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 H H
At pH 7, Some bases
(U, T, G) will be
protonated and
others (A, C) will be
deprotonated
Nucleotides come from aminoacids

CO2 Glycine

C N
Aspartic acid
N C
Formate
C
C C
Formate
N N

Glutamine Glutamine
 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
Methyl nucleotides
methylation is part of
the restriction
modification system in
bacteria, in which DNA
is methylated.

Foreign DNAs which

are not methylated are
degraded by
sequence-specific
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

 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
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

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 Twist

Slide Roll
A to B to Z
A DNA and B DNA
Hairpins and cruciforms
 Replication

The magic of anti-parallel

strands-
Perfect duplication of DNA

Synthesis of DNA chain ONLY

occurs in 5’ to 3’ direction
 5’ to 3’ 3’
5’

3’
5’ 5’
3’
3’5’

3’
5’

5’

Direction of replication
Leading and lagging strands

3’ 5’

Lagging Leading
strand strand

Okazaki
5’ fragments 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 +non-histones 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 phos
 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