Ch.

4 Nucleic Acids

Lectured by Sun-Mi Lee

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Outline
4.1 Nucleic Acids—Informational Macromolecules
4.2 Primary Structure of Nucleic Acids
4.3 Secondary and Tertiary Structures of Nucleic Acids
4.4 The Helix-to-Random Coil Transition: Nucleic Acid
Denaturation
4.5 The Biological Functions of Nucleic Acids
: A Preview of Genetic Biochemistry
4.6 Tools of Biochemistry

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4.1 Nucleic Acids—Informational
Macromolecules

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Two Types of Nucleic Acid: DNA and RNA
(1 of 3)

Chemical structure of
ribonucleic acid (RNA)
and deoxyribonucleic
acid (DNA)

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Two Types of Nucleic Acid: DNA and RNA
(2 of 3)

Purine-containing nucleosides and nucleotides

monomers of
nucleic acids

5’ monophosphorylated derivative of nucleosides

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Two Types of Nucleic Acid: DNA and RNA
(3 of 3)

Pyrimidine-containing nucleosides and nucleotides

DNA is chemically more stable than RNA,

which permits the generation and maintenance of longer genomes.

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Properties of Nucleotides (1 of 2)
• Ionization (or deprotonation) of nucleotides

• Primary ionization of phosphate at pH values close to 1.0

• Secondary ionization of phosphate and protonation or
deprotonation of amino group on the bases at neutral pH

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Properties of Nucleotides
• Purine and pyrimidine bases are capable of conversion
between tautomeric forms (amino vs. imino or keto vs. enol)
• The conjugated double-bond system in the base rings
observe light in UV range (around 260 nm)

UV light absorption is used

to detect and quantify nucleic acids

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Phosphodiester Linkage
• Nucleic acid biosynthesis is driven by the cleavage of the
energy-rich anhydride bond and the hydrolysis of the
pyrophosphate product(PPi) to orthophosphate or
inorganic phosphate (Pi)

The use of favorable reactions

to drive thermodynamically unfavorable ones

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4.2 Primary Structure of Nucleic Acids

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Nature and Significance of Primary Structure
• Primary structure
: the linear order of nucleotides in polymeric nucleic acids

5’

5’- ACGTT -3’

• Important features of polynucleotides

1) A polynucleotide chain has 5′-to-3′ direction
2) A polynucleotide chain has individuality,
3’
determined by the nucleotide sequence
Genetic information is stored in the primary structure of DNA

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4.3 Secondary and Tertiary Structures of
Nucleic Acids

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The DNA Double Helix (1 of 5)

• Secondary: 3D arrangements of nucleotide residues with

respect to one another, and short-term folding interactions
such as the double helix
• Tertiary: Longer range 3D interactions such as
supercoiling

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The DNA Double Helix (2 of 5)

X-ray fiber diffraction image of J. Watson and F. Crick in front

DNA generated by R. Franklin of their DNA double helix model

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The DNA Double Helix - base paring (3 of 5)

• DNA base composition: mole % A = T and G = C

• The two strands in the double helix were stabilized by
hydrogen bonding between A and T and between G and C

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The DNA Double Helix – stacking of base pairs
• 36° angle between stacked base pairs means 10 bp/turn
• The rise of the helix (0.34 nm) is the distance between
successive base pairs (twice the van der Waals thickness of
a planar ring)

Stacking interaction

10 bp/turn: 360°

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The DNA Double Helix (5 of 5)
• The Watson-Crick model for
DNA is a two-stranded,
antiparallel double helix with
10 base pairs per turn. Pairing
is A-T and G-C.
• The complementary, two-
stranded structure of DNA
explains how the genetic
material can be replicated
Direct access
to the bases

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Semiconservative Nature of DNA Replication

Each strand of the parent

molecule acts as a template
Three initial models of
for a new, complementary replication mechanism
strand
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Semiconservative Nature of DNA Replication
The Meselson-Stahl experiment (1958) proved that DNA
replicates semiconservatively

Hybrid molecule

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Alternative Nucleic Acid Structures
Crystal structure of DNA
The secondary structure involves local
variations in the angle of rations between
base pairs, the sugar confirmation, the tilt
of the bases, and even the rise distance.
→ Nucleic acid secondary structure is not
homogeneous. It can be changed by
interaction with other molecules.

Nucleic acids are dynamic molecules.

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DNA and RNA Molecules In Vivo (1 of 2)
Electron microscopic presentation of a DNA molecule

Supercoiled
form

Relaxed
form

Supercoiled form

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DNA and RNA Molecules In Vivo (2 of 2)
Gel electrophoresis demonstrating DNA supercoiling

Topoisomerases are
enzymes that modify state of
DNA supercoiling. Their
action can be followed by
agarose gel electrophoresis
in the presence of ethidium
bromide (a fluorescent dye).
The smaller or more
compact a DNA molecule,
the more rapid its migration

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Single-Stranded Polynucleotides (1 of 2)
Common conformations
Most DNA molecules found in cells are double-stranded, but
most naturally occurring RNA molecules are single-stranded

Random coil form stacked-base helix Hairpin structure

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Single-Stranded Polynucleotides (2 of 2)
Most RNA is single-stranded and could be a random coil
except for areas of internal complementarity. Transfer RNA
molecules used in protein synthesis (75-80 nucleotides long)
have extensive regions of intramolecular complementarity,
which fold in upon one another

Self-
complementarity
Fig. 4.25

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4.4 The Helix-to-Random Coil Transition:
Nucleic Acid Denaturation

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Separation of Double-Stranded into
Single-Stranded DNA (1 of 3)

ΔG = ΔH − TΔS

ΔH > 0 & ΔS > 0

at low temperature
: ΔG > 0 ( the helix is stable)

at high temperature
: ΔG < 0 ( the helix becomes unstable)

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Separation of Double-Stranded into
Single-Stranded DNA (2 of 3)
Native DNA absorbs less light than denatured DNA

Melting
(an abrupt transition)

The change in absorbance can be used to follow DNA

denaturation, that is, separation of double-stranded DNA

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Separation of Double-Stranded into
Single-Stranded DNA (3 of 3)
• The loss of secondary structure over large regions is called
denaturation.
• DNA denaturation is reversible: renaturation promoted by
slow cooling by allowing time for complementary strands to
find one another and pair up, or renature (annealing)
• Rapid cooling creates a population of single-stranded
random coils

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4.6 The Biological Functions of Nucleic
Acids: A Preview of Genetic Biochemistry

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Storage of Genetic Information, Replication,
Transcription, and Translation
• DNA is used to store the total genetic
information of an organism (genome)
• Replication passes on the genetic
information from cell to cell and from
generation to generation
• Transcription converts DNA-based
information of genes into messenger
RNA (mRNA) molecules
• Translation produces proteins based
on information in mRNA at ribosomes

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4.7 Tools of Biochemistry

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Manipulating DNA (1 of 2)

• Common biochemical applications to manipulate genetic

information encoded within DNA are:
1) gene cloning
2) chemical synthesis of oligonucleotides of defined
sequence
3) DNA sequence analysis
4) site-directed mutagenesis
5) polymerase chain reaction (PCR)

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Manipulating DNA (2 of 2)
• Creation of a
recombinant DNA
molecule in vitro

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Cloning a DNA Fragment into a Plasmid
Vector
• Cloning a fragment
of DNA into a
plasmid vector and
introducing the
recombinant
molecule into
bacteria

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Chemical Synthesis of Oligonucleotides
• Providing gene fragments used for synthetic biology
• Enabling the generation of materials useful in biomedicine
or electronics
Design and synthesis of 3D nanostructures

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Dideoxynucleotide Sequence Analysis (1 of 2)
• DNA sequencing by the
Sanger method

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Dideoxynucleotide Sequence Analysis (2 of 2)
Automated DNA sequencing

All four base-specific termination reactions are analyzed in

one gel by use of ddNTPs modified with fluorescent dyes;
each ddNTP receives a unique color

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Site-Directed Mutagenesis
Site-directed mutagenesis using
M13 phage as a cloning vector.
M13 is a single-strand DNA
phage that replicates via a
double-stranded DNA replicative
intermediate

Mutations include single-base substitutions,

short deletions, or insertions

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Chapter 4 Summary (1 of 3)

• There are two types of biologically relevant nucleic acid:

DNA and RNA
– RNA has the sugar ribose, while DNA has deoxyribose
– RNA uses the pyrimidine uracil, while DNA uses
thymine
– Both, RNA and DNA, are polymers of four kinds of
nucleoside 5′-phosphates connected by links between
3′-hydroxyls and 5′-phosphates
• Each nucleic acid has a defined sequence, the primary
structure

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Chapter 4 Summary (2 of 3)

• DNA is the polymer of genetic information

– DNA has a secondary structure of a double helix
– The purine adenine basepairs via two hydrogen bonds
with the pyrimidine thymine, while the purine guanine
basepairs via three hydrogen bonds with the pyrimidine
cytosine
– The replication mechanism is semiconservative
– Energy needs to be invested to separate the double
helical DNA structure into single strands

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Chapter 4 Summary (3 of 3)

• Both, DNA and RNA, form various tertiary structures such

as supercoils, or hairpins
• Nucleic acids have several important biological functions:
1) DNA contains stored genetic information
2) DNA is transcribed into messenger RNA (mRNA)
3) mRNA is translated into proteins at the ribosome
• Several tools and techniques have been developed to
manipulate DNA such as gene cloning, automated
oligonucleotide synthesis, dideoxynucleotide sequencing,
and site-directed mutagenesis

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