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In this section we will cover DNA structure, replication, and repair. In addition, we will cover the process
of transcription.
This material is covered in chapters 11 and 12 of the text.
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DNA is a polymer of nucleotides.
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Nucleotides contain:
Pentose sugar
Phosphate group
Nitrogen containing base
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Pentose sugar
RNA - ribose
DNA - deoxyribose
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Ribonucleic Acid (RNA)
OH
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Deoxyribonucleic Acid (DNA)
H
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Nitrogen containing bases:
Adenine and Guanine – purines
Thymine and Cytosine –pyrimidines
(Uracil in RNA)
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5
OH
O
CH2
O
3
Base
P OH
-O
OP OH
-O
OP OH
-O
O
dNTPdeoxy (aNy base) Tri Phosphate
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Adenine basepairs with Thymine
A-T/U
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Guanine basepairs with Cytosine
G-C
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Basepairing = Hydrogen bonding
also referred to as hybridizing
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RNA can hybridize with DNA.
5’ ATGCCT GCT TA C GAGAGTCT C T TA 3’3’ UACGGACGAAUGCUCUCAGAGAAU 5’
RNA
DNA
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DNA
RNA
DNA
DNA
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The phosphate group links the 5’ carbon of one sugar to the
3’carbon of the next sugar to produce a strand with a sugar-phosphate backbone.
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5
OH
O
CH2
O
3
Base
P OH
-O
OP OH
-O
OP OH
-O
O OH
O
CH2
O
3
Base
P OH
-O
OP OH
-O
OP OH
-O
O
+
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5
OH
O
CH2
O
3
Base
P OH
-O
OP OH
-O
OP OH
-O
O OH
O
CH2
O
3
Base
P OH
-O
OP OH
-O
OP OH
-O
O
+ H2O
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OH
O
CH2
O
3
Base
P O
-O
O
P OH
-O
O
5
O
CH2
OBase
P OH
-O
OP OH
-O
OP OH
-O
O
+ H2OP OH
-O
O
+
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OH
O
CH2
O
3
Base
P O
-O
O
5
O
CH2
OBase
P OH
-O
OP OH
-O
OP OH
-O
O
Phosphodiester Linkage
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OH
O
CH2
OBase
P O
-O
O
O
CH2
OBase
P OH
-O
OP OH
-O
OP OH
-O
O
5
3
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OH
O
CH2
OBase
P O
-O
O
O
CH2
OBase
P OH
-O
OP OH
-O
OP OH
-O
O
OH
O
CH2 OBase
PO
-O
O
O
CH2 OBase
POH
-O
O
POH
-O
O
POH
-O
O
3
3 5
5
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OH
O
CH2
OBase
P O
-O
O
O
CH2
OBase
P OH
-O
OP OH
-O
OP OH
-O
O
OH
O
CH2 OBase
PO
-O
O
O
CH2 OBase
POH
-O
O
POH
-O
O
POH
-O
O
3
3 5
5
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The DNA is double stranded, each strand is held together by the hydrogen bonds that form between the bases of opposite
strands.
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The two strands run antiparallel, one runs in the 5’- 3’ direction while the other runs in the 3’- 5’
direction.
The strands are twisted to form a double helix.
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DNA stores an organism’s genetic information, genes.
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A gene is a unit of heredity.
or
A gene is a unit of genetic function, which carries the information for a
single protein or RNA.
This is not entirely true.
Some genes encode for more than 1 protein.
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Genes are a sequence of nucleotides.
The genetic information is contained in the sequence of nucleotides.
5’ ATGCCT GCT TACGAGAGTCTC TTA 3’3’ TACGGACGAATGCTCTCAGAGAAT 5’
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This sequence of nucleotides can be on either strand.
5’ ATGCCT GCT TACGAGAGTCTC TTA 3’3’ TACGGACGAATGCTCTCAGAGAAT 5’
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Nucleic Acids
5’ AUGCCUGCUUACGAAAGUCUCUUA 3’
M R SA LEY stopProtein
5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
transcription
translation
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Replication
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To replicate is to duplicate, copy, reproduce, or repeat
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Replication DNA makes a copy of itself.
Transcription DNA makes an RNA copy.
Translation The process in which genetic information (sequence of
nucleotides) is translated into a sequence of amino acids (protein synthesis).
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When a cell divides,
the DNA must replicate before cell division.
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Since, there is one copy of DNA in a cell,
the existing DNA must serve as a template for the new DNA.
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Theoretically, DNA could serve as its own template in one of three different ways:
Semiconservative replication uses each parent strand as a template for
a new strand.
Conservative replication would build an entirely new double helix based on the template of the old double helix.
Dispersive replication would use fragments of the original DNA molecule as
templates for assembling two molecules.
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DNA replicatesby a semiconservative mechanism
only.
(Each parent strand is a template for a new strand.)
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OH
O
CH2
OBase
P O
-O
O
O
CH2
OBase
P OH
-O
OP OH
-O
OP OH
-O
O
OH
O
CH2 OBase
PO
-O
O
O
CH2 OBase
POH
-O
O
POH
-O
O
POH
-O
O
3
3 5
5
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A
T
C
G
T
A
A
T
A
T
T
A
G
C
G
C
G
C3’
3’ 5’
5’
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A
T
C
G
T
A
A
T
A
T
T
A
G
C
G
C
G
C3’
3’ 5’
5’
A C T A ATG GG3’ 5’
T G A T TAC CC3’5’
+
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Because A always basepairs with T and G always basepairs with C,
if we know the sequence to one strand we automatically know the sequence to the
complimentary strand ( the other strand).
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A C T A ATG GG3’ 5’
T G A T TAC CC3’5’
+
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A
T
C
G
T
A
A
T
A
T
T
A
G
C
G
C
G
C3’
3’ 5’
5’
+
A
T
C
G
T
A
A
T
A
T
T
A
G
C
G
C
G
C3’
3’ 5’
5’
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DNA replication is semiconservative.
Each new molecule of DNA contains and “old” strand and a
“new” strand.
Each “old” strand serves as a template for the “new” strand.
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DNA replication takes place in two steps:
First,
the hydrogen bonds between the two strands are broken.
This opens the double helix and makes each strand available for base pairing to
new nucleotides.
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Then,
the new nucleotides are covalently bonded to each growing strand.
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In DNA replication,
nucleotides are alwaysadded to the 3’ end of the growing strand
by an enzyme called polymerase.
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There are several different types of polymerases BUT
ALL polymerases add nucleotides to the 3’ end.
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5’
3’
H
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ATP, GTP, CTP, and TTP basepair with the existing strand and polymerase will
covalently attach the nucleotide to the new strand.
Energy for synthesis of nucleotides to the growing chain comes from breaking the bonds between terminal 2 phosphates.
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A
T
C
G
T
A
A
T
A
T
T
A
G
C
G
C
G
C
3’
3’5’
5’
-P-P-P
P-P-P-
HO-HO-
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T G A T TAC CC
3’5’
-P-P-PHO-
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A C TG
3’5’
P-P-P-
A
P-P-P-
T G A T TAC CC
3’5’
-P-P-PHO-
HO-
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A C TG
3’5’
P-P-P-A
P-P-P-
T G A T TAC CC
3’5’
-P-P-PHO-
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A C TG
3’5’
P-P-P-A
P-
T G A T TAC CC
3’5’
-P-P-PHO-
P-P
+
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
P-P -> P + P
+
HO-
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5
OH
O
CH2
O
3
Base
P OH
-O
OP OH
-O
OP OH
-O
O OH
O
CH2
O
3
Base
P OH
-O
OP OH
-O
OP OH
-O
O
+ H2O
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OH
O
CH2
O
3
Base
P O
-O
O
P OH
-O
O
5
O
CH2
OBase
P OH
-O
OP OH
-O
OP OH
-O
O
+ H2OP OH
-O
O
+
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
G
P-P-P-
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
G
P-P-P-
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
GT
P-P-P-
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
GT
P-P-P-
T
P-P-P-
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
GG
P-P-P-
T
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
G T G
P-P-P-
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
G T G
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
G AP-P-P-
T G
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A C TG
3’5’
P-P-P-A
T G A T TAC CC
3’5’
-P-P-PHO-
G AT G
HO-
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DNA replication requires a replication complex, which contains
DNA helicase
Single strand binding proteins
Primase
DNA polymerase III
DNA polymerase I
DNA ligase
Nucleic Acids
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DNA helicase
an enzyme that unwinds DNA.
Nucleic Acids
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Single strand binding proteins bind to the single strand of DNA to
keep it open during replication.
Nucleic Acids
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DNA polymerase III is an enzyme that
adds nucleotides to the 3’ end of a new DNA strand.
BUT
it can’t start a new strand.
Nucleic Acids
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Primaseis an enzyme that starts the new strand.
However, it starts the new strand with RNA.
This segment of RNA is called an RNA primer.
Nucleic Acids
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DNA polymerase I is an enzyme that removes the RNA primer and replaces it with DNA by
adding nucleotides to the 3’ end of a growing strand.
Nucleic Acids
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DNA ligase
an enzyme that links fragments of DNA together.
Nucleic Acids
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DNA replication:
DNA helicase unwinds the DNA to expose the single strands, each strand will serve as a template
for the new strand being made.
Nucleic Acids
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Single stranded binding proteins bind to the DNA to keep it unwound.
Nucleic Acids
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Helicase
P-P-P-
A C T A ATG GG3’ 5’
T G A T TAC CC3’5’
+
A C T A ATG GG
3’
5’
T G A T TAC CC
3’
5’-P-P-P
HO-
HO-
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T G A T TAC CC3’5’
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T G A T TAC CC3’5’
DNA POL III cannot start a new strand.
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A C UG
3’5’
P-P-P-
Primase
T G A T TAC CC3’
5’-P-P-P
RNA
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A C UG
3’5’
P-P-P-
T G A T TAC CC3’
5’-P-P-P
RNA
A
P-P-P-
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A C UG
3’5’
P-P-P-
T G A T TAC CC3’
5’-P-P-P
RNA
AP-
DNA Pol III
P-P+
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A C UG
3’5’
P-P-P-
T G A T TAC CC3’
5’-P-P-P
RNA
A
DNA Pol III
P+
P+
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A C UG
3’5’
P-P-P-
T G A T TAC CC3’
5’-P-P-P
RNA
A
DNA Pol III
G
P-P-P-
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A C UG
3’5’
P-P-P-
T G A T TAC CC3’
5’-P-P-P
RNA
A
DNA Pol III
AT GG
DNA
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A C UG
3’5’
P-P-P-
T G A T TAC CC3’
5’-P-P-P
RNA
A AT GG
DNA
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3’5’
T G A T TAC CC3’
5’-P-P-P
A AT GG
DNA
DNA Pol I
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3’5’
T G A T TAC CC3’
5’-P-P-P
A AT GG
DNA
DNA Pol I
A C TG
P-P-P-
DNA
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3’5’
T G A T TAC CC3’
5’-P-P-P
A AT GGA C TG
P-P-P-
Ligase
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A
T
C
G
T
A
A
T
A
T
T
A
G
C
G
C
G
C3’
3’ 5’
5’
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http://www.youtube.com/watch?v=teV62zrm2P0
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double stranded DNA
5’3’
5’ 3’
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Helicase unwinds the DNA
5’3’
5’ 3’
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5’3’
5’ 3’
The area in a DNA molecule where unwinding is occurring is called a replication fork.
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Single-stranded binding proteins loosely attach to the single stranded DNA to prevent the duplex
from reforming.
5’3’
5’ 3’
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For demonstration purposes the single-stranded binding proteins are removed.
5’3’
5’ 3’
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Primase begins synthesis of the complementary strand with an RNA primer.
5’3’
5’ 3’
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The direction of synthesis is from 5’ to 3’ for the new strand.
5’3’
5’ 3’3’ 5’
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The primase dissociates.
5’3’
5’ 3’
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DNA Polymerase III attaches.
5’3’
5’ 3’
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DNA Polymerase III extends the RNA primer, adding nucleotides to the 3’ end.
5’ 3’
5’3’
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The nucleotides are added according to the rule: A opposite T and G opposite C.
5’ 3’
5’3’
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DNA Pol III proofreads the newly added nucleotide.
5’ 3’
5’3’
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If an error has been made, Pol III removes
the nucleotide and replaces it with
the correct nucleotide.
5’ 3’
5’3’
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Leading StrandLagging Strand
5’ 3’
5’3’
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On the lagging strand DNA Pol III
completes that segment and dissociates.
5’ 3’
5’3’
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Helicase unwinds another segment.
5’ 3’
5’3’
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On the leading strand, DNA Pol III continues to add nucleotides.
5’ 3’
5’3’
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On the lagging strand, primase adds another RNA primer.
5’ 3’
5’3’
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On the lagging strand, primase adds another RNA primer.
5’ 3’
5’3’
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After adding the RNA primer, primase dissociates.
5’ 3’
5’3’
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DNA Pol III adds nucleotide to the 3’ end of the RNA primer.
5’ 3’
5’3’
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DNA Pol III adds nucleotide to the 3’ end of the RNA primer.
5’ 3’
5’3’
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DNA Pol III adds nucleotide to the 3’ end of the RNA primer.
5’ 3’
5’3’
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3’ 5’
The process is repeated until the end of the existing DNA.
5’
5’
3’
3’
5’
3’
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DNA Pol I removes the RNA primers and replaces them with DNA.
3’ 5’
5’
5’
3’
3’
5’
3’
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DNA Pol I removes the RNA primers and replaces them with DNA.
3’ 5’
5’
5’
3’
3’
5’
3’
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But DNA Pol I can only add nucleotides to the 3’ end. This leaves gaps in the DNA backbone.
3’ 5’
5’
5’
3’
3’
5’
3’
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Ligase makes the phosphodiester bond that DNA Pol I could not do.
3’ 5’
5’
5’
3’
3’
5’
3’
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The DNA replication is complete.
3’ 5’
5’
5’
3’
3’
5’
3’
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3’ 5’
5’
5’
3’
3’
5’
3’
Leading StrandLagging Strand
There is a problem with the end of the
lagging strand.The details will be addressed in the telomere section.
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On the leading strand:
Primase binds to the DNA and makes a small sequence of RNA
complementary to the DNA.
DNA polymerase III adds nucleoside triphosphates to the
3’ end of the new strand.
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When DNA polymerase III adds a nucleotide to the new DNA strand, the
bond between the first two phosphates is broken releasing pyrophosphate
(2 phosphate groups covalently bonded).
The bond in the pyrophosphate is also broken to supply additional energy.
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DNA polymerase III can add nucleotides but it can’t start the
process so primase is necessary to initiate replication
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As the DNA is unwound, DNA Pol III keeps adding nucleotides.
The DNA builds the leading strand in the 5’ – 3’ direction
The nucleotides are added to the 3’ end.
or
The template is read in the 3’-5’ direction.
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The lagging strand:
Because DNA POL III can add nucleotides to the 3’ end only,
the lagging strand is built in segments.
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Primase adds several RNA primers along the lagging strand.
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DNA polymerase III adds DNA nucleotides complementary to the
template strand in a 5’ – 3’ direction, filling in the gaps between RNA
primers.
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These DNA fragments synthesized by DNA polymerase III are called
Okazaki fragments.
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DNA polymerase I removes the RNA primers on the lagging strand
and replaces them with DNA.
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DNA Ligase links all the DNA fragments together.
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Overall the lagging strand is built in a 3’-5’ direction, but it
is done by creating small fragments in a 5’-3’ direction and linking them together.
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The DNA builds the leading strand
in the 5’ – 3’ direction as
1 continuous strand.
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DNA polymerase III synthesizes base pairs at a rate of around
1000 nucleotides per second!!!
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The DNA replication complex binds to the origin of replication on the chromosome.
The origin of replication is a sequence of DNA recognized by the replication complex.
Several origins of replication exist on each chromosome so several complexes replicate
the DNA at the same time.
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A mammalian cell typically has 1.2 meters (when completely outstretched) of double stranded DNA. The total time to duplicate
the DNA is 5 hours.
How many origins of replication are there if the rate of duplication is 16 µmeters/min ?
250
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DNA replication occurs in both directions at the same
time, once the complex binds, two replication forks are created and the existing DNA is threaded through the
complex.
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Mutations
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Since DNA contains the genetic information of an organism or a cell
and
since an organism or cell passes its genetic information to its offspring or
daughter cell,
the DNA must be replicated precisely.
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However,
for an organism to evolve,
there must be changes in its genes
(there must be changes in its DNA).
Therefore, when DNA is replicated, it is better to have the replication be a
little less than perfect.
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The DNA must be replicated precisely
but not exactly.
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A change in DNA sequence is called a mutation.
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
M R SA LGY stop
5’ ATGCCTGCTTGCGAAAGTCTCTTA 3’
M R SA LEC stop
During replication, A is mutated to G
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A
AA
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B
A
A
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BB
B
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
M R SA LGY stop
5’ ATGCCTGCTTGCGAAAGTCTCTTA 3’
M R SA LEC stop
During replication, A is mutated to G
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Mutations are heritable changes in DNA that are passed on to
daughter cells.
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Multicellular organisms have two types of mutations:
Somatic mutations are passed on during mitosis, but not to
subsequent generations.
Germ-line mutations are mutations that occur in cells that
give rise to gametes.
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Germ line mutations occur in the eggs and sperm and can be passed on to offspring,
while somatic mutations occur in body cells and are not passed on.
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All mutations are alterations of the DNA nucleotide sequence and are
of two types:
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Point mutations are mutations of single genes.
Chromosomal mutations are changes in the arrangements of chromosomal DNA segments.
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Point mutations result from the addition or subtraction of a base
or the substitution of one base for another.
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Point mutations can occur as a result of mistakes during DNA
replication or can be caused by environmental mutagens.
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Mutations can result from DNA copying mistakes made during cell division (replication), exposure to ionizing radiation,
exposure to chemicals called mutagens, or infection by viruses.
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5’ AUGCCUGCUUACGAAAGUCUCUUA 3’
M R SA LEY stopProtein
5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
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Some mutations,
called missense mutations,
cause an amino acid substitution.
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M R SA LGY stop
M R SA LGC
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
M R SA LGY
5’ ATGCCTGCTTGCGAAAGTCTCTTA 3’
M R SA LGC
During replication, A is mutated to G
stop
stop
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Missense mutations may reduce the functioning of a protein or disable it
completely.
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An example in humans is
sickle-cell anemia.
The -globin in sickle-cell differs from the normal by only one amino acid.
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Because of redundancy in the
genetic code,
some point mutations,
called silent mutations,
result in no change in the
amino acid sequence of the protein.
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
M R SA LGY
5’ ATGCCTGCTTATGAAAGTCTCTTA 3’
M R SA LGY
During replication, C is mutated to T
stop
stop
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Nonsense mutations are base substitutions that change an amino acid codon into a
stop codon.
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
M R SA LGY stop
5’ ATGCCTGCTTAAGAAAGTCTCTTA 3’
M R A stop
During replication, C is mutated to A
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This causes the protein synthesis to terminate before the full protein is synthesized.
Shortened proteins are usually not functional.
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A frame-shift mutation consists of the insertion or deletion of a
single base.
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This type of mutation shifts the code, changing many of the codons to different codons.
These shifts almost always lead to the production of nonfunctional
proteins.
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
M R SA LGY stop
During replication, a base is inserted or deleted.
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
5’ ATGCCTAGCTTACGAAAGTCTCTTA 3’
With an insertion the sequence becomes
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
5’ ATGCCTAGCTTATGAAAGTCTCTTA 3’
Now when the mRNA is translated, the codons are shifted.
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5’ ATGCCTGCTTACGAAAGTCTCTTA 3’
M R SA LGY stop
M R ES SGL
When the protein is synthesized,
5’ ATGCCTAGCTTATGAAAGTCTCTTA 3’
P
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Mutations in non-coding DNA is not well understood.
Mutations in the binding sequence for a transcription factor will alter the level
of expression of that protein.
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Differences in nucleotide sequence among individuals are known as DNA
polymorphisms.
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Chromosomal Mutations
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DNA molecules are long sequences of nucleotides that can break.
Proteins inside the cell recognize the free ends of a broken DNA molecules
and re-attach them.
However, the repair is not always correct.
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Four different types of mutations can result from this repair
mechanism:
Deletions are a loss of a chromosomal segment.
Duplications are a repeat of a segment.
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Inversions result from breaking and rejoining when segments get reattached in the opposite
orientation.
Translocations result when a portion of one chromosome
attaches to another.
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Deletion
Duplication
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Inversion
Translocation
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RNA
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RNA is a polymer of ribonucleotides.
Nucleotide consists of
Phosphate
Ribose (pentose sugar)
Nitrogen Base (A,U,G,C)
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RNA (ribonucleic acid) differs from DNA in three ways:
RNA is single stranded
The sugar in RNA is ribose, not deoxyribose.
RNA has uracil base instead of thymine.
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RNA
can base-pair with
single-stranded DNA
(adenine pairs with uracil instead of thymine)
and
also can fold over and base-pair with itself.
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Types of RNA
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)
Telomerase RNA (TERC)
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HIV and certain tumor viruses
(called retroviruses)
have RNA as their infectious information molecule;
they convert it to a DNA copy inside the host cell and then use it to make
more RNA.
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Recently, new classes of small RNA molecules have been discovered.
The RNA molecules are generally shorter than 200 nucleotides and their functions are not entirely understood.
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MicroRNAs (miRNAs)
are single-stranded RNA molecules of approximately 22 nucleotides.
They regulate protein expression by binding to and suppressing translation
of messenger RNAs.
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Mammalian RNA Polymerases
RNA polymerase I transcribes genes encoding ribosomal RNA.
RNA polymerase II transcribes genes encoding messenger RNA and certain
small nuclear RNAs.
RNA polymerase III transcribes genes encoding tRNAs and other small RNAs.
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Transfer RNA (tRNA)
is the link between the code of the mRNA and the amino acids of the
protein.
Each tRNA binds a specific amino acid.
Each tRNA has an anticodon that is complementary to the codon on mRNA.
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Ribosomal RNA (rRNA)
contributes to ribosome structure
has enzymatic activity (ribozyme)
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Transcription makes a single-stranded RNA copy of a
segment of DNA. DNA RNA
Translation uses information encoded in the RNA to make a
polypeptide.
RNA Protein
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Messenger RNA (mRNA)contains the genetic information
that codes for the amino acid sequence of a protein.
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Genetic code
every three base sequence on mRNA is called codon.
Each codon codes for one amino acid.Start codon = AUG codes for methionine
All proteins start with methionine3 stop codons exist.
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Redundancy of genetic code:
There are 20 amino acids and 64 codons, so there are multiple codons for some
amino acids.
Genetic code is universal among different species.
This is good evidence of evolution .
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In normal prokaryotic and eukaryotic cells, transcription requires the following:
a DNA template
the appropriate ribonucleoside triphosphates
(ATP, GTP, CTP, and UTP)
and
the enzyme RNA polymerase II
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TranscriptionJust one DNA strand
(the template strand)
is used to make the RNA.
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For different genes in the same DNA molecule, the roles of these strands may
be reversed.
The DNA double helix partly unwinds to serve as template.
As the RNA transcript forms, it peels away, allowing the already transcribed DNA to
reform the double helix.
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The polymerase that reads the DNA and makes the mRNA is RNA polymerase II.
BUT
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Eukaryotic RNA polymerases do NOT bind efficiently to DNA.
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We have a number of problems.
RNA polymerase II must first find the gene and its start site (the correct ATG) then it must bind to
the DNA.
In addition, when to express a gene and how much to express the gene must be determined.
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These problems are dealt with in the formation of the initiation complex.
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Three steps are involved in transcription:
Initiation
Elongation
Termination
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Initiation
The latest estimates are that
a human cell contains
20,000–25,000 genes.
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Initiation
Some of these are expressed in all cells all the time, housekeeping genes are
responsible for the metabolic functions common to all cells.
(glycolysis, Kreb’s cycle, respiration)
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Initiation
Some are expressed as a cell differentiates.
For example, a plasma cell continuously expresses the genes for antibody
synthesis.
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Initiation
Some are expressed only as conditions around and in the cell change.
For example, the arrival of a hormone may turn on (or off) certain genes in that cell.
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Initiation
Every gene has a promoter which is a sequence of DNA that regulates
the transcription of that gene.
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Initiation
When the cell decides that a particular gene needs to be transcribed,
special proteins (transcription factors) bind to the promoter.
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Transcription factors bind to specific sequences of DNA adjacent to the genes.
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Transcription factors direct RNA polymerase to the correct strand of DNA and the correct ATG
(start codon).
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Transcription factors are a major determinant for when and how much a particular gene is
expressed.
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There are approximately 2600 proteins in the human genome that contain DNA-binding
domains, and most of these are presumed to function as transcription factors.
(That’s approximately 10% of our genes.)
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In addition, there are coactivators, chromatin remodelers, histone acetylases, deacetylases,
kinases, and methylases.
All of which play crucial roles in gene regulation, but lack DNA-binding domains, and, therefore,
are not classified as transcription factors.
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InitiationA transcription complex forms around the bound
transcription factors.
This transcription complex has over 100 proteins.
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InitiationThe initiation complex then recruits the
RNA polymerase
and directs it to the correct strand and to correct start of the gene (ATG).
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InitiationMost eukaryotic promoters
contain a sequence TATAAA which is known as a TATA box.
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Initiation
The TATA box (sequence TATAAA) binds a TATA binding protein which assists in the
formation of the RNA polymerase transcriptional complex.
The TATA box typically lies very close to the transcriptional start site(often within 50 bases).
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Initiation
The TATAAA is a consensus sequence.
Promoters from different genes may have variations of this sequence.
Differences in the TATA box sequence from the consensus sequence will indicate how
strong the promoter is for a particular gene.
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Promoters also contain sequences that bind proteins called transcription factors which proteins involved in the formation of the
transcriptional complex.
A transcription factor (sometimes called a sequence-specific DNA-binding factor) is a
protein that binds to specific DNA sequences, thereby controlling the transcription of genetic
information from DNA to mRNA.
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ATCGCATAGCGCGACGCAGCTATAAAGCAGTCTATCACTGATGCATAC
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ATCGCATAGCGCGACGCAGCTATAAAGCAGTCTATCACTGATGCATAC
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ATCGCATAGCGCGACGCAGCTATAAAGCAGTCTATCACTGATGCATAC
RNAPOL
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ATCGCATAGCGCGACGCAGCTATAAAGCAGTCTATCACTGATGCATAC
RNAPOL
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ATCGCATAGCGCGACGCAGCTATAAAGCAGTCTATCACTGATGCATAC
RNAPOL
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ATCGCATAGCGCGACGCAGCTATAAAGCAGTCTATCACTGATGCATAC
RNAPOL
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ATCGCATAGCGCGACGCAGCTATAAAGCAGTCTATCACTGATGCATACRNAPOL
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Possible configuration for transcription factors mediating RNA polymerase II binding to a TATA-less promoter containing an Sp1-binding site.
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http://www.youtube.com/watch?v=41_Ne5mS2ls
http://www.youtube.com/watch?v=983lhh20rGY
This is good for ribosomes and nucleus
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ElongationThe RNA polymerase complex
unwinds the DNA and synthesizes a strand of RNA that is complementary
to the DNA.The DNA template is read in the
3’ to 5’ direction.The new RNA strand is synthesized
in the 5’ to 3’ direction (same as in DNA replication).
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Elongation
RNA polymerase is like DNA polymerase, it can only add nucleotides to the 3’ end of
a growing RNA strand
Energy for the synthesis of RNA is produced by splitting the phosphate bonds
in the ribonucleoside triphosphates
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TerminationA termination sequence on the gene
instructs the RNA polymerase to stop making RNA.
Sorry to be so vague but termination in eukaryotic cells is not well understood.
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After termination the RNA undergoes processing
which includes
capping
polyadenylation
and
splicing.
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Dystrophin is the longest gene at the DNA level, covering 2.4 megabases
(2,400,000 bases, 0.08% of the human genome).
The primary transcript measures about 2,400 kilobases and takes 16 hours to transcribe; the mature mRNA measures 14.0 kilobases. The 79 exons code for a protein of over 3500
amino acid residues.
However, it does not encode the longest protein known in humans.
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Titin is the largest known single polypeptide ((pronounced tɪ-tɪn).
The gene for titin contains the largest number of introns (363) discovered in any single gene.
The largest isoform of titin has 34,350 amino acid residues with a
mw=3,816,188.13 Da.
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Nucleosomes
Nucleic Acids
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The human genome has approximately
2,900,000 bases.
That is divided into 46 chromosomes (23 pairs).
Each chromosome is a linear double stranded DNA molecule.
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Our body contains approximately 100 trillion cells.
If the DNA from all our cells were stretched out, it would be over 113 billion miles [182 billion kilometers] long.
That is enough material to reach to the sun and back 610 times.
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species.
The largest genome (150,000,000,000 bases) is found in Paris japonica, a slow growing plant
native to Japan.
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The human genome (over 6 feet long) is packed into the nucleus of each cell in a
manner that still allows for gene regulation.
This remarkable feat is accomplished by the wrapping of DNA around histone proteins in
repeating units of nucleosomes to form a structure known as chromatin.
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Interphase chromatin is wrapped around proteins called histones.
These wraps of DNA and histone proteins are called nucleosomes and resemble beads on a string.
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The more condensed chromatin is less accessible to transcription factors and
polymerases.
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Nucleic Acids
There are 5 types of histones.
H1, H2, H3, H4, and H5
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The core of a nucleosome contains eight histone molecules, two each from four of
the histone classes except H1.
H1 clamps the DNA to the core.
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H5H4
H3H2
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H1
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Nucleic Acids
Histones have several functions.
They pack proteins so that they'll fit inside cell nuclei.
Packed DNA are 50,000 times shorter than unpacked ones.
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Histones also perform a function in gene expression.
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Transcription factors cannot bind to their promoter if the promoter is blocked by a
nucleosome.
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H1
promoter coding
atg………………………..taa
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Histones can be methylated,
causing DNA to be bound more tightly, which will down-regulate or even inhibit gene
transcription.
OR
Histones can be acetylated,
causing DNA to be bound more loosely, which will encourage transcription.
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Nucleic Acids
Histones can also be methylated phosphorylated, ubiquitinated, and
ADP-ribosylated.
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transcriptionactive inactive
Normally the chromatin will transition between the active and inactive fibers.
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transcriptionactive inactive
transcriptionactive inactive
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But diet and our environment can alter the ability of the chromatin to go from active to inactive or
from inactive to active.
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The changes occur at the level of the histones.
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For example, the histones may be methylated (a methyl group is covalently attached to the
histones).
CH3 CH3CH3
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Methylation (the addition of methyl groups) now prevents or hinders the transition from inactive to
active chromatin.
CH3 CH3CH3
transcriptionactive inactive
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This methylation is to the histones. It is different from DNA methylation.
CH3 CH3CH3
transcriptionactive inactive
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The net result is that certain genes would be underexpressed.
CH3 CH3CH3
transcriptionactive inactive
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A similar modification could happen to the 10nm fiber. Now the modification would prevent or
hinder the gene from transitioning from active to inactive.
transcriptionactive inactive
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Now genes would be overexpressed.
transcriptionactive inactive
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In the final analysis, there is a change in gene
expression without a change in DNA.
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Epigenetics
Epigenomics
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The Greek prefix epi- in epigenetics implies features that are "on top of" or "in addition to"
genetics;
thus epigenetic traits exist on top of or in addition to the traditional molecular basis for
inheritance (the sequence of bases).
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Epigenetics is an emerging frontier of science that involves the study of changes in the
regulation of gene activity and expression that are not dependent on the sequence of bases.
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Epigenetics focuses on processes that regulate how and when certain genes are
turned on and turned off.
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The molecular basis of epigenetics is complex.
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It involves modifications of the activation of certain genes, but not the basic structure of
DNA.
Proteins associated with DNA may be activated or silenced.
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The best example of epigenetic changes in eukaryotic biology is the process of cellular
differentiation.
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During morphogenesis, totipotent stem cells become fully differentiated cells.
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In other words,
a single fertilized egg cell - the zygote - changes into the many cell types including neurons, muscle cells, epithelium, blood
vessels etc as it continues to divide.
It does so by activating some genes while silencing others.
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What this means is that every cell in your body has the same instruction manual, but different cell types are using different
chapters.
Your neurons, for example, contain the DNA instructions on how to make your fingernails- but in neurons, those genes are turned off.
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Most importantly, epigenetic changes are preserved when cells divide.
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Most epigenetic changes only occur within the course of one individual organism's lifetime, but some epigenetic changes are inherited
from one generation to the next.
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Epigenomics includes any process that alters gene activity without changing the DNA
sequence.
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Researchers believe some epigenomic
changes can be passed on
from generation to generation.
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Several studies have documented that children born to mothers who did not get
adequate nutrition during pregnancy were more likely to develop type 2 diabetes and
coronary heart disease later in life.
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Among other factors,
diet and exposure to environmental chemicals
throughout all stages of human development
can cause epigenomic changes
that may turn on or turn off certain genes.
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Regulation of gene activity is critically important for normal functioning of the
genome.
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Changes in gene expression include
increased expression,
decreased expression,
or
improperly timed expression.
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Chromatin structure is subject to various modifications that may have profound
influences on gene expression.
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One way that genes are regulated is through the remodeling of chromatin.
Chromatin is the complex of DNA and the histone proteins with which it associates.
If the way that DNA is wrapped around the histones changes, gene expression can
change as well.
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Changes in chromatin structure include
changes in
the higher order of chromatin folding,
attachment to the nuclear matrix,
packaging of DNA around nucleosomes,
covalent modifications of histone tails,
and
DNA methylation.
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Assembly, mobilization and disassembly of nucleosomes can influence the regulation of
gene expression.
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Gene expression can be altered without changing the sequence of nucleotides.
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