UNIT-4

NUCLEIC ACIDS

There are two types of nucleic acids, namely deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). Primarily, nucleic acids serve as repositories and
transmitters of genetic information.

Functions of nucleic acids

DNA is the chemical basis of heredity and may be regarded as the reserve bank of
genetic information. DNA is exclusively responsible for maintaining the identity of
different species of organisms over millions of years. Further, every aspect of cellular
function is under the control of DNA. The DNA is organized into genes, the
fundamental units of genetic information. The genes control the protein synthesis
through the
mediation of RNA, The interrelationship of these three classes of biomolecules (DNA,
RNA and proteins) constitutes the central dogma of molecular biology or more
commonly the central dogma of life.

Components of nucleic acids

Nucleic acids are the polymers of nucleotides (polynucleotides) held by 3􀁣 and 5􀁣

phosphate bridges. In other words, nucleic acids are built up by the monomeric units
—nucleotides (It may be recalled that protein is a polymer of amino acids).

Nucleotides:

Nucleotides are composed of a nitrogenous base, a pentose sugar and a phosphate.

Nucleotides perform a wide variety of functions in the living cells, besides being the
building blocks or monomeric units in the nucleic acid (DNA and RNA) structure.
These include their role as structural components of some coenzymes of B-complex
vitamins (e.g. FAD, NAD+), in the energy reactions of cells (ATP is the energy
currency), and in the control of metabolic reactions

STRUCTURE OF NUCLEOTIDES

As already stated, the nucleotide essentially consists of nucleobase, sugar and

phosphate. The term nucleoside refers to base + sugar. Thus, nucleotide is nucleoside
+ phosphate

Purines and pyrimidines

The nitrogenous bases found in nucleotides (and, therefore, nucleic acids) are
aromatic heterocyclic compounds. The bases are of two types—purines and
pyrimidine. Purines are numbered in the anticlockwise direction while pyrimidines are
numbered in the clockwise direction. And this is an internationally accepted system to
represent the structure of bases.

Major bases in nucleic acids

The structures of major purines and pyrimidines found in nucleic acids .DNA and
RNA contain the same purines namely adenine (A) and guanine (G). Further, the
pyrimidine cytosine (C) is found in both DNA
and RNA. However, the nucleic acids differ with respect to the second pyrimidine
base. DNA contains thymine (T) whereas RNA contains uracil (U). As is observed
in the thymine and uracil differ in structure by the presence (in T) or absence (in U) of
a methyl group.

The purine—guanine and pyrimidinescytosine, thymine and uracil exhibit

tautomerism. The lactam and lactim forms of [Link] physiological pH, the lactam
(keto) tautomeric forms are predominantly present.

Minor bases found in nucleic acids : Besides the bases described above, several
minor and unusual bases are often found in DNA and RNA. These include 5-
methylcytosine, N4-acetylcytosine, N6-methyladenine, N6, N6-dimethyladenine,
pseudouracil etc. It is believed that the unusual bases in nucleic acids will help in the
recognition of specific enzymes.

Other biologically important bases : The bases such as hypoxanthine, xanthine

and uric acid are present in the free state in the cells. The former two are the
intermediates in purine synthesis while uric acid is the end product of purine
degradation.

Purine bases of plants : Plants contain certain methylated purines which are of
pharmacological interest. These include caffeine (of coffee), theophylline (of tea) and
theobromine (of cocoa).

Sugars of nucleic acids

The five carbon monosaccharides (pentoses) are found in the nucleic acid structure.
RNA contains D-ribose while DNA contains D-deoxyribose. Ribose and deoxyribose
differ in structure at C2. Deoxyribose has one oxygen less at C2 compared to ribose.

Nomenclature of nucleotides
The addition of a pentose sugar to base produces a nucleoside. If the sugar is ribose,
ribonucleosides are formed. Adenosine, guanosine, cytidine and uridine are the
ribonucleosides of A, G, C and U respectively. If the sugar is a deoxyribose,
deoxyribonucleosides are produced. The term mononucleotide is used when a single
phosphate moiety is added to a nucleoside. Thus adenosine monophosphate (AMP)
contains adenine + ribose + phosphate. The principal bases, their respective
nucleosides and nucleotides found in the structure of nucleic acids. Note that the
prefix ‘d’ is used to indicate if the sugar is deoxyribose (e.g. dAMP).

The binding of nucleotide components

The atoms in the purine ring are numbered as 1 to 9 and for pyrimidine as 1 to 6 The
carbons of sugars are represented with an associated prime (􀁣) for differentiation.
Thus the pentose carbons are 1􀁣 to 5􀁣. The pentoses are bound to nitrogenous bases
by 􀁅-N-glycosidic bonds. The N9 of a purine ring binds with C1(1􀁣) of a pentose
sugar to form a covalent bond in the purine nucleoside. In case of pyrimidine
nucleosides, the glycosidic linkage is between N1 of a pyrimidine and C􀁣1 of a
pentose. The hydroxyl groups of adenosine are esterified with phosphates to produce
5􀁣- or 3􀁣-monophosphates. 5􀁣-Hydroxyl is the most commonly esterified, hence 5􀁣
is usually omitted while writing nucleotide names. Thus AMP represents adenosine
5􀁣-monophosphate. However, for adenosine 3􀁣-monophosphate, the abbreviation
3􀁣-AMP is used. The structures of two selected nucleotides namely AMP and TMP
are depicted.

Nucleoside di- and triphosphates

Nucleoside monophosphates possess only one phosphate moiety (AMP, TMP). The
addition of second or third phosphates to the nucleoside results in nucleoside
diphosphate (e.g. ADP) or triphosphate (e.g. ATP), respectively.

STRUCTURE OF DNA
DNA is a polymer of deoxyribonucleotides (or simply deoxynucleotides). It is
composed of monomeric units namely deoxyadenylate (dAMP), deoxyguanylate
(dGMP), deoxycytidylate (dCMP) and deoxythymidylate (dTMP) (It may be noted
here that some authors prefer to use TMP for deoxythymidylate, since it is found only
in DNA). The details of the nucleotide structure are given above.

Schematic representation of polynucleotides

The monomeric deoxynucleotides in DNA are held together by 3􀁣,5􀁣-

phosphodiester bridges. DNA (or RNA) structure is often represented in a short-
hand form. The horizontal line indicates the carbon chain of sugar with base attached
to C1􀁣. Near the middle of the horizontal line is C3􀁣 phosphate linkage while at
the other end of the line is C5􀁣 phosphate linkage

Chargaff’s rule of DNA composition

Erwin Chargaff in late 1940s quantitatively analysed the DNA hydrolysates from
different species. He observed that in all the species he studied, DNA had equal
numbers of adenine and thymine residues (A = T) and equal numbers of guanine and
cytosine residues (G = C). This is known as Chargaff’s rule of molar equivalence
between the purines and pyrimidines in DNA structure. The significance of
Chargaff’s rule was not immediately realised. The double helical structure of DNA
derives its strength from Chargaff’s rule.

Single-stranded DNA, and RNAs which are usually single-stranded, do not obey
Chargaff’s rule. However, double-stranded RNA which is the genetic material in
certain viruses satisfies Chargaff’s rule.

DNA DOUBLE HELIX

The double helical structure of DNA was proposed by James Watson and Francis
Crick in 1953 (Nobel Prize, 1962). The elucidation of DNA structure is considered as
a milestone in the era of modern biology. The structure of DNA double helix is
comparable to a twisted ladder. The salient features of Watson-Crick
model of DNA (now known as B-DNA) are described next (Fig.5.9).

1. The DNA is a right handed double helix. It consists of two

polydeoxyribonucleotide chains (strands) twisted around each other on a common
axis.
2. The two strands are antiparallel, i.e., one strand runs in the 5􀁣 to 3􀁣 direction
while the other in 3􀁣 to 5􀁣 direction. This is comparable to two parallel adjacent
roads carrying traffic in opposite direction.
3. The width (or diameter) of a double helix is 20 A° (2 nm).
4. Each turn (pitch) of the helix is 34 A° (3.4 nm) with 10 pairs of nucleotides, each
pair placed at a distance of about 3.4 A°.

5. Each strand of DNA has a hydrophilic deoxyribose phosphate backbone (3􀁣-5􀁣

phosphodiester bonds) on the outside (periphery) of the molecule while the
hydrophobic bases are stacked inside (core).
6. The two polynucleotide chains are not identical but complementary to each other
due to base pairing.
7. The two strands are held together by hydrogen bonds formed by complementary
base pairs (Fig.5.10). The A-T pair has 2 hydrogen bonds while G-C pair has 3
hydrogen bonds. The G 􀁻 C is stronger by about 50% than A = T.
8. The hydrogen bonds are formed between a purine and a pyrimidine only. If two
purines face each other, they would not fit into the allowable space. And two
pyrimidines would be too far to form hydrogen bonds. The only base arrangement
possible in DNA structure, from spatial considerations is A-T, T-A, G-C and
C-G.
9. The complementary base pairing in DNA helix proves Chargaff’s rule. The
content of adenine equals to that of thymine (A = T) and guanine equals to that of
cytosine (G = C).
10. The genetic information resides on one of the two strands known as template
strand or sense strand. The opposite strand is antisense strand. The double helix has
(wide) major grooves and (narrow) minor grooves along the phosphodiester backbone.
Proteins interact with DNA at these grooves, without disrupting the base pairs and
double helix.

Conformations of DNA double helix

Variation in the conformation of the nucleotides of DNA is associated with

conformational variants of DNA. The double helical structure of DNA exists in at
least 6 different forms-A to E and Z. Among these, B, A
and Z forms are important. The B-form of DNA double helix, described by Watson
and Crick (discussed above), is the most predominant form under physiological
conditions. Each turn of the B-form has 10 base pairs spanning a distance of 3.4 nm.
The width of the double helix is 2 nm. The A-form is also a right-handed helix. It
contains 11 base pairs per turn. There is a tilting of the base pairs by 20° away from
the central axis. The Z-form (Z-DNA) is a left-handed helix and contains 12 base pairs
per turn.

OTHER TYPES OF DNA STRUCTURE

It is now recognized that besides double helical structure, DNA also exists in certain
unusual structures. It is believed that such structures are important for molecular
recognition of DNA by proteins and enzymes.
This is in fact needed for the DNA to discharge its functions in an appropriate manner.
Some selected unusual structures of DNA are briefly described.
Bent DNA
In general, adenine base containing DNA tracts are rigid and straight. Bent
conformation of DNA occurs when A-tracts are replaced by other bases or a collapse
of the helix into the minor groove of A-tract. Bending in DNA structure has also been
reported due to photochemical damage or mispairing of bases.
Certain antitumor drugs (e.g. cisplatin) produce bent structure in DNA. Such changed
structure can take up proteins that damage the DNA.

Triple-stranded DNA
Triple-stranded DNA formation may occur due to additional hydrogen bonds between
the bases. Thus, a thymine can selectively form two Hoogsteen hydrogen bonds to
the adenine of A-T pair to form T-A-T. Likewise, a protonated cytosine can also form
two hydrogen bonds with guanine of G–C pairs that results in C–G–C. An outline of
Hoogsteen triple helix is depicted in Fig.5.11. Triple-helical structure is less stable
than double helix. This is due to the fact that the three negatively charged backbone
strands in triple helix results in an increased electrostatic repulsion.

Four-stranded DNA
Polynucleotides with very high contents of guanine can form a novel tetrameric
structure called G-quartets. These structures are planar and are connected by
Hoogsteen hydrogen bonds (Fig.5.12A). Antiparallel four-stranded DNA structures,
referred to as G-tetraplexes have also been reported (Fig.5.12B). The ends of
eukaryotic chromosomes namely telomeres are rich in guanine, and therefore form G-
tetraplexes. In recent years, telomeres have become the targets for anticancer
chemotherapies.

G-tetraplexes have been implicated in the recombination of immunoglobulin genes,

and in dimerization of double-stranded genomic RNA of the human
immunodeficiency virus (HIV).

THE SIZE OF DNA MOLECULE —UNITS OF LENGTH

DNA molecules are huge in size. On an average, a pair of B-DNA with a thickness of
0.34 nm has a molecular weight of 660 daltons. For the measurement of lengths, DNA
doublestranded structure is considered, and expressed in the form of base pairs (bp).
A kilobase pair (kb) is 103 bp, and a megabase pair (Mb) is 106 bp and a gigabase
pair (Gb) is 109 bp. The kb, Mb and Gb relations may be summarized as follows :
1 kb = 1000 bp
1 Mb = 1000 kb = 1,000,000 bp
1 Gb = 1000 Mb = 1,000,000,000 bp
It may be noted here that the lengths of RNA molecules (like DNA molecules) cannot
be expressed in bp, since most of the RNAs are single-stranded. The length of DNA
varies from species to species, and is usually expressed in terms of base pair
composition and contour length. Contour length represents the total length of the
genomic DNA in a cell. Some examples of organisms with bp and contour lengths are
listed. l 􀁏 phage virus — 4.8 􀁵 104 bp — contour length 16.5 􀁐m. l E. coli — 4.6 􀁵
106 bp — contour length
1.5 􀁐m. l Diploid human cell (46 chromosomes) — 6.0 􀁵 109 bp — contour length 2
meters. It may be noted that the genomic DNA size is usually much larger the size of
the cell or nucleus containing it. For instance, in humans, a 2-meter long DNA is
packed compactly in a nucleus of about 10􀁐m diameter. The genomic DNA may exist
in linear or circular forms. Most DNAs in bacteria exist as closed circles. This
includes the DNA of bacterial chromosomes and the extrachromosomal DNA of
plasmids. Mitochondria and chloroplasts of eukaryotic cells also contain circular
DNA. Chromosomal DNAs in higher organisms are mostly linear. Individual human
chromosomes contain a single DNA molecule with variable sizes compactly packed.
Thus the smallest chromosome contains 34 Mb while the largest one has 263 Mb.

STRUCTURE OF RNA

RNA is a polymer of ribonucleotides held together by 3􀁣,5􀁣-phosphodiester

bridges. Although RNA has certain similarities with DNA structure, they have
specific differences

1. Pentose : The sugar in RNA is ribose in contrast to deoxyribose in DNA.

2. Pyrimidine : RNA contains the pyrimidine uracil in place of thymine (in DNA).
3. Single strand : RNA is usually a singlestranded polynucleotide. However, this
strand may fold at certain places to give a doublestranded structure, if complementary
base pairs are in close proximity.
4. Chargaff’s rule—not obeyed : Due to the single-stranded nature, there is no
specific relation between purine and pyrimidine contents. Thus the guanine content is
not equal to cytosine (as is the case in DNA).
5. Susceptibility to alkali hydrolysis : Alkali can hydrolyse RNA to 2􀁣,3􀁣-cyclic
diesters. This is possible due to the presence of a hydroxyl group at 2􀁣 position. DNA
cannot be subjected to alkali hydrolysis due to lack of this group.
6. Orcinol colour reaction : RNAs can be histologically identified by orcinol colour
reaction due to the presence of ribose.
TYPES OF RNA
The three major types of RNAs with their respective cellular composition are given
below
1. Messenger RNA (mRNA) : 5–10%
2. Transfer RNA (tRNA) : 10–20%
3. Ribosomal RNA (rRNA) : 50–80%

Besides the three RNAs referred above, other RNAs are also present in the cells.
These include heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA),
small nucleolar RNA (snoRNA) and small cytoplasmic RNA (scRNA).
The RNAs are synthesized from DNA, and are primarily involved in the process of
protein biosynthesis. The RNAs vary in their structure and function. A brief
description on the major RNAs is given.

Messenger RNA (mRNA)

The mRNA is synthesized in the nucleus (in eukaryotes) as heterogeneous nuclear

RNA (hnRNA). hnRNA, on processing, liberates the functional mRNA which enters
the cytoplasm to articipate in protein synthesis. mRNA has high molecular weight
with a short half-life. In general, mRNA of eukaryotes is more stable with longer half-
life, compared to prokaryotic mRNA. The eukaryotic mRNA is capped at the 5􀁣-
terminal end by 7-methylguanosine triphosphate. It is believed that this cap helps to
prevent the hydrolysis of mRNA by 5􀁣-exonucleases. Further, the cap may be also
involved in the recognition of mRNA for protein synthesis. The 3􀁣-terminal end of
mRNA contains a polymer of adenylate residues (20-250 nucleotides) which is known
as poly (A) tail. This tail may provide stability to mRNA, besides preventing it from
the attack of 3􀁣-exonucleases. mRNA molecules often contain certain modified bases
such as 6-methyladenylates in the internal structure.

Transfer RNA (tRNA)

Transfer RNA (soluble RNA) molecule contains 71-80 nucleotides (mostly 75) with a
molecular weight of about 25,000. There are at least 20 species of tRNAs,
corresponding to 20 amino acids present in protein structure. The structure of tRNA
(for alanine) was first elucidated by Holley. The structure of tRNA, depicted in
Fig.5.16, resembles that of a clover leaf. tRNA contains mainly four arms, each arm
with a base paired
stem.
1. The acceptor arm : This arm is capped with a sequence CCA (5􀁣 to 3􀁣). The
amino acid is attached to the acceptor arm.
2. The anticodon arm : This arm, with the three specific nucleotide bases
(anticodon), is responsible for the recognition of triplet codon of mRNA. The codon
and anticodon are complementary to each other.
3. The D arm : It is so named due to the presence of dihydrouridine.

4. The T􀀼C arm : This arm contains a sequence of T, pseudouridine (represented by

psi, 􀀼) and C.
5. The variable arm : This arm is the most variable in tRNA. Based on this
variability, tRNAs are classified into 2 categories :
(a) Class I tRNAs : The most predominant (about 75%) form with 3-5 base pairs
length.
(b) Class II tRNAs : They contain 13-20 base pair long arm.

Base pairs in tRNA : The structure of tRNA is maintained due to the complementary
base pairing in the arms. The four arms with their respective base pairs are given
below
The acceptor arm – 7 bp
The T􀀼C arm – 5 bp
The anticodon arm – 5 bp
The D arm – 4 bp

Ribosomal RNA (rRNA)

The ribosomes are the factories of protein synthesis. The eukaryotic ribosomes are
composed of two major nucleoprotein complexes–60S subunit and 40S subunit. The
60S subunit contains 28S rRNA, 5S rRNA and
5.8S rRNA while the 40S subunit contains 18S rRNA. The function of rRNAs in
ribosomes is not clearly known. It is believed that they play a significant role in the
binding of mRNA to ribosomes and protein synthesis.

Other RNAs

The various other RNAs and their functions are

CATALYTIC RNAs—RIBOZYMES

In certain instances, the RNA component of a ribonucleoprotein (RNA in association

with protein) is catalytically active. Such RNAs are termed as ribozymes. A selected
list of ribozymes along with their biochemical functions . Ribonuclease P (RNase P)
is a ribozyme containing protein and RNA component. It cleaves tRNA precursors to
generate mature tRNA molecules. RNA molecules are known to adapt tertiary
structure just like proteins (i.e. enzymes). The specific conformation of RNA may be
responsible for its function as biocatalyst. It is believed that ribozymes (RNAs) were
functioning as catalysts before the occurrence of protein enzymes, during the
course of evolution.

Recombinant ribozymes (rribozymes)

It is now possible to design recombinant ribozymes that will cleave any RNA. These
ribozymes are now being considered as therapeutic agents to cure diseases.
Theoretically it is possible to selectively degrade faulty RNAs (mutated or
inappropriately expressed RNAs in diseases) by rribozymes. This way specific RNAs
can be eliminated from the cell that will help to inhibit the disease process.