CHAPTER 8
Nucleotides and Nucleic Acids
Ribonucleotide
Purine and pyrimidine
ATGCU
H
H
A - pKa=3.5
C- pKa= 4.2
T- pKa= 9
G- pKa= 9.2
U- pKa=9.2
Protonated Form (red)
dominates below pKa
At pH 7, Some bases
(U, T, G) will be
protonated and others
(A, C) will be
deprotonated
H
H
Nucleotides come from aminoacids
Glycine
CO2
C
Aspartic acid
N
N
C
C
Formate
C
C
N
Glutamine
N
Glutamine
Formate
Nomenclature
nucleoside = sugar +base
nucleotide = sugar + base + phosphate
Nucleotides
Linear to ring
In solution, the straight-chain (aldehyde) and ring (b-furanose)
forms of free ribose are in equilibrium. RNA contains only the
ring form, b-D-ribofuranose. Deoxyribose undergoes a similar
interconversion in solution, but in DNA exists solely as β-2′deoxy-D-ribofuranose.
Adenine or cytosine
methylation is part of the
restriction modification
system in bacteria, in
which DNA is methylated.
Foreign DNAs which are
not methylated are
degraded by sequencespecific restriction
enzymes.
Neurospora crassa has a
well characterized
methylation system.
Genome has very little
repeated DNA,
methylation occurs in
repeated DNA –transposon
60% and 90% of all CpGs
are methylated in
mammals. Unmethylated
CpGs are grouped in
clusters called CpG islands
that are present in the
promoters of genes.
Methyl nucleotides
Nucleotide functions
Energy for metabolism in cells
ATP
Cofactors for enzymes
NAD
Signal transduction
cAMP
Cyclic nucleotides
Cyclic adenosine monophosphate (cAMP, cyclic AMP or 3'-5'-cyclic adenosine monophosphate) is
a second messenger important in many biological processes. cAMP is derived from adenosine
triphosphate (ATP)
cAMP is a second messenger, used for intracellular signal transduction, such as transferring the
effects of hormones like glucagon which cannot pass through the cell membrane. It is involved in
the activation of protein kinases
Asthma and bronchodilation
The b-adrinergic receptors are the targets for treatment of asthma. They are located in many organs of
the body, but the ones that are pertinent to asthma are the b-receptors located in the bronchial smooth
muscle and arterioles of the lungs which are especially important in the body’s airflow to and from the
lungs. When these receptors are stimulated they cause smooth muscle relaxation resulting in bronchial
dilation and vasodilation.
Beta2 receptors are serpentine receptors, meaning the protein crosses the cellular membrane seven
times. They are activated primarily by epinephrine. The carboxy-terminal end is on the intracellular
side and the amino-terminal end is on the extracellular side. These are coupled to G proteins which
have three subunits a,b, g. The alpha subunit of the G protein is activated by GTP, and the GTP
activated a-subunit activates adenylate cyclase. Adenylate cyclase converts ATP to cAMP which
serves as a second messenger leading to physiologic effects.
Beta-2 adrenergic receptor agonists
These drugs work to dilate the bronchial airways during an acute asthma attack. The b2-adrenergic
receptor agonists work by binding to the receptor and activating adenylyl cyclase. Adenylyl cyclase,
in turn, increases the production of cyclic adenosine monophosphate (cAMP). Bronchodilation is
supported by this increase in cAMP.
Phosphodiester
linkage
RNA hydrolysis
(alkaline)
Hydrolysis of RNA under alkaline conditions. The 2’ hydroxyl acts as a nucleophile in an intramolecular
displacement. The 2’,3’-cyclic monophosphate derivative is further hydrolyzed to a mixture of 2’- and 3’monophosphates.
DNA, which lacks 2’ hydroxyl, is stable under similar conditions.
Base pairing
DNA
Base pairing
Anti-parallel strands
Major groove is 22A wide.
Minor groove is 13A wide
Rotation
a
b
g
d
e
c
z
Endo and Exo
four of the five atoms are in a single plane. The fifth atom (C-2′ or C-3′) is on either the same (endo) or the opposite
(exo) side of the plane relative to the C-5′ atom.
Syn and Anti
Adjacent Bases
Stacked
Slide
Twist
Roll
A to B to Z
A DNA and B DNA
Hairpins and cruciforms
Replication
The magic of anti-parallel strandsPerfect duplication of DNA
Synthesis of DNA chain ONLY occurs
in 5’ to 3’ direction
5’ to 3’
5’
3’
3’
5’
3’
5’
3’5’
3’
5’
5’
Direction of replication
Leading and lagging strands
3’
Lagging
strand
5’
Okazaki
fragments
5’
Leading
strand
3’
Semi conservative
Dispersive
Semi conservative
Conservative
Semi conservative
Semi-conservative
Conservative
Dispersed
RNA primed DNA replication
Steps in SV40 replication
T antigen double hexamer Binds to origin
Unwinding origin
T ag recruit RP-A
T ag and RP-A recruit polaPrimase
Replication initiates
RNA synthesis followed by DNA synthesis
RF-C, PCNA, poldrecruited
polato poldswitch
Replication elongation
Replication Fork
Basic scheme
Chromatin
Chromatin
The single chromosome of the prokaryote Escherichia coli is about 1.3 mm of DNA.
A human cell contains about 2 m of DNA (1 m per chromosome set)
The human body consists of approximately 1013 cells and therefore contains a total of
about 2 × 1013 m of DNA.
Distance from the earth to the sun is 1.5 × 1011 m
The DNA in your body could stretch to the sun and back about 50 times.
The diameter of the nucleus is 5x10-6 meters
How is the DNA packaged?
Chromatin=
DNA
+histones
1g
+1g
+non-histones
+1g
43
Chromatin
44
Nucleosome- Histones
Four histone proteins
H2A
H2B
H3
H4
Very highly conserved
DNA is wrapped around the outside of the histone octamer
166 bp of DNA wraps around the histones
Linker DNA connects nucleosomes
7 fold compaction
Histone H1
45
Nucleosomes
2 mol H2A
2 mol H2B
2 mol H3
2 mol H4
1 mol H1
~200 bp DNA
46
Ionic interactions between basic positively charged histones and negatively charged phosphates in DNA
Sequence recognition
Each base pair can be identified
by characteristic chemical groups
that lie along the edge of the base
pair exposed in the major or minor
groove
Lambda repressor
The lambda repressor is a dimer also called cI protein. It binds DNA via helix-turn-helix motif.
Regulates transcription of cI and Cro protein.
Absence of cI protein, cro gene may be transcribed.
In the presence of cI, only cI gene may be transcribed.
Lambda repressor Sequence recognition
Diameter of major groove=22A
Diameter of minor grooe =13A
Diameter of alpha helix= 12A
Why a dimer? Co-operativity!
A single operator binds one dimer
Non-cooperative would be hyperbolic curve
Cooperative would be sigmoid curve
30x fold less repressor is needed to
reach 99% occupancy with two operator
sites compared to single site.
Sangamo Pharmaceuticals
ZFP TFs are novel transcription factors designed and engineered by Sangamo scientists to
regulate the expression of target endogenous genes.
Different Zinc finger genes can be engineered that recognize specific DNA sequences to
turn on or turn off specific genes in the cell.
Amyotrophic Lateral Sclerosis (ALS) (SB-509)
ALS, commonly referred to as “Lou Gehrig’s disease,” progressive neurodegenerative disease - affects nerve cells.
Progressive degeneration of the motor neurons- fatal because ability of the brain to control muscle movement is lost.
Animal and clinical data suggest that a defect or deficiency in VEGF (growth factor) expression plays a key role in ALS.
In an ongoing Phase 2 trial (SB-509-801), Sangamo is evaluating whether a regional muscle or systemic effect of SB-509 delivery will result in
a therapeutic effect in ALS.
Ss RNA
RNA secondary structures
RNA prefers A form.
De and Renaturation
Heat denaturation of DNA. The denaturation, or melting curves of
two DNA specimens.
The temperature at the midpoint of the transition (tm) is the melting
point;
it depends on pH
ionic strength
base composition of the DNA.
DNA hybridization
Basis for
DNA fingerprinting
Cloning
PCR amplification of specific DNA fragments
Genomics
Deamination converts Cytosine to thymine
via methyl-C