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Nucleotides and Nucleic Acids

Contents of Lecture
Structure and composition of nucleotides
DNA as genetic material
Structure of DNA and RNA
Mutation of DNA
DNA sequencing technology

Discovery of Nucleic Acids


In 1868, Friedrich Miescher isolated nuclein,
a phosphorus-containing substance, from the nuclei
of pus cells. The nuclein consists of an acidic and
a basic portion.
Nuclein
Protein

Nucleic acids
Nucleotide

Nucleoside
Sugar
(Ribose or
Deoxyribose)

Phosphate

Base
(Purine or
Pyrimidine)

Classification of Nucleic Acids


Nucleic acids can be divided into two major classes:
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
DNA also exists in chloroplast and mitochondria.
RNA can be further divided into messenger RNA
(mRNA, 5%), transfer RNA (tRNA, 10-15%),and ribosomal
RNA (rRNA, 80-85%).
In recent years, there were two new classes of RNA
have been found: Small interfering RNA (SiRNA) and
microRNA (miRNA). They play important roles in regulating
gene expression.

Nucleotides are the building blocks of nucleic acids.

General Structure of a Nucleotide

A nucleotide has three components: a base,


a pentose, and a phosphate.

The Parent Compounds of Bases

Pentose

Pentose

Major bases of nucleic acids

Nucleotide and Nucleic Acid Nomenclature

The Structure of Deoxyribonucleotides

The Structure of Deoxyribonucleotides

The Structure of Ribonucleotides

The Structure of Ribonucleotides

Minor bases of DNA


Plants and
animals

Bacteria

Plants and
animals

Bacteria
(infected
wtih phage)

Minor Bases of tRNA

1981116
13

Other Structure of Adenylate

Phosphodiester Linkages in DNA and RNA

Hydrolysis of RNA under Alkaline Conditions

Writing a Nucleic Acid

pA-C-G-T-AOH
pApCpGpTpA
pACGTA
5-ACTGA-3
A nucleic acid containing 50 or fewer
nucleotides is generally called oligonucleotide.

Nucleotides Have a Strong UV Absorption

Some key points we have learned


Nucleotides are the building blocks for nucleic acids.
A nucleotide consists of a nitrogen base (purine or
pyrimidine), a pentose, and one or more phosphate
group.
There are two types of nucleic acid: RNA and DNA.
The nucleotides in RNA contain ribose, and the common
pyrimidine bases are uracil and cytosine. In DNA, the
nucleotides contain 2-deoxyribose, and the common
pyrimidine bases are thymine and cytosine. The primary
purines are adenine and guanine in both RNA and DNA.

The Griffiths Early Experiments (1936)

Bacterium: Streptococus pneumoniae

The Griffiths Early Experiments (1936)

Heat can kill


virulent bacteria

Heat-killed virulent
strain can transform
nonvirulent to virulent
one

The Avery Experiments (1944)

The Hershey-Chase Experiment (1952)

Schrodinger and His Influence

In 1944, Erwin Schrodinger published his


book of What is life?. This book influenced
a generation of physicists and chemists, and
drawn many of them into the field of biology.

Mautice Wilkins

Cavendish Lab of Cambridge

James Waston

Francis Crick

Rosalind Franklin and Her Contributions

Rosalind Franklin
(1920-1958)

X-Ray Diffraction of DNA


Molecular

The Milestone in Biology

Waston and Crick


Page 737-738

Wilkins
Page 738-740

Nature, (1953), Vol. 171

Franklin
Page 740-741

The Milestone in Biology

We wish to suggest a structure for the


salt of deoxyribose nucleic acid (DNA). This
structure has novel features which are of
considerable biological interest.

It has been not escaped our notice that


the specific paring we have postulated immediately suggests a possible copying mechanism for the genetic material.
Waston and Crick,
Nature, (1953), 171:737-738

By James Waston

By Brenda Maddox

Waston-Crick DNA Model of DNA

10.5 base pairs per turn

The Structure of DNA

Hydrogen-bonding patterns in the base


pairs defined by Waston and Crick.

Replication of DNA

Semiconservative
replication as
suggested by
Watson and Crick

Three Possible Models for DNA Replication

The Meselson-Stahl Experiment (1958)

14N14N

15N15N

15N14N

14N14N

15N14N

Structural Variation of Nucleotides

DNA is a remarkably flexible molecular. Thermal


fluctuation can produce bending, stretching, and
unparing (melting) of the strands.
In A, B forms, all bases are in the anti orientation;
In Z-form, pyrimidines are always in anti, and purines
are always in syn form.

A, B, and Z forms of DNA

These structural variations generally do not


affect the key properties of DNA: strand complementarity, antiparallel strands, and the requirement for A-T and G-C base pairs.

Comparison of A, B, and Z forms of DNA

The B form is the most stable structure of DNA.


Whether A-DNA occurs in cells is uncertain, but
there is evidence for some short stretches of ZDNA in both prokaryotes and eukaryotes.

Palindromes and Mirror Repeat of DNA

Hairpin Structure of DNA or RNA

Hairpin formation occurs when one


strand of DNA (or RNA) is involved

Cruciform Structure of DNA

Cruciform formation occurs


when two strands are involved.

Many palindromic sequences


are the recognition site for DNA
restriction enzymes and binding
sites for transcription factors.

Structural Basis for Triple Helix

Structural Basis for DNA with Four Strands

Structure of H-DNA

H-DNA, a triple helix structure of DNA, is


found in polypyrimidine or polypurine tracts.

mRNA, tRNA, and rRNA


RNA generally can be divided into three
categories: messenger RNA (mRNA), transfer
RNA (tRNA), and ribosomal RNA (rRNA).
mRNA encodes proteins.

In prokaryotes and chloroplast, one mRNA


can encode several proteins.

Structure of RNAs

Secondary structure
of RNA
Typical right-handed
stacking structure of
RNA

Base-paired helical structure in an RNA

In RNA, G=U pairing can also be formed

Three Dimensional Structure in RNA

a) Yeast Phe tRNA


b) A hammerhead
ribozyme from
plant virus
c) An intron from
Tetrahymena

Some key points we have learned


Many lines have indicated that DNA bears genetic
information.
The Waston-Crick model of DNA shows that DNA
consists of two antiparallel chains in a right-handed
double-helical arrangement. Complementary base
pairs, A=T and G=C , are formed by hydrogen bonding
within the helix. The base pairs are stacked perpendicular
to the long axis of the double helix.
DNA can exist in A, B, and Z forms. DNA strands with
proper sequence can form hairpin/cruciform structures
or triplex or tetraplex DNA. RNA molecule has more
diverse structures.

Double-Helical DNA and RNA Can Be Denatured

Reversible denaturation
(melting) and renaturation
(annealing) of DNA

Heat Denaturation of DNA

Melting
point

Denaturation of a double-stranded nucleic


acid result in an increase of UV absorption. The
temperature at the midpoint of the transition (tm)
is the melting point. It depends on pH, ionic strength,
and size and base composition of the DNA.

tm and the GC Content of a DNA

There is a positive correlation between


tm and the GC content of a DNA.

DNA Denaturation Starts with AT Rich Regions

DNA Hybridization

Southern Hybridization

This technique was invented by Jeremy Southern

Mutations of Nucleic Acids


Nonenzymatic mutations of DNA
Deamination
Depurination

UV-induced pyrimidine dimers


Chemical agents that cause DNA
damage
Methylation

Deamination of DNA
Deamination causes the
transformation of cytosine
to uracil.
In a mammalian cell, deamination of cytosine occurs
about 100 times per day.
Deamination Of A and G
occurs at about 1/100 of this
rate.
However, normally uracil is
removed by a repair system.

Depurination of DNA

Depurination is not considered physiologically


significant

UV-induced mutations on DNA

Formation of pyrimidine dimers


induced by UV light

Chemicals that cause mutations on DNA

Precursors of nitrous acid (HNO2), which


promotes deamination.

Chemicals that cause mutations on DNA

Alkylating agents can cause


methylation of G (O6-methylguanine), thus renders it
unable to form base-pairing
with cytosine.

Methylation of DNA
In a living organism, some nucleotide bases
are enzymatically methylated using S-adenosylMethionine (SAM) as methyl group donor.
In bacteria, methylation is used for defense
purpose and for mismatch repair.
In eukaryotic cells, about 5% of cytidines in
DNA are methylated, usually occurs at CpG
sequences. Methylation can suppress the activity
of transposons. Ectopic methylation can lead to
developmental abnormality.

Hypermethylation of SUPERMAN Gene in Arabidopsis


Causes Developmental Abnormality

Determination of DNA sequence

DNA polymerization reaction requires a free


3-hydoxyl group.

Determination of DNA sequence

The Dideoxy method or Sanger method


(invented in 1977)

Automation of DNA sequencing

Automation of DNA sequencing

Chemical Synthesis of DNA

Today, the cost for synthesis of an oligonucleotide


is only about 1.2 Yuan per base

Some key points we have learned


DNA undergoes reversible unwinding and separation
on heating or at extremes of pH.
Denatured single-stranded DNAs from two species can
form a hybrid duplexs, the degree of hybridization
depending on the extent of sequence similarity.
DNA is relatively stable polymer. Spontaneous mutations
occur at very low rate. UV light and some chemicals
can induce the mutations on DNA.
DNA sequences can be determined and DNA polymers
can be synthesized.