Nucleic acids.pdf

Nucleic Acid:
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
• A segment of a DNA molecule that contains the
information required for the synthesis of a functional
biological product, whether protein or RNA, is referred
to as a gene.
• A cell typically has many thousands of genes, and DNA
molecules.
• The storage and transmission of biological information
are the only known functions of DNA.
Dr. Shiny C Thomas, Department of Biosciences, ADBU

RNAs have a broader range of functions, and several
classes are found in cells.
• Ribosomal RNAs (rRNAs) are components of ribosomes,
the complexes that carry out the synthesis of proteins.
• Messenger RNAs (mRNAs) are intermediaries, carrying
genetic information from one or a few genes to a
ribosome, where the corresponding proteins can be
synthesized.
• Transfer RNAs (tRNAs) are adapter molecules that
faithfully translate the information in mRNA into a specific
sequence of amino acids.

Nucleotides and Nucleic Acids Have Characteristic
Bases and Pentoses
Nucleotides have three characteristic components:
(1) a nitrogenous (nitrogen-containing) base, (2) a
pentose, and (3) a phosphate (Fig. 8–1).
The molecule without the phosphate group is called a
nucleoside. The nitrogenous bases are derivatives of two
parent compounds, pyrimidine and purine.
• The bases and pentoses of the common nucleotides are
heterocyclic compounds.

Structure of nucleotides. (a) General structure showing
the numbering convention for the pentose ring. This is a
ribonucleotide.
In deoxyribonucleotides the OH group on the 2 carbon
(in red) is replaced with H.

• The Pentoses of nucleotides and nucleosides the carbon
numbers are given a prime (‘) designation to distinguish
them from the numbered atoms of the nitrogenous
bases.
• The base of a nucleotide is joined covalently (at N-1
of pyrimidines and N-9 of purines) in an N—glycosyl bond to
the 1 carbon of the pentose, and the phosphate is esterified
to the 5 carbon.
• The N--glycosyl bond is formed by removal of the
elements of water (a hydroxyl group from the pentose
and hydrogen from the base), as in O-glycosidic bond
formation

• Both DNA and RNA contain two major purine bases,
adenine (A) and guanine (G).
• In both DNA and RNA two major pyrimidines one of the
pyrimidines is cytosine (C), but the second major
pyrimidine is not the same in both: it is thymine (T) in
DNA and uracil (U) in RNA.

Structures of the five major bases

ATP, its diphosphate, and
its monophosphate.

Heterocylic N -Glycosides Exist as Syn and Anti Conformers
Steric hindrance by the heterocycle dictates that there is no
freedom of rotation about the -N-glycosidic bond of
nucleosides or nucleotides. Both therefore exist as non
interconvertible syn or anti conformers.
Unlike tautomers, syn and anti conformers can only be
interconverted by cleavage and reformation of the glycosidic
bond. Both syn and anti conformers occur in nature, but the
anti conformers predominate.

single-stranded DNA sequence is written in the 5' to 3' direction (ie, pGpCpTpA, where G,
C, T, and A represent the four bases and p represents the interconnecting phosphates).

• A diagrammatic representation of
the Watson and Crick model of the
double-helical structure of the B
form of DNA.
• The horizontal arrow indicates the
width of the double helix (20 ), and
the vertical arrow indicates the
distance spanned by one complete
turn of the double helix (34 ). One
turn of B-DNA includes 10 base
pairs (bp), so the rise is 3.4 per bp.
• The central axis of the double helix
is indicated by the vertical rod. The
short arrows designate the polarity
of the antiparallel strands.
• The major and minor grooves are
depicted. (A, adenine; C, cytosine;
G, guanine; T, thymine; P,
phosphate; S, sugar [deoxyribose].)
Hydrogen bonds between A/T and
G/C bases indicated by short, red,
horizontal lines.

• This common form of DNA is said to be right-handed
because as one looks down the double helix, the base
residues form a spiral in a clockwise direction.
• In the double-stranded molecule, restrictions imposed
by the rotation about the phosphodiester bond, the
favored anticonfiguration of the glycosidic bond (Figure
32–5), and the predominant tautomers (see Figure 32–
2) of the four bases (A, G, T, and C) allow A to pair only
with T and G only with C, as depicted in Figure.

DNA Nucleotides and Tautomeric Form
• DNA consists of two strands of phosphate and sugar
coiled around each other in a helical manner and held
together by hydrogen bonding between pairs of
nitrogenous bases.
• There are four bases: adenine (A) and guanine (G), which
are purines, and thymine (T) and cytosine (C), which are
pyrimidines. Guanine and thymine can have alternate
molecular structures based on different locations of a
particular hydrogen atom.
• A keto structure occurs when the hydrogen atom bonds to
a nitrogen atom within the ring. An enol structure occurs
when the hydrogen atom bonds to an nearby oxygen atom
that sticks out from the ring.
• These two types of structures are known as tautomers.

• Both guanine and thymine can switch easily from one
tautomer to another.
• The change in shape affects the three-dimensional shape of
the molecule.
• In the early 1950s, guanine and thymine were generally
portrayed in the enol form, although there was little data to
support the predominance of one form over the other.
• James Watson and Francis Crick discovered that by using
the keto forms instead of the enol forms, they could "form"
two base pairs, an adenine thymine pair and a guanine-
cytosine pair, that had the same overall size and shape.
• These base pairs formed the basis for Watson and Crick's
model of DNA.

Tautomers of Adenine, Cytosine, Guanine, and Thymine
• The four bases of DNA can exist in at least two tautomeric
forms as shown below.
• Adenine and cytosine (which are cyclic amidines) can exist
in either amino or imino forms, and guanine, thymine, and
uracil (which are cyclic amides) can exist in either lactam
(keto) or lactim (enol) forms.
• The tautomeric forms of each base exist in equilibrium but
the amino and lactam tautomers are more stable and
therefore predominate under the conditions found inside
most cells.
• The rings remain unsaturated and planar in each tautomer.

• The two strands, in which opposing bases are held
together by inter strand hydrogen bonds, wind around a
central axis in the form of a double helix.
• In the test tube double stranded DNA can exist in at least
six forms (A–E and Z).
• The B form is usually found under physiologic conditions
(low salt, high degree of hydration).
• A single turn of BDNA about the long axis of the molecule
contains ten base pairs.
• The distance spanned by one turn of B-DNA is 3.4
nm (34 ). The width (helical diameter) of the double helix in
B-DNA is 2 nm (20 ).

• The two strands of the double-helical molecule, each of
which possesses a polarity, are antiparallel; ie, one
strand runs in the 5' to 3‘ direction and the other in the
3' to 5' direction.
• In the double-stranded DNA molecules, the genetic
information resides in the sequence of nucleotides on
one strand, the template strand.
• This is the strand of DNA that is copied during
ribonucleic acid (RNA) synthesis. It is sometimes
referred to as the noncoding strand.

• The opposite strand is considered the coding strand
because it matches the sequence of the RNA transcript
(but containing uracil in place of thymine; see Figure
34–8) that encodes the protein.

• The relationship between the sequences of an RNA
transcript and its gene, in which the coding and template
strands are shown with their polarities.
• The RNA transcript with a 5' to 3' polarity is
complementary to the template strand with its 3' to 5‘
polarity.
• Note that the sequence in the RNA transcript and its
polarity is the same as that in the coding strand, except
that the U of the transcript replaces the T of the gene.

• DNA base pairing between
adenosine and thymidine
involves the formation of
two hydrogen bonds.
• Three such bonds form
between cytidine and
guanosine.
• The broken lines represent
hydrogen bonds.

• As depicted in Figure, three hydrogen bonds, formed by
hydrogen bonded to electronegative N or O atoms, hold
the deoxyguanosine nucleotide to the deoxycytidine
nucleotide, whereas the other pair, the A–T pair, is held
together by two hydrogen bonds.
• Thus, the G–C bonds are more resistant to
denaturation, or strand separation, termed "melting,"
than A–T-rich regions of DNA.

• Nucleic acids have two kinds of pentoses.
• The recurring deoxyribonucleotide units of DNA contain
2- deoxy-D-ribose, and the ribonucleotide units of RNA
contain D-ribose.
• In nucleotides, both types of pentoses are in their -
furanose (closed five-membered ring form.

Phosphodiester Bonds Link Successive Nucleotides
in Nucleic Acids
• The successive nucleotides of both DNA and RNA are
covalently linked through phosphate-group “bridges,” in
which the 5-phosphate group of one nucleotide unit is
joined to the 3-hydroxyl group of the next nucleotide,
creating a phosphodiester linkage

• Thus the covalent backbones of nucleic acids consist of
alternating phosphate and pentose residues, and the
nitrogenous bases may be regarded as side groups
joined to the backbone at regular intervals.
• The backbones of both DNA and RNA are hydrophilic.
The hydroxyl groups of the sugar residues form
hydrogen bonds with water.

Hydrolysis:
• The covalent backbone of DNA and RNA is subject to
slow, nonenzymatic hydrolysis of the phosphodiester
bonds.
• In the test tube, RNA is hydrolyzed rapidly under
alkaline conditions, but DNA is not; the 2-hydroxyl
groups in RNA (absent in DNA) are directly involved in
the process.
• Cyclic 2,3-monophosphate nucleotides are the first
products of the action of alkali on RNA and are rapidly
hydrolyzed further to yield a mixture of 2- and 3-
nucleoside monophosphates.

Hydrolysis of RNA under alkaline conditions.
• The 2 hydroxyl acts as a nucleophile in an
intramolecular displacement.
• The 2,3-cyclic monophosphate derivative is further
hydrolyzed to a mixture of 2- and 3-monophosphates.
• DNA, which lacks 2 hydroxyls, is stable under similar
conditions.

The nucleotide sequences of nucleic
acids represented schematically: The
phosphate groups are symbolized by
(P) and each deoxyribose is
symbolized by (S) beta furanose form,
from C-1 at the top to C-5 at the
bottom.

A short nucleic acid is referred to as an oligonucleotide. A
longer nucleic acid is called a polynucleotide.
Summary
• A nucleotide consists of a nitrogenous base (purine or
pyrimidine), a pentose sugar, and one or more
phosphate groups.
• Nucleic acids are polymers of nucleotides, joined
together by phosphodiester linkages between the 5-
hydroxyl group of one pentose and the 3- hydroxyl group
of the next.

■ 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.

• In all cellular DNAs,
regardless of the species, the
number of adenosine
residues is equal to the
number of thymidine
residues (that is, A T), and
the number of guanosine
residues is equal to the
number of cytidine residues
(G C).
• From these relationships it
follows that the sum of the
purine residues equals the sum
of the pyrimidine residues; that
is, A + G = T + C.

DNA Is a Double Helix
• In 1953 Watson and Crick postulated a three dimensional
model of DNA structure.
• It consists of two helical DNA chains wound around the
same axis to form a right handed double helix.
• The hydrophilic backbones of alternating deoxyribose
and phosphate groups are on the outside of the double
helix, facing the surrounding water.
• The purine and pyrimidine bases of both strands are
stacked inside the double helix, with their hydrophobic
and nearly planar ring structures very close together
and perpendicular to the long axis.

• The pairing of the two strands creates a major groove
and minor groove on the surface of the duplex.
• Each nucleotide base of one strand is paired in the
same plane with a base of the other strand.
• Watson and Crick found that the hydrogen-bonded
base pairs G with C and A with T, are those that fit best
within the structure.
• In any DNA, G = C and A = T. I
• It is important to note that three hydrogen bonds can
form between G and C, symbolized , but only two can
form between A and T, symbolized
The strands of DNA are antiparallel; 5,3-phosphodiester
bonds run in the opposite directions

• The bases inside the double helix would be 3.4 Å apart;
the secondary repeat distance of about 34 Å was
accounted for by the presence of 10 base pairs in each
complete turn of the double helix.
• The two antiparallel polynucleotide chains of double-
helical DNA are not identical in either base sequence or
composition.
• Instead they are complementary to each other.
• Wherever adenine occurs in one chain, thymine is found
in the other; similarly, wherever guanine occurs in one
chain, cytosine is found in the other.

• The DNA double helix, or duplex, is held together
by two forces, as described earlier: hydrogen bonding
between complementary base pairs and base-stacking
interactions.
• The essential feature of the model is the
complementarity of the two DNA strands.
• This structure could logically be replicated
by (1) separating the two strands and (2) synthesizing
a complementary strand for each.

• The Watson-Crick structure is also referred to as B-
form DNA, or B-DNA. The B form is the most stable
structure and standard point of reference in any study
of the properties of DNA.
Two structural variants that have been well characterized
in crystal structures are the A and Z forms.

Comparison of A, B, and Z forms of DNA.

A- Form:
• The A form is favored in many solutions that are
relatively devoid of water.
• The DNA is still arranged in a right-handed double
helix, but the helix is wider and the number of base pairs
per helical turn is 11, rather than 10.5 as in B-DNA.
• The plane of the base pairs in A-DNA is tilted about 20
with respect to the helix axis.
• These structural changes deepen the major groove while
making the minor groove shallower.

Z-form DNA
• is a more radical departure from the B structure
• the most obvious distinction is the left handed helical
rotation.
• There are 12 base pairs per helical turn, and the
structure appears more slender and elongated.
• The DNA backbone takes on a zigzag appearance.
• The major groove is barely apparent in Z-DNA, and the
minor groove is narrow and deep.

Palindrome
• A palindrome is a word, phrase, or sentence
that is spelled identically read either forward or backward;
two examples are ROTATOR and NURSES RUN.
• The term is applied to regions of DNA with inverted
repeats of base sequence having two fold symmetry
over two strands of DNA.

• Such sequences are self-complementary within each
strand and therefore have the potential to form hairpin
or cruciform (cross-shaped) structures (Fig.).
When only a single DNA (or RNA) strand is involved, the
structure is called a hairpin

When the inverted repeat
occurs within each
individual strand of the
DNA, the sequence is called
a mirror repeat.
When both strands of a
duplex DNA are involved, it
is called a cruciform

This cruciform structure blocks DNA
motif from binding to some proteins

Some nucleases cut cruciform DNA structure and DNA breakage
brebrebre

This causes cancer, genetic disorder etc………

H-DNA: Triple-stranded DNA is a DNA structure in which
three oligonucleotides wind around each other and form
a triple helix. In this structure, one strand binds to a B-
form DNA double helix through Hoogsteen or reversed
Hoogsteen hydrogen bonds.

The N-7, O6, and N6 of purines, the atoms that participate
in the hydrogen bonding of triplex DNA, are often referred
to as Hoogsteen positions, and the non-Watson-Crick
pairing is called Hoogsteen pairing, after Karst Hoogsteen,
who in 1963 first recognized the potential for these unusual
pairings.
Hoogsteen pairing allows the formation of triplex DNAs.
A simple example is a long stretch of alternating T and C
residues

The H-DNA structure features the triple-stranded form

a) A sequence of alternating T and C residues can be
considered a mirror repeat centered about a central T or
C.
(b) These sequences form an unusual structure in which the
strands in one half of the mirror repeat are separated and
the pyrimidine containing strand (alternating T and C
residues) folds back on the other half of the repeat to form
a triple helix.
The purine strand (alternating A and G residues) is left
unpaired.

Slipped strand mispairing (SSM), (also known as replication
slippage), is a mutation process which occurs during DNA
replication.
• It involves denaturation and displacement of
the DNA strands, resulting in mispairing of the
complementary bases.
• Slipped strand mispairing is one explanation for the
origin and evolution of repetitive DNA sequences.
• SSM events can result in either insertions or deletions.
Insertions are thought to be self-accelerating: as repeats
grow longer, the probability of subsequent mispairing
events increases.

• Insertions can expand simple tandem repeats by one or
more units.
• In long repeats, expansions may involve two or more
units.
• For example, insertion of a single repeat unit in GAGAGA
expands the sequence to GAGAGAGA, while insertion of
two repeat units in [GA]6 would produce [GA]8.
• Genomic regions with a high proportion of repeated
DNA sequences (tandem repeats, microsatellites) are
prone to strand slippage during DNA replication.

The Denaturation of DNA
• The double-stranded structure of DNA can be separated
into two component strands in solution by increasing the
temperature or decreasing the salt concentration.
• Not only do the two stacks of bases pull apart but the
bases themselves unstack while still connected in the
polymer by the phosphodiester backbone.
• Concomitant with this denaturation of the DNA molecule
is an increase in the optical absorbance of the purine and
pyrimidine bases—a phenomenon referred to as
hyperchromicity of denaturation.

• Because of the stacking of the bases and the hydrogen
bonding between the stacks, the double-stranded DNA
molecule exhibits properties of a rigid rod and in
solution is a viscous material that loses its viscosity upon
denaturation.
• The strands of a given molecule of DNA separate over a
temperature range. The midpoint is called the melting
temperature, or Tm .
• The Tm is influenced by the base composition of the DNA
and by the salt concentration of the solution. DNA rich in
G–C pairs, which have three hydrogen bonds, melts at a
higher temperature than that rich in A–T pairs, which
have two hydrogen bonds.

Renaturation of DNA
• Importantly, separated strands of DNA will renature or
reassociate when appropriate physiologic temperature
and salt conditions are achieved; this reannealing process
is often referred to as hybridization.
• The rate of reassociation depends upon the concentration
of the complementary strands.
• Reassociation of the two complementary DNA strands of a
chromosome after transcription is a physiologic example
of renaturation (see below).
• At a given temperature and salt concentration, a
particular nucleic acid strand will associate tightly only
with a complementary strand.

• Hybrid molecules will also form under appropriate
conditions. For example, DNA will form a hybrid with a
complementary DNA (cDNA) or with a cognate
messenger RNA (mRNA; see below).
• When combined with gel electrophoresis techniques
that separate nucleic acids by size coupled with
radioactive or fluorescent labelling to provide a
detectable signal, the resulting analytic techniques are
called Southern (DNA/DNA) and Northern (RNA-DNA)
blotting, respectively.
• These procedures allow for very distinct, high sensitivity
identification of specific nucleic acid species from complex
mixtures of DNA or RNA

• In some organisms such as bacteria, bacteriophages,
many DNA-containing animal viruses, as well as
organelles such as mitochondria (see Figure 35–8), the
ends of the DNA molecules are joined to create a closed
circle with no covalently free ends.
• This of course does not destroy the polarity of the
molecules, but it eliminates all free 3' and 5' hydroxyl
and phosphoryl groups.
• Closed circles exist in relaxed or supercoiled forms.
Supercoils are introduced when a closed circle is twisted
around its own axis or when a linear piece of duplex
DNA, whose ends are fixed, is twisted.

DNA PROVIDES A TEMPLATE FOR REPLICATION &
TRANSCRIPTION
• The genetic information stored in the nucleotide
sequence of DNA serves two purposes.
• It is the source of information for the synthesis of all
protein molecules of the cell and organism, and it
provides the information inherited by daughter cells or
offspring.
• Both of these functions require that the DNA molecule
serve as a template—in the first case for the transcription
of the information into RNA and in the second case for
the replication of the information into daughter DNA
molecules.

THE CHEMICAL NATURE OF RNA DIFFERS FROM THAT OF
DNA
• Ribonucleic acid (RNA) is a polymer of purine and
pyrimidine ribonucleotides linked together by 3',5'-
phosphodiester bonds analogous to those in DNA (Figure
34–6).
• Although sharing many features with DNA, RNA
possesses several specific differences:

1. In RNA, the sugar moiety to which the phosphates and
purine and pyrimidine bases are attached is ribose
rather than the 2'-deoxyribose of DNA.
2. The pyrimidine components of RNA differ from those of
DNA. Although RNA contains the ribonucleotides of
adenine, guanine, and cytosine, it does not possess thymine.
Instead of thymine, RNA contains the ribonucleotide of
uracil.
3. RNA typically exists as a single strand, whereas DNA exists
as a double-stranded helical molecule.

However, given the proper complementary base sequence
with opposite polarity, the single strand of RNA—as
demonstrated in Figure—is capable of folding back on itself
like a hairpin and thus acquiring double stranded
characteristics: G pairing with C, and A with U.
4. Since the RNA molecule is a single strand complementary
to only one of the two strands of a gene, its guanine content
does not necessarily equal its cytosine content, nor does its
adenine content necessarily equal its uracil content.

5. RNA can be hydrolyzed by alkali to 2',3' cyclic diesters of
the mononucleotides, compounds that cannot be formed
from alkali-treated DNA because of the absence of a 2'-
hydroxyl group.
The alkali lability of RNA is useful both diagnostically and
analytically.

Diagrammatic
representation of the
secondary structure of a
single-stranded RNA
molecule in which a stem
loop, or "hairpin,"
has been formed.
Formation of this structure
is dependent upon the
indicated intramolecular
base pairing (colored
horizontal lines between
bases). Note that A forms
hydrogen bonds with U in
RNA.

• Information within the single strand of RNA is contained
in its sequence ("primary structure") of purine and
pyrimidine nucleotides within the polymer.
• The sequence is complementary to the template strand
of the gene from which it was transcribed.
• Because of this complementarity, an RNA molecule can
bind specifically via the base-pairing rules to its template
DNA strand; it will not bind ("hybridize") with the other
(coding) strand of its gene.
• The sequence of the RNA molecule (except for U
replacing T) is the same as that of the coding strand of
the gene

The expression of genetic information in DNA into the
form of an mRNA transcript. This is subsequently
translated by ribosomes into a specific protein molecule.

The cap structure
attached to the 5'
terminal of most
eukaryotic messenger
RNA molecules. A 7-
methylguanosine
triphosphate (black) is
attached at the 5'
terminal of the mRNA
(shown in color),
which usually also
contains a 2'-O -
methylpurine
nucleotide. These
modifications (the
cap and methyl
group) are added
after the mRNA is
transcribed from
DNA.

• Eukaryotic mRNAs have unique chemical characteristics.
The 5' terminal of mRNA is "capped" by a 7-
methylguanosine triphosphate that is linked to an adjacent
2'-O -methyl ribonucleoside at its 5'-hydroxyl through
the three phosphates (Figure).
• The cap is involved in the recognition of mRNA by the
translation machinery, and also helps stabilize the mRNA
by preventing the attack of 5'-exonucleases.

• The protein synthesizing machinery begins translating
the mRNA into proteins beginning downstream of the 5'
or capped terminal.
• The other end of mRNA molecules, the 3'-hydroxyl
terminal, has an attached polymer of adenylate
residues 20–250 nucleotides in length.
• Both the mRNA "cap" and "poly(A) tail" are added post-
transcriptionally.
• mRNA represents 2–5% of total eukaryotic cellular RNA.

Messenger RNA
It accounts for 1-5% of cellular RNA.
Structure
1. Majority of mRNA has primary structure. They are single-
stranded linear molecules.
They consist of 1000-10,000 nucleotides (Figure 16.7a).
2. mRNA molecules have free or phosphorylated 3’ and 5’
end.
3. mRNA molecules have different life spans. Their life span
ranges from few minutes to days.
4. Eukaryotic mRNA are more stable than prokaryotic
mRNA.
5. The mRNA nucleotide sequence is complementary from
which it is synthesized or copied.

6. Some eukaryotic mRNA molecules are capped at 5’ end.
The cap is methylated GTP (mGTP). Some mRNA contain
internal methylated nucleotides. Capping protects mRNA
from nuclease attack.

7. At 3' end of most of eukaryotic mRNA, a polymer of
adenylate (poly A) is found as tail.
Poly A tail protects mRNA from nucleaes attack.
8. In prokaryotes 5' end of mRNA contains a sequence rich in
A and G. Such sequence is known as Shine-Dalgarno
sequence. It helps attachment of mRNA with ribosome during
protein synthesis.
9. Some prokaryotic mRNA has secondary structure.
Intrastrand base paring among complementary
bases allows folding of liner molecule. As a result hairpin, or
loop like secondary structure is formed. (Figure).

Functions
1. mRNA is direct carrier of genetic information from the
nucleus to the cytoplasm.
2. Usually a molecule of mRNA contains information
required for the formation of one protein molecule.
3. Genetic information is present in mRNA in the form of
genetic code.
4. Some times single mRNA may contain information for the
formation of more than one protein.

Transfer RNA
t-RNA accounts for 10-15% of total cell RNA.
Structure
• They are the smallest of all the RNAs. Usually they consist
of 50-100 nucleotides.
• They are single strand molecules. t-RNA molecules contain
many unusual bases 7-15 per molecule.
• They are methylated adenine, guanine, cytosine and
thymine, dihydrouracil, pseudo uridine, isopentenyl
adenine etc.
• These unusual bases are important for binding of t-RNA to
ribosomes and interaction of t-RNA with aminoacyl-t-RNA
synthetases.

• About half of the nucleotides in t-RNA are involved in
intrachain base pairing. As a result, double helical
segments are formed in t-RNA.
• Further some bases are not involved in the base pairing
resulting in loops and arms formation in t-RNA.
• Thus, folding in primary structure generate secondary
structure.
Though t-RNAs differ in chain lengths they have some
common features with regard to secondary structure.

Secondary structure of t-RNA
Secondary structure of all the t-RNAs is in the form of clover
leaf (Figure). The important features of clover-leaf structure
are

1. An amino acid arm where amino acid is attached to 3'-OH
of adenosine moiety of t-RNA. ACC is the common base
sequence at this 3'-end.
2. Tϕc arm, which contains sequence of ribothymidine-
pseudouridine-cytidine. Greek alphabet ϕ (Psi) stands for
pseudo uridine. Thymine and pseudouracil are the two
unusual bases found in this arm.
3. An anti-codon arm, which recognizes codon on mRNA.
4. DHU arm, which contains many dihydrouridine (UH2)
residues.
5. The 5' end of t-RNA is phosphorylated and residue is
guanosine.
6. About 75% t-RNA molecules have extra arm. It consist of 3-
5 base pairs. It is found between TϕC and anti-codon arm.

Tertiary structure of t-RNA
• X-ray diffraction analysis indicated complex three-
dimentional structure for t-RNA molecule.
• Three-dimentional structure of t-RNA looks like inverted
or tilted L.
• The anti-codon arm is at the tip of the vertical arm of
tilted L. The acceptor arm is at the tip of horizontal arm
of tilted L. The D loop and TϕC loop are pushed into corner of
tilted L (Figure 16.8b).

Functions
1. It is the carrier of amino acids to the site of protein
synthesis.
2. There is at least one t-RNA molecule to each of 20 amino
acids required for protein synthesis.
3. Eukaryotic t-RNAs are less stable where as prokaryotic
RNAs are more stable.

Ribosomal RNA
• Ribosomal RNA or r-RNA accounts for 80% of total
cellular RNA.
• It is present in ribosomes.
• In ribosomes, r-RNA is found in combination with
protein. It is known as ribonucleoprotein.
• The length of r-RNA ranges form 100-600 nucleotides.
Both prokaryotic and eukaryotic ribosomes contain r-
RNA molecules.
• r-RNAs differ in sedimentation coefficients (S).
• There are four types of r-RNAs in eukaryotes. They are 5,
5.8, 18 and 28S r-RNA molecules.
• Prokaryotes contains 3 types of r-RNA molecules. They
are 5, 16 and 23S r-RNA molecules.

Structure
• r-RNA molecules have secondary structure. Intra strand
base pairing between complementary base generates
double helical segments or loops.
• They are known as domains. 16S r- RNA with 1500
nucleotides has four major domains (Figure 16.8c).
• The three-dimentional tertiary structure of r-RNA is highly
complex.

Functions
1. r-RNAs are required for the formation of ribosomes.
2. 16S RNA is involved in initiation of protein synthesis.

hnRNA OR Precursor mRNA (pre-mRNA) is an immature
single strand of messenger ribonucleic acid (mRNA).
• Pre-mRNA is synthesized from a DNA template in
the cell nucleus by transcription.
• Pre-mRNA comprises the bulk of heterogeneous
nuclear RNA (hnRNA).
• The term hnRNA is often used as a synonym for pre-
mRNA, although, in the strict sense, hnRNA may include
nuclear RNA transcripts that do not end up as
cytoplasmic mRNA.
Once pre-mRNA has been completely processed, it is
termed "mature messenger RNA", or simply "messenger
RNA".

• Pre-mRNA is the first form of RNA created through
transcription in protein synthesis.
• The pre-mRNA lacks structures that the messenger RNA
(mRNA) requires.
• First all introns have to be removed from the transcribed
RNA through a process known as splicing.
• Before the RNA is ready for export, a Poly(A)tail is added
to the 3' end of the RNA and a 5' cap is added to the 5'
end.

siRNA :
• Small (or short) interfering RNA (siRNA) is the most
commonly used RNA interference (RNAi) tool for inducing
short-term silencing of protein coding genes.
• siRNA is a synthetic RNA duplex designed to specifically
target a particular mRNA for degradation.
• Small interfering RNA (siRNA), sometimes known
as short interfering RNA or silencing RNA, is a class
of double-stranded RNA molecules, 20-25 base pairs in
length, similar to miRNA, and operating within the RNA
interference (RNAi) pathway.
• It interferes with the expression of specific genes with
complementary nucleotide sequences by degrading
mRNA after transcription, preventing translation.

Molecular hybridization
• Molecular hybridization in molecular biology, formation of a p
artially or wholly complementary nucleic acid duplex by
association of single strands,
• usually between DNA and RNA strands
• or previously unassociated DNA strands, but also
between RNA strands; used to detect and isolate specific sequ
ences, measure homology, or define other characteristics
of one or both strands.

NUCLEIC ACID HYBRIDIZATION:
If DNA from two different species are mixed, denatured and
allowed to cool slowly so that reannealing can occur, artificial
hybrid duplexes (hetero duplex) may form, provided the DNA
from one species is similar in nucleotide sequence to the DNA of
the other. This phenomenon is referred as nucleic acid
hybridization.
• Since DNA involved in hybridization this is otherwise known as
DNA hybridization.
• Hybridization can occur between DNA and DNA, DNA and
RNA, or RNA and RNA and may be intra molecular or

• Hybridization can occur between nucleic acids in solution or
where one is in solution and the other immobilized either on
a solid support or fixed in situ in cell.
• Hybridization depends upon intrinsic factors and extrinsic
factors.
• Intrinsic factors include number of hydrogen bonds, the
length of duplex, its GC content and the degree of mismatch.
• Extrinsic factors include temperature and chemical
environment.

Cot analysis
• C0t analysis, a technique based on the principles of DNA
reassociation kinetics, is a biochemical technique that
measures how much repetitive DNA is in a DNA sample such as a
genome.
• It is used to study genome structure and
organization and has also been used to simplify the sequencing of
genomes that contain large amounts of repetitive sequence.
• The procedure involves heating a sample of genomic DNA
until it denatures into the single stranded-form, and then
slowly cooling it, so the strands can pair back together.

• While the sample is cooling, measurements are taken of how
much of the DNA is base paired at each temperature.
Analysis
• Since a sequence of single-stranded DNA needs to find its
complementary strand to reform a double helix, common
sequences renature more rapidly than rare sequences.
• Indeed, the rate at which a sequence will reassociate is
proportional to the number of copies of that sequence in the
DNA sample.
• A sample with a highly-repetitive sequence will renature
rapidly, while complex sequences will renature slowly.

• The amount of renaturation is measured relative to a C0t
value.
• The C0t value is the product of C0 (the initial
• concentration of DNA), t (time in seconds), and a constant
that depends on the concentration of cations in the buffer.
• Repetitive DNA will renature at low C0t values, while
complex and unique DNA sequences will renature at high C0t
values.
• The fast renaturation of the repetitive DNA is because of the
availability of numerous complementary sequences.

Repetitive
DNA
sequences
renature at
lower C0t
values than
single-copy
sequences.

Hyperchromicity
• Hyperchromicity is the increase of absorbance (optical
density) of a material.
• The most famous example is the hyperchromicity of
DNA that occurs when the DNA duplex is denatured. The
UV absorption is increased when the two single DNA
strands are being separated, either by heat or by
addition of denaturant or by increasing the pH level.
• The opposite, a decrease of absorbance is called
hypochromicity.

• Heat denaturation of DNA, also called melting, causes the
double helix structure to unwind to form single stranded DNA.
• When DNA in solution is heated above its melting
temperature (usually more than 80 °C), the double-stranded
DNA unwinds to form single-stranded DNA.
• The bases become unstacked and can thus absorb more light.
In their native state, the bases of DNA absorb light in the 260-
nm wavelength region.
• When the bases become unstacked, the wavelength of
maximum absorbance does not change, but the amount
absorbed increases by 37%.

• Hyperchromicity can be used to track the condition of DNA as
temperature changes.
• The transition/melting temperature (Tm) is the temperature
where the absorbance of UV light is 50% between the
maximum and minimum, i.e. where 50% of the DNA is
denatured.
• The hyperchromic effect is the striking increase in absorbance
of DNA upon denaturation. The two strands of DNA are bound
together mainly by the stacking interactions, hydrogen bonds
and hydrophobic effect between the complementary bases.

• A double strand DNA dissociating to single strands
produces a sharp cooperative transition.
• When the DNA double helix is treated with denatured
agents, the interaction force holding the double
helical structure is disrupted.
• The double helix then separates into two single strands
which are in the random coiled conformation.
• At this time, the base-base interaction will be reduced,
increasing the UV absorbance of DNA solution because
many bases are in free form and do not form hydrogen

• As a result, the absorbance for single-stranded DNA will be
37% higher than that for double stranded DNA at the same
concentration.

DENSITY:
During denaturation, density of DNA solution
increases single stranded DNA denser than double helical DNA.
VISCOSITY:
The solution of native DNA possesses a high viscosity
because of the relatively rigid double helical structure and long,
rod like character of DNA. Disruption of the hydrogen bonds
causes a decreased in viscosity.

BUOYANT DENSITY OF DNA:
• G: C rich DNA has significantly higher density than A: T rich
DNA. Furthermore a linear relationship exists between the
buoyant densities of DNA from different sources and their
GC content.
• The density of DNA, as a function of its G:C content is given
by the equation rho=1.660+0.098(GC) where GC= is the mole
fraction of G+C in the DNA.
• Because of its relatively high density, DNA can be purified
from cellular material by a form of density gradient
centrifugation known as isopycnic centrifugation.

Density gradient centrifugation
Density gradient centrifugation can be used to isolate DNA.
The densities of DNAs are about the same as concentrated
solutions of cesium chloride, CsCl (1.6 to 1.8 g/mL).
Centrifugation of CsCl solutions at very high rotational speeds,
where the centrifugal force becomes 105 times stronger than the
force of gravity, causes the formation of a density gradient within
the solution.

Isopycnic centrifugation.
• This gradient is the result of a balance that is established
between the sedimentation of the salt ions toward the bottom
of the tube and their diffusion upward toward regions of lower
concentration.
If DNA is present in the centrifuged CsCl solution, it moves to a
position of equilibrium in the gradient equivalent to its buoyant
density.
Caesium chloride is used because at a concentration of 1.6 to
1.8 g/mL it is similar to the density of DNA. For this reason, this
technique is also called Isopycnic centrifugation.

The net movement of solute particles in an ultracentrifuge is the
result of two processes: diffusion (from regions of higher
concentration to regions of lower concentration) and
sedimentation due to centrifugal force (in the direction away from
the axis of rotation).
• In general, diffusion rates for molecules are inversely
proportional to their molecular weight
— larger molecules diffuse more slowly than smaller ones.
On the other hand, sedimentation rates increase with increasing
molecular weight.

• A macromolecular species that has reached its position of
equilibrium in isopycnic centrifugation has formed a
concentrated band of material.
• Cesium chloride centrifugation is an excellent means of
removing RNA and proteins in the purification of DNA.
• The density of DNA is typically slightly greater than1.7 g/cm3
• (1.70+ 0.01), while the density of RNA is more than 1.8 g/cm3
• Proteins have densities less than 1.3 g/cm3
• In CsCl solutions of appropriate density, the DNA bands near
the centre of the tube, RNA pellets to the bottom, and the
proteins float near the top.

Nucleic acids.pdf

Recommended

Recommended

More Related Content

Similar to Nucleic acids.pdf

Similar to Nucleic acids.pdf (20)

More from shinycthomas

More from shinycthomas (20)

Recently uploaded

Recently uploaded (20)

Nucleic acids.pdf