Nucleotides and Nucleic Acids

Deoxyribonucleic Acid (DNA) and

Ribonucleic Acid (RNA)

Nucleotide
Purine and Pyrimidine Bases in DNA and
RNA
Deoxyribonucleotides
Ribonucleotides
Tautomerization of the Bases
The Bases Absorb UV Light
Avery, McLeod
and McCarty,
1944
“Waring
Blender
Experiment”

Hershey and
Chase,
1952
 Incorrect Triple-Helical Structure

Pauling and Corey, 1953

Chargaff’s Rules:
A=T and G=C
purines = pyrimidines
 April 25, 1953
MOLECULAR STRUCTURE OF NUCLEIC ACIDS
A Structure for Deoxyribose Nucleic Acid

We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This
structure has novel features which are of considerable biological interest.
…
It has not escaped our notice that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material.
…
J. D. WATSON F. H. C. CRICK
Medical Research Council Unit for the Study of Molecular Structure of Biological
Systems, Cavendish Laboratory, Cambridge.
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Watson-Crick Base Pairs
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 Genetic Information Flow:

DNA RNA Protein

“The Central Dogma”

Francis Crick, 1957
Replication, Transcription and Translation

Replication and
transcription
occur in the nucleus

Translation occurs
in the cytoplasm
DNA Replication
Three Possible Models of DNA Replication
 Meselson-Stahl Experiment:
Semiconservative DNA Replication
DNA Transcription into RNA
 RNA Translation into Protein

Aside: ribozymes or catalytic RNA; example: the peptidyl transferase activity of the
ribosome.
Stability of Nucleic Acids
The DNA Duplex Can Be Reversibly
Denatured (Melted)
 Factors Determining DNA Duplex Stability

In order for the double helix to form under given conditions, forces favoring duplex formation
must outweigh disfavoring factors, so that the ∆G for duplex formation under those conditions is
negative.
 Factors Stabilizing the DNA Duplex
1. Base stacking and hydrophobic interactions (vertical base stacking
interactions make duplex formation enthalpically favored, although
entropically opposed, unlike the classic hydrophobic effect involved
in protein folding and lipid bilayer formation)

2. Ionic interactions (duplex becomes more stable as ionic strength

increases, since presence of positive counterions partially neutralize
negative charges of backbone phosphates)

3. Hydrogen bonding between base pairs

Under conditions in which the duplex forms spontaneously, stabilizing forces outweigh
destabilizing forces, so that ∆G of duplex formation is negative.
London Dispersion Forces in Base Stacking (Pi-Pi
Stacking)

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 Factors Destabilizing the DNA Duplex

1. Higher entropy of denatured random coil (denaturation is

temperature-dependent since relevant term is -T∆S in the Gibb’s free
energy equation, ∆G = ∆H - T∆S)

2. Charge-charge repulsion of backbone phosphates (so absence of

positive counterion would favor denaturation)

Under denaturing conditions, destabilizing factors dominate over stabilizing, so that ∆G of

denaturation becomes negative (and ∆G of duplex formation now has a positive sign).
 Denaturation of DNA Duplex

Increasing T
makes
denaturation
favorable

Denaturation and
renaturation
of DNA duplex are
cooperative
processes.

Hyperchromic Shift
 Instability Due to Reactivity:
Individual RNA Strands Chemically Less
Stable than DNA
2’ -OH can
participate in
intramolecular
nucleophilic attack on
phosphorus, breaking
the phosphodiester
backbone and
forming a 2’,3’ cyclic
phosphate product.
Transfer RNA (tRNA) Structure
 Palindromic DNA Sequences:
Potential to Form Cruciform Structures (Double Hairpins)
Some Palindromes:
Madam, I’m Adam
And E.T. saw waste DNA.
No, Mel Gibson is a casino's big lemon.
Sanger Dideoxy Method of DNA Sequencing

The most commonly used of two methods for DNA sequencing.

The other method, not as used today, is the Maxam-Gilbert
chemical cleavage method.
 NTP, dNTP and ddNTP (Dideoxynucleoside
Triphosphate) Structures

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Sanger Dideoxy Method of DNA Sequencing

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Automated DNA Sequencing
Formation of DNA Supercoil
 Supercoiling of DNA Shown by Gel
Electrophoresis
Agorose Gel Stained with Ethidium Bromide

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Supercoiled circular DNA migrates faster

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Ethidium = fluorescent
intercalating agent used to stain
during agarose gel electrophoresis than does DNA in agarose gels
relaxed circular DNA (or linear DNA).
 Negative Supercoiling of DNA is Important
Biologically

• Negatively supercoiled DNA is equivalent to underwound DNA.

This makes it easier to separate strands during replication and
transcription.

• In eukaryotes, formation of nucleosomes results in torsional strain in the

DNA molecule (equivalent to ~1.5-1.8 supercoils/nucleosome particle
theoretically; actual value is ~1), which is relieved by topoisomerases. This
results in DNA that is negatively supercoiled once histone proteins are
removed.

• In prokaryotes, an enzyme called DNA gyrase (a type II

topoisomerase) generates negative supercoils.