOGY – DNA replication, transcription
snRNAs of snurps recognize 3 types of splice signal in the
pre-mRNA
5’ splice site
‘branch site’
site
3’ splice
This adenosine is
critical to successful
splicing
The recognition of these 3
splice signal sequences
directs the correct assembly
of the spliceosome and the
correct joining of the exons
Figure 6-28 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Spliceosome reaction mechanism
Cowboy’s lariat
2’ - 5’
phosphodiester
bond
Figure 6-26a Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
Where do the snRNPs fit
in?
N.B. the dynamics in the composition of the
spliceosome between catalysing the first and
second phosphoryl-transfer reactions
Figure 6-29 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
snRNP & snRNA interactions in the two forms of
spliceosome active site
Formation of the lariat with the
conserved adenosine (A) in the
branch site (i.e catalysis of the 2’ -5’
phosphodiester bond)
Figure 6-30c Molecular Biology of the Cell (© Garland Science 2008)
Excision of the lariat and joining of
the two exons (i.e. a conventional 3’
-5’ phosphodiester bond)
MOLECULAR BIOLOGY – DNA replication, transcription
General splicing video/ tutorial
http://bcs.whfreeman.com/thelifewire/content/chp14/1402001.html
MOLECULAR BIOLOGY – DNA replication, transcription
Self splicing introns
Secondary structure formation within the intron caused by complementary base-pairing between its
nucleotides forms inherent enzymatic activities (i.e. ‘ribozymes’) that catalyse the removal of the
intron and the joining of exons
As with spliceosome
assisted splicing a
conserved adenine
(A) nucleotide in the
intron participates in
the reaction and the
intron is excised as a
lariat
Unicorporated
guanine (G)
particpates as a
cofactor and intron
excised as a linear
moleclue
Some lower eukaryotic species e.g.
tetrahymena
Some fungi and plants species
Figure 6-36 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – DNA replication, transcription
ALTERNATIVE SPLICING
Alternative splicing is a mechanism by which one gene can code for more than one version of a
protein depending upon which exons make it into the mature mRNA and are translated.
Therefore provides an evolutionary mechanism for extra diversity!
Figure 6-31 Molecular Biology of the Cell (© Garland Science 2008)