:

Nucleic Acids

DNA and RNA

Objectives

• At the end of the session, students can:

1. Identify the genetic material of DNA/RNA
2. Describe the structure and components
nucleic acids
3. Describe the physical properties of nucleic
acids
• Table of contents

01 DNA/RNA is Genetic
Material 03 DNA Topoisomerase
Catalytic Mechanisms

02 04
Components, Structure and Kinetics of RNA
Properties of Nucleic Acids folding
 01
•DNA/RNA is Genetic Material•
 Introduction •
• In 1928, Frederick Griffith
identified bacterial transformation
• He used both virulent and avirulent
Streptococcus pneumoniae in his
research.
• DNA is the Genetic Material for Bacteria

● In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty

published what is now considered a classic paper in the field of
molecular genetics.
● They demonstrated that DNA is the transforming principle.
● In their experiments, they removed the protein from the transforming
extract through organic solvent extraction.
● After this treatment, proteins were absent from the transforming extract.
● They found that the transforming principle was still active, which meant
the heat-killed bacteria were still able to convert live avirulent cells to
virulent cells
• DNA is the Genetic Material for Bacteriophage

● The second major piece of evidence supporting DNA as the genetic

material was through experiments conducted by A. D. Hershey and
Martha Chase in 1952.
● Hershey and Chase used T2 bacteriophage in their experiment to identify
whether DNA or protein is the genetic material.
● The phage attaches to the bacterial cell, and the genetic component of
the phage enters the bacterial cells.
● Following infection, the viral genetic component dominates the cellular
machinery of the host cells and leads to viral reproduction.
● Subsequently, many new phages are constructed, and the bacterial cell is
lysed, releasing the progeny viruses. This process is normally referred to
as the lytic cycle
• RNA is the Genetic Material for Viruses

●It has been demonstrated that the other type of

nucleic acid, RNA can also be a genetic
material
●It was first demonstrated that when purified
RNA from tobacco mosaic virus was spread on
tobacco leaves.
●Retroviruses utilizes RNA as their genetic
material.
 02
•Components, Structure and
Properties of Nucleic Acids•
• DNA and RNA are composed of
various combinations of nucleic
acids.
• These are macromolecules that
exist as polymers called
polynucleotides.
• Composed of many monomers
called nucleotides.
• Three components of nucleic acids
• Phosphate group
• Nitrogenous base
• Pentose sugar
• Nitrogenous base
• The five-carbon sugar ring and the content of the nitrogenous base
between DNA and RNA are slightly different from each other.
• Four different types of nitrogenous bases are found in DNA:
• adenine (A),
• thymine (T),
• cytosine (C), and
• guanine (G).
• In RNA, the thymine is replaced by uracil (U).
• Because of their structural similarity, we usually refer the nine-
member double rings adenine and guanine as purines, and six-
member single-ring thymine, uracil, and cytosine are pyrimidines.
• Pentose Sugar
• It depicts the structure of sugar found in nucleic acids.
• The difference between DNA and RNA lies in the C-20-
position of the ribose sugar ring.
• In RNA, the carbon at the C-2 position is attached to a
hydroxyl (OH) group.
• In DNA, the carbon at the C-2 does not contain this hydroxyl
group; rather it is replaced by a hydrogen (H) atom.
• Therefore, the pentose ring in DNA is considered a
deoxyribose (it is a deoxygenated five-carbon sugar ring).
• In the absence of the C-20 hydroxyl group of DNA, the sugar
is more specifically named 2-deoxyribose.
• Phosphate Group
• If a molecule is composed of a purine or pyrimidine base and
a ribose or deoxyribose sugar, this chemical unit is called a
nucleoside.
• The nitrogenous base and the pentose sugar are linked by
glycosidic bond between C-10-position of sugar and
nitrogenous base.
• If the base is a purine, the N-9 atom is covalently bonded to
the sugar. If the base is a pyrimidine, the N-1 atom bonds to
the sugar.
• When a phosphate group attaches to a nucleoside through a
phosphoester bond, the entire complex becomes a nucleotide.
• Chargaff’s Rules
• In 1953, James Watson and Francis Crick proposed the
theory that DNA exist as a double helix.
• The double helix model was mainly based on the X-ray
diffraction data collected by Rosalind Franklin and Maurice
Wilkins.
• The DNA composition studies observed by Erwin Chargaff
• Chargaff’s Rules
1. Two long polynucleotide chains are coiled around a central axis, forming a right
handed double helix.
2. The two DNA strand are antiparallel, that is, their 5’-3’ orientation runs in opposite
direction.
3. The base of both chains lie perpendicular to the axis, and they are stacked on one
another.
4. The nitrogenous bases of opposite chains are paired as the result of the formation of
a hydrogen bond in DNA.
5. Each complete turn 34 angstroms long.
6. The double helix has a diameter of 20 angstroms.
7. The amount of adenine residues is proportional to the amount of thymine residues in
DNA. Also, the amount of guanin residues is proportional to the amount of cytosine.
8. The sum of the purines equal to the sum of pyrimidine.
9. The percentage of (G+C) is not necessarily equal the percentage of (A+T)
• The arrangement of each subunit in the DNA structure
• Watson-Crick model places the sugar-phosphate backbones
on the outside of the double helix and carries the negative
charges on the phosphate group.
• The two polypeptide chains run in opposite directions known
as antiparallel.
• The nitrogenous bases are on the inside the double helix.
• Alternative forms of DNA
• It is the least common type of structural conformation
that a DNA can adopt under dehydrating conditions.

FEATURES
• Wider and flatter than B-DNA
• Has an axial hole at the center
• Has narrow and deep major
grooves
• Contains 11.6 base pairs per
turn
• 20-25% shorter than B-DNA
due to smaller rise per turn
• Alternative forms of DNA
• It is the common type of structural conformation of
DNA in the cells.

FEATURES
• Each base pair has the same
width
• Has a solid central core
• Right-handed helix
• Major groove is wide and deep
• Each turn consists of 10 base
pairs
• Alternative forms of DNA
• It is one of the biologically active forms of DNA
found in vivo, although its exact biological function
is not clear.

FEATURES
• Nucleotide pairs in Z-DNA
occur as nucleotide dimers
• Has a solid core at the center
• Left-handed helix
• Has a more or less flat major
groove
• Each helical turn contains 12
nucleotides
• Various Forms of RNA
• Several kinds of RNA play an important role in cellular
activities.
• Three major classes of cellular RNA molecules function
during the expression of genetic information:
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
• Messenger RNA (mRNA)
• Ribosomal RNA
• Constitutes about 80% of all RNA
in E. coli cells.
• They are important structural
components of ribosome, which
functions as non-specific sites of
protein synthesis during
translation.
• Transfer RNA

• Accounts for up to 15% of the RNA in

a typical cell.
• It carries amino acids to the ribosome
during translation, aiding in the
translation of DNA to mRNA to
protein.
• Although ribosomal RNA and transfer
RNA molecules are also synthesized by
transcription of DNA sequences, unlike
mRNA molecules, these RNAs are not
subsequently translated to form
proteins, and they remain in RNA form
 • Messenger RNA

• Carry genetic information from

the DNA of the gene.
• The mRNAs vary in size,
reflecting the range in the sizes
of the proteins encoded
• The other important RNA
• Small Nuclear RNA (snRNA)
• Participates in processing hnRNAs into mRNA and
snoRNA primarily functions as RNA chaperones in the
processing of ribosomal RNA (rRNA).
• Both are neither tRNA nor small rRNA molecules,
although they are similar in size (100–200 nucleotides)
to these species.
• The snRNA is always found in a stable complex with
specific proteins forming small nuclear
ribonucleoprotein particles (snRNP).
• The other important RNA
• Micro RNA and Small interfering RNA (miRNA & siRNA)
• Both miRNA and siRNA are involved in gene regulation
• The siRNAs disrupt gene expression by blocking specific
protein production hence their name “interfering,” even
though the mRNA encoding the protein has been
synthesized.
• The miRNAs control developmental timing by base
pairing with and preventing the translation of certain
mRNAs, thus, blocking synthesis of specific proteins.
• However, unlike siRNAs, miRNAs (22 nucleotides long)
do not cause mRNA degradation.
• Physical Properties of Nucleic Acids: DNA Denaturation
• An important feature of double helix DNA is the ability to separate the
two strands (a process called denaturation) and to base pair the two
strands together, a process called renaturation, without disrupting the
covalent bonds that make up the sugar-phosphate backbone.
• These processes occur at the very rapid rate needed to sustain genetic
functions.
• Because the complementary strands are held by hydrogen bonds,
the lack of covalent bonds makes it possible to denature and
renature double helix DNA without affecting its properties.
• The hydrogen bonds can be disrupted by high temperature, low salt
concentration, or high pH in vitro
• Denaturation and renaturation can occur with the combinations of
DNA-DNA, DNA-RNA, and RNA-RNA as the intermolecular or
intramolecular interaction.
• Secondary Structure of DNA
• It has been shown that DNA molecules can have dynamic changes in
secondary and tertiary structures, so that they can regulate expression of
their linear sequence information.
• Unusual secondary structures, such as slipped structures, cruciform, and
triple-helix DNA, are generally sequence-specific.
• Slipped structures usually occur at tandem repeats and are usually
found upstream of regulatory sequences in vitro.
• Cruciform structures are paired stem-loop formations.
• They can be found in vitro for many inverted repeats in plasmids
and bacteriophages.
• In a triplex helix, a third strand of DNA joins the first two to form
triplex DNA.
• Triplex helix DNA occurs at purine-pyrimidine stretches in DNA
and is favored by sequences containing a mirror repeat symmetry.
• Tertiary Structure of DNA
• Many naturally occurring DNA molecules are circular, with no
free 50 or 30 end.
• For example, prokaryotic DNA is circular, and this DNA forms
a supercoil.
• To understand how supercoiled and relaxed circular DNA differ,
you can take a covalently closed, circular DNA molecule, cleave
both strands in one place, and then rotate the DNA end around
several times before covalently closing the circle again.
• You can imagine this by taking a rubber band with two hands
and twisting it together.
• The resulting DNA circle is supercoiled and will wind around
itself.
• Secondary and Tertiary Structure of RNA
• RNA molecules are typically single-stranded.
• The course of a single-stranded RNA in three-dimensional
space conceivably would have six degrees of freedom per
nucleotide, represented by rotation about each of the six
single bonds along the sugar-phosphate backbone per
nucleotide unit.
• Compare this situation with DNA, whose separated strands
would obviously enjoy the same degrees of freedom.
However, the double-stranded nature of DNA imposes great
constraint on its conformational possibilities.
• Compared with dsDNA, an RNA molecule has a much
greater number of conformational possibilities
• Secondary Structure of RNA
• Usually refers to the portion of the RNA molecule that forms
short double-helical stretches.
• Secondary structure of RNA can be predicted accurately by
computer analysis.
• Common secondary structures that form the building blocks
of RNA architecture are bulges, stems single-stranded
hairpin/internal loops, and junctions
• Tertiary Structure of RNA
• RNA chains can fold into unique three-dimensional
structures that act similarly to globular proteins.
• For example, the functional transfer RNA (tRNA) can twist
into an L-shaped three-dimensional structure in the cell.
• The tRNA is about 76 nt long, and all of the different tRNAs
of a cell fold into the same general shape.
• The ribosomal RNA (rRNA) is another example of the RNA
that can fold into three-dimensional structure.
• DNA TOPOISOMERASE CATALYTIC MECHANISMS
• Forms of DNA that have the same sequences yet differ in their linkage
number are referred to as topological isomers, or topoisomers.
• Topoisomers can be visualized by their differing mobilities when separated
by gel electrophoresis.
• Topoisomerases are enzymes that modulate the degree of DNA
supercoiling, and they can convert one isomer of DNA to another.
• Topoisomerases are divided broadly into two families:
• Type I enzymes transiently cleave and reseal one strand of duplex
DNA in the absence of ATP
• Type II enzymes cleave and religate both DNA strands in the presence
of ATP.
• These two families are divided further into subfamilies, which can be
distinguished on the basis of protein architecture (monomer versus
oligomer), DNA substrate preference (duplex versus single-strand),
reaction outcomes (net loss or gain of supercoils; complete or partial
supercoil removal), and requirements for metals and ATP.
• Type 1 topoisomerase
• Type I topoisomerases can be subdivided according to their
structure and reaction mechanisms:
• type IA (TopIA; bacterial and archaeal topoisomerase I, and
topoisomerase III)
• Transiently cleave a single strand of supercoiled DNA to
form a 50- phosphotyrosyl intermediate.
• These enzymes include E. coli TopA, which
preferentially relaxes negatively supercoiled DNA, and
Topoisomerase III, which efficiently unknots and
decatenates single-stranded or nicked DNA.
• TopIA enzymes have a clamp-like structure with a large
central cavity in which DNA binds.
• Cleavage yields a covalent enzyme-DNA intermediate, in
which TopA bridges the nick that it created in the DNA.
• Type 1 topoisomerase
• Type IB (TopIB; eukaryotic topoisomerase I)
• TopIB enzymes form a 30-phosphotyrosyl intermediate and are
structurally unrelated to TopIA.
• TopIB can switch the DNA back and forth between a nicked and a
religated state, with a preference for the religated state over the nicked
state.
• TopIB can relax both negative and positive supercoils.
• When the DNA is cleaved, torsional energy present in the molecule
dissipates by rotation of the DNA about its intact strand.
• This mechanism is generally referred to as the “swivel” mechanism.
TopIB enzymes engage the DNA duplex as a C-shaped protein clamp.
• The tightly closed clamp may hinder DNA swiveling.
• Thus, DNA strand rotation within the covalent enzyme-DNA complex
requires at least some opening of the flexible TopIB protein clamp
• Type 1 topoisomerase

• Type IC (topoisomerase V)
• Archaeal topoisomerase V is the sole member of the
newly established TopIC family.
• Although structurally distinct, this enzyme
functionally resembles TopIB in terms of its
mechanism of action; it forms a 30-phosphotyrosyl
intermediate and relaxes positive and negative
supercoils
• Type 2 topoisomerase
• Type II topoisomerases transiently cleave both strands of
a DNA duplex to allow the unidirectional passage of
another DNA duplex through the protein-linked DNA
gate.
• Cleavage of the phosphodiester backbone in one
segment of duplex DNA (termed the gate or G-segment)
by the two active site tyrosines is accomplished by the
formation of a covalent 50-phosphotyrosyl-enzyme
adduct on each DNA strand, separated by 4 nucleotides.
• Type II topoisomerases are divided into two subfamilies:
IIA and IIB.
• Type 2 topoisomerase (IIA

• In eukaryotes, IIA enzymes catalyze the

relaxation of positively or negatively
supercoiled DNA, as well as the decatenation
and unknotting of DNA helices.
• Bacterial IIA topoisomerases, including DNA
gyrase and topoisomerase IV (TopIV), can also
decatenate and unknot DNA, but they also
possess unique activities.
• Type 2 topoisomerase (IIB)
• Topoisomerase VI (TopVI) exemplifies the IIB
subfamily, which is found in archaea, plants, and algae.
• It is distinguished from IIA enzymes on the basis of its
primary structure and domain architecture.
• In particular, TopVI lacks the third dimerization
interface present in IIA enzymes.
• TopVI is a homodimer, cleaves two strands of DNA by
staggered 50-phosphotyrosyl linkages, and uses ATP to
drive a second DNA duplex through the protein-linked
DNA gate to change Lk by steps of two.
• TopVI can also catalyze DNA decatenation and the
relaxation of positive and negative supercoils.
• The Cellular Roles of the Various Topoisomerases
• All DNA exists in the negative supercoiled state within both
prokaryotic and eukaryotic cells.
• If the DNA is unrestrained, there is equilibrium between
tension and unwinding of the helix.
• If the supercoiled is restrained with proteins, they are stabilized
by the energy of interaction between the proteins and the DNA.
• It has been suggested that DNA supercoiling plays an important
role in many genetic processes, such as replication,
transcription and recombination.
• These cellular activities require topoisomerases to remove
constraints and stabilize the DNA.
• All topoisomerases catalyze changes in the linkage of DNA
strands or helices by a conserved mechanism of transient DNA
strand cleavage and religation, yet the different types of
topoisomerases carry out distinct roles inside the cell.
• KINETICS OF RNA FOLDING

• Although DNA in cells is normally in a helical, double-

stranded form, RNA has more versatile compact
structures in cells.
• This is because single-stranded RNA molecules can fold
to form compact three dimensional structures. As a
result, they
• carry out structural, catalytic and regulatory roles in
many cellular processes including protein synthesis.