Molecular Biology II Nucleic Acids 1: Structure and Function

DNA Purine: Pyrimidine:

RNA Purine: Pyrimidine:
Keto: Guanine Thymine
Keto: Guanine Uracil
Amino: Adenine Cytosine Amino: Adenine Cytosine

Other minor bases do exist, but are far more rare

In DNA, all bases are in keto form
DNA is more stable to hydrolysis than RNA

Nucleoside = Nitrogenous base + sugar

Nucleotide = Nucleoside + phosphate

DNA Double Helix:

 Consists of 2 antiparallel polynucleotide chains

 Bases are on the inside
 Sugar-phosphate backbone is on the outside
 Bases interact via H-bonding (A-T / G-C)
 Contains a major and minor groove
 Right handed helix
 Sugar is found on the minor groove side of the base pair

Can exist in A, B (most common) and Z forms

C/D/E forms exist but function is unknown
Relative humidity, sequence and ionic conditions will dictate form
B-form DNA – 92% humidity, Alkali metal ions
Puckering of sugar is different between DNA species

Table on slide 11

The number of secondary structures that DNA can form is limited due to its reduced flexibility and
due to the lower complexity of its chemical groups (cf. proteins).
Heat will denature DNA, resulting in a hyperchromic effect (UV absorbance increases by around
40%)
The midpoint of the ‘melting profile’ of DNA is defined as Tm (Melting Temperature)
The ring structure of bases allows us to study them using absorbance measurements.
The optic properties of bases change when they unstack.

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Molecular Biology II Nucleic Acids 1: Structure and Function

Factors Affecting Tm:

 Solvent
 Ions
 pH
  % G-C pairs =  Tm
  Mismatching =  Tm (1C for 1% mismatch)
  Length =  Tm
  [Formamide] =  Tm

Approximate Tm (C) = 68 + 0.4 x (%GC)

or 2C for AT and 3C for GC (for short strands only)

If hybridisation occurs too quickly or at a low temperature then there will only be partial pairing.
The sugar-phosphate conformations make dsDNA more rigid than ssDNA
Studies show that species that live in high temperature environments have a higher GC content,
and therefore their DNA has a higher Tm.

Base Pairing:

Responsible for the structure of dsDNA

Geometric and electrostatic complementarity
A-T / G-C have higher affinities for one another than non-Watson/Crick base pairs, forming more
stable structures. The H-bonds do not contribute greatly to the overall stability of DNA (2-
3Kcal/mol/bp), but they orientate the bases correctly for stacking which has a much greater
influence on stability.

Base pairing is more important in RNA secondary and tertiary structure.

Base Stacking / Hydrophobic Interactions:

Contribute the most to stability

Stacks are stabilised by hydrophobic forces (4-15Kcal/mol/pair)

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Molecular Biology II Nucleic Acids 1: Structure and Function

Hydrophobic solvents will increase the solubility of the free base, promoting denaturation of
dsDNA and reducing the Tm.

Ionic / Polar Interactions:

When in water, DNA becomes more rigid due to repulsive forces acting between the phosphates
in the backbone.
[cation] increases stability of DNA by masking the phosphate groups from one another, thereby
reducing repulsion and increasing hybridisation.
Dipole-diploe interactions between stacked bases stabilise the structure.

Chemical Effects:

Organic solvents (formamide, ethanol etc.) will disrupt stacking and cause denaturation
RNA is more susceptible t base catalysed hydrolysis than DNA
High pH will cause tautomerisation of the base (keto  enol) disrupting base pairin3g
Chaotropic chemicals (urea) disrupt the solvent and will disrupt the 3D structure of the RNA/DNA
These chemicals can be used to separate RNA/DNA

Physical Effects:

A260nm can be used to measure nucleic acid concentration

When A260 = 1…
[dsDNA] = 50g/ml
[RNA] = 50g/ml
[Oligonucleotides] = 50g/ml

A260/A280 is used to measure DNA purity

1.8 < Pure
1.8 > has protein contamination

DNA Structures:

Supercoiled DNA is less accessible

Catenation – Linking of rings to form a ‘chain’
 Type II topo makes a ds break in one ring allowing the other to link around it
L = Linking number (= Twisting number + Writhing number)
 Refers to the state of supercoiling

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Molecular Biology II Nucleic Acids 1: Structure and Function

 DNA molecules that differ only by L are known as topoisomers (topological isomers)

Type I Topoisomerases – break one strand only

Found in both prokaryotes and eukaryotes
Relax negative supercoils using a nick-close mechanism (cannot relax positive supercoils)
Active site contains a Tyr residue which forms a phosphodiester bond with the 3’-OH of the DNA
Contains 4 domains and a hole containing + charges (for the DNA)

Type II Topoisomerases – Break both strands

DNA Gyrase is only found in bacteria – decreases L, therefore introduces negative supercoiling
The negative supercoils store energy for DNA reactions
Requires ATP (when no ATP is present DNA Gyrase slowly relaxes supercoils)
Do not negatively supercoil DNA in eukaryotes

Balanced activity of each of these enzymes maintains the correct level of supercoiling
In eukaryotes, topoisomerases maintain negative supercoils by wrapping DNA around proteins

Therapeutics
Some antibiotics (novobiocin / oxolinic acid) inhibit DNA gyrase resulting in inhibition of DNA
replication and transcription. Eukaryotes do not have DNA gyrase so they are unaffected.

Drugs can be used to kill tumor cells as they have elevated topoisomerase activity
 Camptothesin targets topo I by inhibiting the enzyme catalysed re-ligation of DNA. The
enzyme is converted into a DNA breaking activity (fatal)
 Doxorubicin targets type II topo

RNA Structure:

RNA contains more minor bases than DNA

2’-OH limits the secondary structures of RNA (can’t take up ‘B-form’)
Similar to A-form DNA (11bp/turn)
More susceptible to enzymatic and chemical degradation

RNA secondary structure:

 Single strands
 Bulges
 Internal Loops
 Hairpins

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Molecular Biology II Nucleic Acids 1: Structure and Function

DNA cannot form these structures

tRNA notes from MBI

rRNA’s have complex secondary and tertiary structures with many domains
rRNA’s are similar across species and have a highly conserved structure.

mRNA is the least stable form of RNA and can have a half life of minutes – hours.
Can have a secondary structure – Trp operon

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