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The Flow of Genetic
Information
replication
MBLG1001 Lecture 7
DNA
DNA
Transcription
Nucleic Acid Properties
RNA
Translation
Protein
Folding, modification,
translocation
Functional protein
History
• First isolated by a Swiss Biochemist
Fredrich Miescher circa 1860 from pus on
bandages.
• The new polymer contained the usual
suspects C, O, H and N and also
Phosphorus (this is not in protein).
History
• It was named nucleic acid because it was
first isolated in the nucleus and it was
acidic
• It was not thought to be the genetic
material. That role was thought to be
fulfilled by proteins as they contained more
potential for variation (20 different amino
acids rather than 4 different nucleotides).
The Role of DNA as genetic
material
• Work by Griffith then MacLeod,
McCartney and Avery.
• Using Pneumococcus they were able to
transform the bacterium from the rough
(R) non-pathogenic form to the smooth (S)
pathogenic form by adding a killed lysate
of S bacteria. By a process of elimination
the active ingredient in the killed S
bacterial lysate was identified as DNA.
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The Role of DNA as genetic
material
• This was further confirmed by the
Waring Blender experiment
(Hersey and Chase).
• The bacteriophage T2 which infects
certain bacteria was labeled with 35S
(which labels protein only) and 32P (which
only labels DNA).
The Chemistry of DNA
The Role of DNA as genetic
material
• The 35S was found in the supernatant, not
the pellet. Hence the protein part of the
phage was not transferred to the bacteria.
• The 32P was found in the pellet, indicating
the DNA was transferred to the bacteria.
The birth of the double helix
Chargaff’s Rules:
• A = T; G = C
• The amount of purine
= pyrimidine.
• The #sugars
(deoxyribose) = #
phosphates = # bases
Sodium deoxyribose nucleate from calf thymus,
Structure B, Photo 51, taken by Rosalind E. Franklin
and R.G. Gosling.
Rosalind Franklin - March 1956
Erwin Chargaff 1930
Watson and Crick
• The original paper
describing the
structure of DNA.
2
Properties of DNA explained by
the Watson and Crick Model
• Ability to store genetic information
• Ability to transfer a faithful copy of this
information to daughter cells
• Physical and chemical stability so that
information can be stored for long periods
of time
• There is potential for small changes, which
could be inherited, without the loss of
parental information
Terminology:
• Base: the purine or pyrimidine like ring
structure; adenine, guanine, cytosine,
thymine, uracil
• Nucleoside: base + sugar
• Nucleotide: base + sugar + phosphate.
B DNA
B DNA
The same piece of
DNA highlighting a
guanine
nucleotide.
The sugar phosphate
backbone is purple
The bases are green
The schematic
diagrams show
the backbone
only
B DNA
B DNA: Backbone
Deoxyribose
(sugar)
•Yellow is Phosphorus
•Red is Oxygen
•White is Carbon
Phosphate
Phosphate (PO4)
Guanine base
Sugar
3
B DNA: Backbone
Base
5’ Phosphate
A schematic
diagram of one
strand of DNA
Purine
P
Phosphodiester
linkage
Base
H
H
Sugar
O
-O
Base
P
P
Pyrimidine
Phosphate
P
OH
H
O
P
O
CH2
H
OOH
O
H
H
OH
H
H
Pyrimidine = Cytosine or Thymine
3’OH
Base
O
O-
Sugar = deoxyribose
Sugar
H
O
H
Purine = Adenine or Guanine
B DNA: Backbone
5’ Phosphate
O
Phosphoester
linkage
N
-O
NH
N
O
P
O
H
Sugar
NH2
Base
OH
P
Pyrimidine
O
H
O-
N
A schematic
diagram of one
strand of DNA
Purine
P
CH2
Base
P
H
Sugar = deoxyribose
Sugar
Pyrimidine = Cytosine or Thymine
H
H
3’OH
Purine = Adenine or Guanine
B DNA: Backbone
The 5' phosphate is attached here via a
phosphoester bond, forming a nucleotide.
The nucleotide is linked here to the 3' hydroxyl
of the ajoining nucleotide in DNA and RNA
by a second phosphoester bond (phosphodiester).
5’ Phosphate
Purine
P
Base
HO
5
OH
CH2
O
4
Reacts with the aldehyde
from C1 forming an acetal
ring.
C
H
3
C
H
2
1
C
P
Pyrimidine
OH
This is the 3' hydroxyl.
The 5' phosphate of the
ajoining nucleotide links
here by a phosphoester bond
Base
P
H
The hydroxyl is absent
at this position in DNA
A schematic
diagram of one
strand of DNA
Sugar
The base is attached here
by an N-glycosidic bond,
forming a nucleoside.
C
H
OH
Phosphate
Sugar
Phosphate
Sugar = deoxyribose
Pyrimidine = Cytosine or Thymine
3’OH
Purine = Adenine or Guanine
4
Purine
The Bases:
• The two purine bases, adenine and
guanine are based on the heterocyclic (a
fancy chemical name for a ring structure
containing both carbon and nitrogen)
compound, purine . Note the numbering
conventions for the ring atoms.
6
C
7
1
5
2
4
N
C
N
8
C
C
3
N
C
9
N
H
attaches to sugar
The amino group (δ+ve)
acts as an H-bond donor
The oxygen is very electronegative (δ-ve)
and acts as an H-bond acceptor
NH2
N
The ring N (pKa ~4)
acts as an H-bond acceptor
at physiological pH
N
N
N
H
O
The pKa of the ring N is ~9.
It will act as an H-bond donor
at physiological pH
The amino group (δ+ve) acts
as an H-bond donor
N
HN
H2 N
N
N
H
Guanine
Adenine
Pyrimidine
The amino group (δ+ve)
is an H-bond donor
4
C
3
5
N
C
2
The ring N has a pKa~4.5.
It acts as an H-bond
acceptor at pH 7.
NH2
N
6
C
C
1
The keto group acts as
an H-bond acceptor
N
attaches to sugar
O
N
H
Cytosine
5
Differences between DNA and
RNA:
The keto group acts as
an H-bond acceptor
O
The ring N has a pKa~9.
It acts as an H-bond donor.
This carbon is
methylated in thymine
HN
N
H
O
The keto group acts as
an H-bond acceptor
Uracil
Methyl added to
5-carbon
O
CH3
HN
-O
P
N
O
O
O
O
OH
H
H
OH
H
H
• The sugar is deoxyribose, which has no
–OH at position 2’
• The uracil is methylated at position 5
forming thymine
• These differences at first seem subtle
but are not accidental.
What forces maintain the double
helix?
• Nucleotides joined by a phosphodiester
bond (5’ P joins to the 3’ –OH)
• Hydrophobic interactions resulting in
base stacking
• Hydrogen bonding
• Ionic interactions
• Van der Waal’s forces
No Hydroxyl (-OH) at 2’
Base Stacking
• The bases are flat and aromatic in
character quite hydrophobic.
• They tend to be “buried” in the interior of
the molecule. Because they are flat the
bases can stack favorably on top of each
other at a distance of ~3.4 A.
Base Stacking
• This interaction is actually more
energetically important than hydrogen
bonding. Van der Waals forces become
significant at this close proximity, with
mutual distortion of the electron clouds.
• The nature of this base stacking
interaction is poorly understood. It is not
quite the same as the hydrophobic
interactions that operate with proteins.
6
Hydrogen bonding
• Hydrogen bonding between the bases when
they are in their correct tautomeric form gives
the double helix its specificity.
• Adenine pairs with thymine; the amine group on
adenine acts as a donor to the keto group of
thymine (provided both bases are in these
forms) and the ring N of adenine acts as an
acceptor; the ring N of thymine (and uracil) is
protonated at physiological pH and acts as a
donor.
NH 2
N
N
Ionic or electrostatic
interactions.
HN
N
N
O
deoxyribose
Adenine base paired to Thymine
O
N
N
deoxyribose
• Likewise the amine groups on guanine
and cytosine act as donors and the keto
groups as acceptors. If the wrong
tautomeric forms of the bases exist then
the base pairing is altered.
O
N
deoxyribose
Hydrogen bonding
H 2N
NH
N
NH 2
O
N
N
deoxyribose
• Theses forces give the DNA its twist. The
phosphate group in each residue is negatively
charged at physiological pH and hence they
actually repel each other. So you have some
conflict here. The bases actually want to
associate together but the phosphates want to
be as far as possible away from each other. The
net result is the twist to accommodate both
forces. If the molecule twists the phosphates can
be separated while still maintaining the base
pairing.
Guanine base paired to Cytosine
The general statistics:
•
•
•
•
Right handed double helix
10 residues per turn
diameter ~20 Å
vertical distance between turns 3.4 nm or
34 Å
• contains a major and minor groove
Major Groove
NH2
CH3
O
N
C
N
H
O
H
N
C
N
H
C
C
C
C
HN
C
C
N
H
O
O
H
H
Minor Groove
H
H
OH
H
HO
H
7
Major and Minor Grooves
Major and Minor Grooves
Major groove
Minor groove
8