CHAPTER 8Nucleotides and Nucleic Acids

Ribonucleotide

Purine and pyrimidine

ATGCU

H H

H H

A - pKa=3.5C- pKa= 4.2T- pKa= 9G- pKa= 9.2U- 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

C

CN

N

C

C

NN

C

Nucleotides come from aminoacids

Glutamine Glutamine

Formate

Aspartic acid

CO2 Glycine

Formate

Nomenclature

nucleoside = sugar +base

nucleotide = sugar + base + phosphate

Nucleotides

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.

Linear to ring

Methyl nucleotidesAdenine 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 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 CpGs are methylated in mammals. Unmethylated CpGs are grouped in clusters called CpG islands that are present in the promoters of genes.

Nucleotide functions

Energy for metabolism in cellsATP

Cofactors for enzymesNAD

Signal transductioncAMP

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

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.

Endo and Exo

Syn and Anti

Stacked Twist

Slide Roll

Adjacent Bases

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’

3’

3’

5’

Direction of replication

5’

3’

3’5’

5’

5’3’

5’ to 3’

Leading and lagging strands

5’

3’ 5’

3’

Laggingstrand

Leadingstrand

Okazakifragments

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 polPrimaseReplication initiates

RNA synthesis followed by DNA synthesis

RF-C, PCNA, polrecruited

polto polswitch Replication elongation

Replication Fork

Basic scheme

Chromatin

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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

1g +1g +1g

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Chromatin

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Nucleosome- Histones

Four histone proteins

H2AH2BH3H4

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

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2 mol H2A2 mol H2B2 mol H32 mol H41 mol H1~200 bp DNA

Nucleosomes

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=22ADiameter of minor grooe =13ADiameter 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 fingerprintingCloningPCR amplification of specific DNA fragmentsGenomics

Deamination converts Cytosine to thymine via methyl-C