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Replication
Requirements:
1) Double helix must open up and stay open.
2) Replication needs to occur on both strands,
3’5’ and 5’3’.
Kornberg (1959)
Discovered the first enzyme that catalyzed the
reaction for adding a nucleotide to another
nucleotide, DNA polymerase I.
Features of DNA polymerase I:
1. Could remove nucleotides in a 5’3’
direction and/or in a 3’5’ direction from
the end of a DNA strand (DNA exonuclease)
2. Could add bases in only one direction,
5’3’. For this to occur an open 3’ end
needed to be present.
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3. Could remove RNA (RNA nuclease).
Problems:
 only adds bases in one direction.
 needed a nucleotide with an open 3’ end
present to start adding bases.
 DNA polymerase I was too slow (did not
add bases fast enough).
Other polymerases were soon identified.
DNA polymerase III proved to be the
principal enzyme for replication.
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DNA polymerases
Pol I
mol. wt.
109,000
 subunit
Pol II
Pol III
120,000
140,000
yes
no
yes
yes
exonuclease activity
3’5’
5’3’
yes
yes
turnover activity
nucleotides
per minute
600
30
9000
function
repair
?
synthesis
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Other enzymes involved in replication:
Topoisomerase - allows DNA to move from a
supercoiled state to a relaxed state.
Enzyme also called DNA gyrase.
Helicase - unwinds the DNA double helix.
Destabilizing enzymes (SSBPs) DNA single
strand binding proteins to keep the
strands separated.
Initiator protein - Binds to the DNA at the
origin of replication.
Primase - Makes a RNA primer. Different
from RNA polymerase.
DNA Polymerase I - Removes RNA primer
and repairs any errors (proofreads).
Ligase - Joins ends of DNA fragments.
Also need - Mg2+ ions and all four dNTPs
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Eukaryotic replication
(mammalian)
DNA Polymerases
location
function

nucleus
replication

nucleus
repair

nucleus
replication

nucleus
repair

mitochondria
mitochondrial
replication
The replication process is similar to system in
prokaryotes except for the 3’ end of the linear
DNA.
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Need an additional enzyme, Telomerase
The problem is that once the primer is
removed from the end of the 3’strand of DNA
there is no way to replicate that section
because there is not an open 3’ end for the
DNA polymerase to start attaching
nucleotides.
The enzyme can synthesize TTGGGG repeats
to the end of a strand by using an internal
RNA sequence as a template.
The internal RNA sequence has a region that
is complementary to the region where the
RNA primer had been.
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The telomerase uses its RNA sequence as a
template to extend the DNA.
A primer can now be attached to the
extended section of DNA and serve as an open
3’ end to allow completion of replication of
the linear DNA.
DNA polymerase fills in the gap in the DNA,
the end is trimmed, and ligase connects the
nick in the DNA.
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Origins of Replication
Site where replication begins on a circular or
linear piece of DNA.
Site is rich in A-T base pairs to facilitate
separation of the DNA strands.
With circular DNA (prokaryotes), there is
usually a single origin of replication.
With linear DNA (eukaryotes ), there are
multiple origins of replication along the DNA.
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Transcription - The formation of an RNA
molecule upon a DNA template by
complementary base pairing, mediated by
RNA polymerase.
Basic components for transcription:
1) RNA polymerase
2) DNA template
3) Nucleotides: ATP, UTP, GTP, CTP
Prokaryotic RNA polymerase
2 units
1) core enzyme
2) sigma factor
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Core enzyme
5 polypeptide subunits
 chain
’ chain
2  chains
 chain
The core enzyme can bind to a DNA template
without the sigma () factor but it binds at
random.
Function of the sigma factor appears to be the
recognition of the promoter regions usually
found at the beginning of a gene.
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Promoters
Usually have a similar sequence and contain a
high percentage of A and T.
example: 5’-TATAAT - 3’
In prokaryotes the sequences are similar for
all genes.
In eukaryotes the sequence depends on the
gene product: protein, tRNA, or rRNA.
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Steps in Transcription
1) Binding of the sigma factor to the core
enzyme.
2) Binding of the RNA polymerase to the
DNA template.
3) Release of the sigma factor.
4) RNA synthesis.
5) Termination of RNA synthesis and release
of the RNA and RNA polymerase from the
DNA template.
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1. Binding of Sigma factor to the core enzyme
Once the sigma factor is attached the RNA
polymerase can bind to the promoter region
of the gene.
2. Binding of the RNA polymerase to a gene’s
promoter region
a) RNA polymerase first binds to the
consensus sequence - 35 base pairs
from the initiation site for
transcription.
example: 5’-TTGACA -3’
b) tighter binding then occurs at the
Pribnow box - 10 base pairs from the
initiation site for transcription. This is
the site where the DNA opens.
Example: 5’-TATAAT-3’
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3. Initiation of RNA synthesis
The first region transcribed is called the
leader region. This region can be 18 to 175
bases long and is used as a recognition site
during translation.
DNA
promoter
leader
gene
4. Elongation or gene transcription
Only one of the DNA strands is transcribed
(called the sense strand). RNA is synthesized
5’  3’ so the DNA sense strand is read
3’  5’.
Pairing of the bases:
DNA
RNA
A
 U
T
 A
G
 C
C
 G
5. Termination of transcription
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 DNA sequence near the end of the gene
codes for a transcript that has two-fold
symmetry (a complementary sequence) that
allows RNA to form a region of base
pairing producing a hairpin loop. This
followed by a series of uracils.
AAU
U
G
C
A
CG
GC
C G region of
C G complementarity
CG
GC
AUUUUUUUUUUU
The hairpin loop and series of uracils is found
with rho-independent termination (intrinsic
termination).
Two types of termination:
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 rho-independent termination
 rho-dependent termination
Rho-independent termination (intrinsic
termination)
After synthesis of the region of two fold
symmetry the RNA polymerase then
transcribes a series of adenines (A’s) on the
DNA sense strand. The association of the
RNA polymerase in this region is weak.
When the hairpin loop forms in the newly
transcribed RNA it folds back interfering
with the RNA polymerase causing it to
release.
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Rho-dependent termination
A separate protein factor rho () interacts
with the RNA transcript and the RNA
polymerase to cause the RNA polymerase to
release from the DNA.
There are regions in the RNA sequence that
signal rho to bind. There are also nucleotide
sequences slightly downstream from these
regions called potential terminators because
they cause the RNA polymerase to pause
letting the rho factor to catch up and
terminate transcription.
They are called ‘potential ‘terminators
because if the rho factor is blocked from
binding then the RNA polymerase will
continue transcribing after pausing. Rho
factor can be blocked from binding by
ribosomes
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Ribosomes can be present to block
attachment of the rho factor in prokaryotes
because transcription and translation can
occur at the same time for a given gene.
Why?
No nuclear membrane separating the site of
transcription from the site of translation in
prokaryotes.
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Transcription in Eukaryotes
3 RNA polymerases have been identified
Polymerase
RNA product
I
ribosomal RNA
II
messenger RNA
III
transfer RNA
small ribosomal RNA
There appears to be no ‘sigma factor’ in
eukaryotic polymerases. Instead the
polymerase specificity is determined by the
promoter sequence of the gene.
RNA polymerase I
 promoter found in the 3’ flanking region of
the DNA.
 sequence 5’-TATTG-3’ or 5’-TTTTGG-3’
 not highly conserved across species.
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RNA polymerase II
 promoter found in the 3’ flanking region of
the DNA.
 promoters:
a) TATA box (Goldberg-Hogress box)
-30 bps 5’-TATAAA-3’
b) CAAT element
-80 bps 5’-GGCCAATCT-3’
c) GC element
1 to 2 copies location varies
5’-GGGCGG
 conserved across species
RNA polymerase III
- promoter located within the gene
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Eukaryotic transcription does involve
additional proteins that facilitate or modulate
initiation of transcription. There are two
types of proteins, transcription factors and
activators.
Transcription factors - These are proteins
that facilitate initiation of transcription by
binding to the TATA region forming a
binding site for RNA polymerase II.
Activators - Are proteins that bind to
enhancer sites upstream or downstream from
the gene increasing the efficiency of initiation
of transcription.
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Termination of Transcription in Eukaryotes
Termination of transcription in eukaryotes is
not understood as well as termination in
prokaryotes.
It appears to vary for the different RNA
polymerases and cleavage of the newly
produced RNA strand may occur without
halting transcription or inducing the release
of the RNA polymerase.
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How can transcription be stopped?
1) Bind to the DNA
a) prevent separation of the DNA strands
b) block the binding site of RNA
polymerase
2) Bind to RNA polymerase
a) change the shape of the RNA
polymerase so it can not function
properly.
a) bind or remove the sigma factor
Transcriptional control occurs:
 naturally: binding of proteins to DNA to
block transcription is a common form of
gene regulation
 chemically: antibiotics are available that
can bind to DNA or RNA polymerase
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Genetic Code
How is the genetic information carried on the
DNA?
Sequence of bases act as a code:
DNA

bases 
RNA

bases 
protein
amino acids
How many nucleotides equal one amino acid?
Have 4 different nucleotides and 20 different
amino acids.
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Want the fewest number of bases per amino
acid for the greatest efficiency.
2 bases per amino acid (42) = 16 combinations
3 bases per amino acid (43) = 64 combinations
4 bases per amino acid (44) = 256
combinations
So 3 bases per amino acid gives enough
combinations for 20 amino acids.
A 3 base sequence codes for one amino acid
and is called a codon.
The 3 base codons for the 20 amino acids is
considered to be universal in that in general
the codons code for the same amino acid
regardless of the species.
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