Nucleotides and Nucleic

Acids
2
 Biomedical Importance of
Nucleotides
1. Precursors of nucleic acids:
Without them, DNA or RNA cannot
be produced

2. Protein synthesis: Proteins cannot

be synthesized or cells cannot
proliferate

3
 Biomedical Importance of
Nucleotides
1. Energy currency: Nucleotides play an
important role as "energy currency" in the cell
– Nucleoside tri- and diphosphates such as
ATP and ADP are the principal donors and
acceptors of phosphoryl group in
metabolism
– By doing this, they play a key role in the
energy transductions

4
 Biomedical Importance
4. Carriers of intermediates: Nucleotides also serve
as carriers of activated intermediates in the synthesis
of some carbohydrates, lipids, and proteins
– The sugar derivatives UDP-glucose and
UDP-galactose participate in sugar
interconversions and in the biosynthesis of starch
and glycogen
– Similarly, nucleoside-lipid derivatives such as
CDP-acylglycerol are intermediates in lipid
biosynthesis
5
 Biomedical Importance
5. Co-enzymes: When linked to vitamins
nucleotides are structural components of several
essential coenzymes, for example, coenzyme A,
FAD, NAD+ and NADP+
6. Nucleotides are used for
– Muscle contraction,
– Transmission of nerve impulse,
– Transports of nutrients across cell membrane
– Motility of spermatozoa

6
 Biomedical Importance
7. Regulatory compounds: Nucleotides are important
regulatory compounds for many of the pathways of
intermediary metabolism, inhibiting or activating key
enzymes
• Roles that nucleotides perform in metabolic
regulation include
– ATP-dependent phosphorylation of key
metabolic enzymes
– Allosteric regulation of enzymes by ATP, AMP,
and CTP
– Control of the rate of oxidative phosphorylation by
ADP 7
 Biomedical Importance
8. Sulfate group donor: Adenosine
3'-phosphate-5'-phosphosulfate (PAPS) is the sulfate
donor for
– sulfated proteoglycans
– sulfate conjugates of drugs
10. Methyl group donor: S-adenosylmethionine (SAM)
is a methyl group donor

e.g Nor-adrenaline → Adrenaline by methylation.

8
 Biomedical Importance
10. Second messengers: Nucleotides, such
as cyclic AMP (cAMP) and cyclic GMP
(cGMP), serve as second messengers in
signal transduction pathways

11. Signal Transduction: GTP and GDP

play key roles in the cascade of events
that characterize signal transduction
pathways
9
 Biomedical Importance
[Link] applications: Specifically medical
applications include the use of synthetic
purine and pyrimidine analogs that
contain halogens, thiols, or additional
nitrogen atoms in
– The chemotherapy of cancer and AIDS
– As suppressors of the immune response
during organ transplantation
10
 Nucleotides
• Nucleotides are essential for all cells

• Nucleotides are the building blocks of nucleic

acids

• Without them, neither DNA nor RNA can be

produced and therefore proteins cannot be
synthesized and cells cannot proliferate

11
 Nucleotides
• They have the ability to store and
transmit genetic information from
one generation to the next which is a
fundamental condition for life

• They have multiple additional

functions

12
 Nucleotide Structure

• Nucleotides are
composed of
– A nitrogenous base
(purine or pyrimidine)

– A pentose
monosaccharide

– One, two, or three

phosphate groups
13
 Nucleosides
• Nucleosides are derivatives of
– bases (purines and pyimidines) and
– a sugar linked to a ring nitrogen of the base
• If the sugar is
– D-ribose, a ribonucleoside is produced
– 2-deoxy D- ribose, a deoxyribonucleoside is
produced

14
 Nucleosides
• Both sugars are linked to the heterocycle by a -
β- N-glycosidic bond, almost always to the
– N-1 of a pyrimidine or
– N-9 of a purine

15
• The N-glycosyl bond is formed by removal of
the elements of water (a hydroxyl group from
the pentose and hydrogen from the base)

16
 Nucleosides
• The ribonucleosides of A, G, C, and U are
named adenosine, guanosine, cytidine, and
uridine, respectively
• The deoxyribonucleoside of A, G, C, and T
have the added prefix, "deoxy-", for example
deoxyadenosine
• The compound deoxythymidine is often
simply called thymidine, with the "deoxy"
prefix being understood
17
18
 Nitrogenous bases (purine or
pyrimidine)
• The
nitrogen-conta
ining bases
belong to two
families of
compounds:

– Purines

– Pyrimidines
19
 Purines
• Both DNA and
RNA contain
the same
purine bases:

– Adenine (A)

– Guanine (G)

20
 Purines
• Adenine is 6-aminopurine
• Guanine is 2-amino,6-oxypurine
• Hypoxanthine and xanthine are also purines

21
 Pyrimidines
• Pyrimidines include:
• Thymine (T)—2,4-dioxy-5-methyl-pyrimidine
• Cytosine (C)—2-oxy-4-amino-pyrimidine
• Uracil (U) —2,4-dioxy-pyrimidine

22
 Pyrimidines
• Both DNA and RNA contain the pyrimidine
cytosine (C)
• They differ in their second pyrimidine base:
– DNA contains thymine (T)
– RNA contains uracil (U)
• T and U differ by only one methyl group,
which is present on T but absent on U

23
 Pentose Sugar
• Pentose sugar for
ribonucleotides is
D- ribose

• The sugar in
deoxyribo-
nucleotides is
2-deoxy-D-ribose
24
 Numbering of Carbon and Nitrogen
Atoms
• The carbon and nitrogen atoms in the
rings of the base and the sugar are
numbered separately

25
 Numbering of Carbon and Nitrogen
Atoms
• The atoms in the
rings of the bases are
numbered
– 1 to 6 in
pyrimidines and
– 1 to 9 in purines,

• whereas the carbons

in the pentose are
numbered 1' to 5'

26
 Numbering of Carbon and Nitrogen
Atoms
• Numerals with a prime (e.g., 2' or 3')
distinguish atoms of the sugar from those
of the hetero cycle

27
 Numbering of Carbon and Nitrogen
Atoms
• Thus, when the 5'-carbon of a nucleoside
(or nucleotide) is referred to, a carbon
atom in the pentose, rather than an atom
in the base, is being specified

28
 Properties of Nucleotide Bases
1. Aromatic: The Nitrogen containing
bases are aromatic i.e. they have
alternate double bonds

2. Heteocyclic: They are heterocyclic

i.e. structures that contain, in
addition to carbon, other atoms such
as nitrogen
29
 Properties of Nucleotide Bases
3. Tautomerism: All these bases can exist
in amine-imine or keto-enol form
•At physiologic pH keto and amine form is
predominant

30
 Properties of Nucleotide Bases
4. Nucleotides Are Polyfunctional Acids:
• The primary phosphoryl group of nucleotides have
pKa values of about 1.0 and hence bear significant
negative charge at physiologic pH
• By contrast, the pKa values of the secondary
phosphoryl groups are about 6.2
• These secondary phosphoryl groups can therefore
serve as proton donors or acceptors at pH values
approximately two or more units above or below
neutrality

31
32
 Properties of Nucleotide Bases
5. Weak Bases: Purines or pyrimidines
with an –NH2 group are weak bases
(pKa =10-11)

6. Hydrophobicity: The purine and

pyrimidine bases are hydrophobic
and relatively insoluble in water at
the near-neutral pH of the cell
33
 Properties of Nucleotide Bases
7. Functional Groups: The most
important functional groups of
pyrimidines and purines are
–Ring nitrogens
–Carbonyl groups
–Exocyclic amino groups
34
• 8. UV light absorbance: The conjugated
double bonds of purine and pyrimidine
derivatives absorb ultraviolet light
• Nucleic acids are characterized by a strong
absorption at wavelengths near 260 nm
• The mutagenic effect of ultraviolet light is due
to its absorption by nucleotides in DNA that
results in chemical modifications

35
 Properties of Nucleotide Bases
9. Stacking Interaction: Hydrophobic stacking
interactions, in which two or more bases are
positioned with the planes of their rings
parallel (like a stack of coins), is important
mode of interaction between bases in nucleic
acids
• Base stacking helps to minimize contact of the
bases with water, and these interactions are
very important in stabilizing the
three-dimensional structure of nucleic acids
(RNA and DNA)
36
37
 Properties of Nucleotide Bases
10. Note that the smaller
pyrimidine molecule
has the longer name
and the larger purine
molecule the shorter
name

[Link] six-atom rings

are numbered in
opposite directions

38
 Minor Bases
• Although nucleotides bearing the major purines and
pyrimidines are most common, both DNA and RNA
also contain some minor bases (that have undergone
some modifications)

– In DNA the most common of these are methylated

forms of the major bases

– In some viral DNAs and RNAs, especially tRNAs,

certain bases may be modified

39
 Unusual Bases
• Base modifications
include
– Methylation,
– Hydroxy
methylation
– Glycosylation
– Acetylation
– Reduction

40
 Unusual Bases
• The presence of an
unusual base in a
nucleotide sequence
may
– aid in its
recognition by
specific enzymes
– protect it from
being degraded
by nucleases
41
 Other examples of Unusual Bases
• 5-methylcytosine of bacterial and human DNA
• 5-hydroxymethylcytosine of bacterial and viral
nucleic acids

42
 Other examples of Unusual Bases
• Mono- and the di-N-methylated adenine and
guanine of mammalian messenger RNAs that
function in oligonucleotide recognition and in
regulating the half-lives of RNAs

43
 Natural structural analogue
• Methylated heterocycles of
plants include:
– Theophylline:
Chemically
1,3-dimethyl xanthine is
found in tea
– Theobromine: Occurs
in cocoa which is
chemically 3,7-dimethyl
xanthine
44
• Methylated
heterocycles of plants
include

– Caffeine: Present
in coffee, is
chemically
1,3,7-trimethyl
xanthine
45
 Synthetic Analogues of
Nucleotides
• Chemically, synthesized analogues of purines and
pyrimidines, their nucleosides and their
nucleotides have therapeutic applications in
medicine.
• An analogue is prepared either by altering the
heterocyclic ring or sugar moiety.
• These are used chemotherapeutically
(treatment of disease using chemical
substances) to control cancer or infections.
• Analogues of purines and pyrimidines used in
the treatment of infections or in cancer
chemotherapy are:
• Cytarabine (arabinosyl cytosine; ara-c)
– In this nucleoside arabinose replaces ribose
– It is used in the chemotherapy of cancer and
viral infections.
• 5 Fluoro or 5 Iododerivatives of uracil
• These compounds get incorporated into DNA and block
cell proliferation.
 Synthetic Analogues of
Nucleotides
• Allopurinol (6-OH pyra Zolopyrimidine)
– This purine analogue used in treatment of
hyperuricemia and gout.
• 5-iododeoxyuridine,
• It is a nucleoside analogue
has antiviral activity and is
used in the treatment of
herpetic keratitis, an
infection of the cornea by
Herpes simplex virus
 Azapurine, Azacytidine, and
8-Azaguanine:
• In these the Purine or pyrimidine rings contain
extra nitrogen atoms.
• They have also been tested and used clinically.
 6– thio-guanine and 6 –
Mercaptopurine:
• In both naturally occurring purines, –OH groups
are replaced by “thiol” (–SH) group at 6 – position
of purine, widely used in clinical medicine.
 Unusual bases of tRNA
• Inosine contains the base
hypoxanthine

56
•Ribose is attach to uracil at
carbon no 5 instead of
Nitrogen no.1 to form unusual
base pseudouridine (Ψ)

57
58
 ?
• Another unusual base is TMP
(thymidine monophosphate):
Methylation by S-adenosylmethionine
(SAM) of a UMP of preformed tRNA
forms TMP, which contains ribose rather
than deoxyribose

59
 Nucleotides
• Nucleotides
are mono
phosphate,
diphosphate,
or
triphosphate
esters of
nucleosides
60
 Nucleotides
• The first phosphate
group is attached by
an ester linkage to
the 5’-OH of the
pentose

• Such a compound is
called a nucleoside
5'-phosphate or a
5'-nucleotide
61
• The second and third phosphates are each
connected to the nucleotide by an acid
anhydride bond which is a
"high-energy" bond

62
• The phosphate
groups are
responsible for the
negative charges
associated with
nucleotides, and
cause DNA and
RNA to be referred
to as "nucleic
acids" 63
 Mononucleotides
• If a phosphate group is added to a nucleoside,
forming an ester linkage with the hydroxyl
group of the pentose sugar, a mononucleotide
is formed e.g. AMP, CMP

64
• In case of ribose there are
three such places at carbon
atoms number 2,3 and 5
where an ester linkage can be
formed which are written as
– nucleoside
2´-monophosphate
– nucleoside
3´-monophosphate
– nucleoside
5´-monophosphate

65
 Deoxyribonucloetides
• In contrast to ribose which has three OH
groups and thus three points for phosphate
group attachment, deoxyribose has two such
places (3´and 5´) as C2 lacks an oxygen atom
and thus named as deoxyribose
• The letter ‘d’ is placed before the name of the
nucleotide containing deoxyribose e.g. dAMP

66
67
68
 Nucleoside Diphosphates
• If a second phosphate is added to the
nucleotide, a nucleoside diphosphate is
formed
• Examples:
– Adenosine diphosphate(ADP)
– Uridine diphosphate (UDP)
69
 ADP
• ADP acts as phosphate acceptor in oxidative
phosphorylation
• It serve as major biologic transducer of free energy
• It has role in
– Cellular respiration
– Muscle contraction
– Enzyme activation

70
 UDP
• UDP Glucose is formed by the reaction of UTP with
glucose-1 phosphate, which provides glucose units to
– Glycogen during glycogen synthesis and
– Lactose synthesis
– Glucosyl disaccharides
– The oligosaccharides of glycoproteins and
proteoglycans
• UDP-sugar derivatives participate in sugar
epimerizations
71
 UDP
• UDP glucose and UDP galactose can be oxidized to
UDP glucuronic acid and UDP galacturonic acid by
NAD dependent dehydrogenase
• UDP glucuronic acid is used for
– conjugation and detoxification of bilirubin,
benzoic acid, sterols and drugs
– for biosynthesis of hyaluronic acid, heparin
– UDP galactronic acid and UDP –L Iduronic acid
for synthesis of chondroitin SO4
72
 Nucleoside triphosphates
• If a third phosphate is added to a nucleoside
diphosphate or nucleoside triphosphate is formed
• Examples:
– Adenosine triphosphate (ATP)
– Guanosine triphosphate (GTP)
– Cytosine triphosphate (CTP)

73
 ATP
• The mean intracellular concentration of
ATP, the most abundant free nucleotide in
mammalian cells, is about 1 mmol/L
• It is the store house of energy for cells
• Acid anhydrides, unlike phosphate
esters, have high group transfer potential

74
 ATP
0
• ΔG ' for the hydrolysis of each of
the two terminal (β and γ)
phosphoryl groups of nucleoside
triphosphates is about –7
kcal/mol or (–30 kJ/mol)

75
76
 ATP

• The high group transfer potential

of purine and pyrimidine nucleoside
triphosphates permits them to
function as group transfer reagents,
most frequently of the
γ-phosphoryl group

77
 ATP
• Cleavage of an acid anhydride bond
typically is coupled with a highly
endergonic process such as covalent bond
synthesis—e.g, polymerization of
nucleoside triphosphates to form a
nucleic acid

78
Structure of ATP

79
 Functions of ATP
• Many synthetic reactions require energy, e.g. arginino
succinate synthetase reaction in the urea cycle
• ATP is also required in
– the synthesis of Phosphocreatine from creatine,
– synthesis of FA from acetyl CoA,
– synthesis of peptides and proteins from amino
acids,
– formation of glucose from pyruvic acid,
– synthesis of glutamine, etc
80
• ATP is an important source of energy for
– Muscle Contraction,
– Transmission Of Nerve Impulses,
– Transport Of Nutrients Across Cell
Membranes,
– Motility Of Spermatozoa

81
• ATP is required for formation of
active methionine (SAM), which is
required for methylation reactions

• ATP donates Phosphate for a variety

of phosphotransferase reactions, e.g.
hexokinase reaction
82
83
• ATP is required for formation of
‘active’ sulphate (PAPS) which is
necessary for incorporation of SO4 in
compounds like formation of
chondroitin SO4

84
85
• In vivo ATP is converted to ADP,
AMP and cyclic nucleotides like
3’,5’-cyclic AMP which have
important role to play in many
biochemical processes

86
 GTP
• GTP is necessary for the formation of
cyclic AMP
• For activation of adenyl cyclase by
some hormones
• GTP is required in rhodopsin cycle

87
 GTP
• GTP serves as
– an allosteric regulator
– an energy source for protein synthesis
• It has role in
– Citric acid cycle (the oxidation of
succinyl-CoA)
– Purine synthesis (formation of AMP from
IMP)
– Gluconeogenesis
88
 CTP
• CTP participates in biosynthesis of
–Phosphoglycerides
–Sphingomyelin
–Other substituted sphingosines

89
 Cyclic nucleotides

• There are two

important
cyclic
nucleotides:
– cAMP
– cGMP

90
 Cyclic nucleotides
• cAMP
– Cyclic AMP is a cyclic
nucleotide and chemically
it is 3´-5´ adenosine
monopohosphate
– It is synthesized in tissues
from ATP
– The intracellular cAMP
concentration is three
orders of magnitude below
that of ATP
91
92
93
94
 Adenyl Cyclase
• The enzyme is widely distributed in
nature and has been identified in
every mammalian tissue studied with
the exception of mature mammalian
erythrocytes.

95
• The profound physiological and metabolic
importance of adenyl cyclase resides in the fact
that its activity responds to a wide variety of
hormones and other pharmacologically active
agents, viz histamine, 5-HT
• (serotonin), ouabain, etc.

96
• The activity of the enzyme is inhibited by
insulin and prostaglandins.
• It has been confirmed that insulin inhibits the
activation of adenylcyclase in liver and fat
cells and PG-E1 that of fat cells.

97
 Phosphodiesterase:
• Cyclic AMP is rapidly inactivated by an
enzyme, a cyclic nucleotide
Phosphodiesterase which opens the 3’,
5’-phosphate bond at the 3’ position leaving
ordinary 5’-AMP as the product and thus
inactivating cyclic AMP.

98
• Certain activators and inhibitors of the enzyme
are known.
• Activators promote degradation of c-AMP and,
thus reduce its level.
• Inhibitors prevent degradation and, thus
increase cyclic-AMP level.

99
• Known activators and inhibitors of the enzyme
are listed below.

100
101
 Formation and Functions of c-AMP
• Acts as second messenger in the cell
• It has role in glycogen metabolism
•↑cAMP, ↑ glycogenolysis
• Triglyceride metabolism
•↑ lipolysis
• It decreases cholesterol synthesis
• It causes activation of protein kinases
• It has role in protein biosynthesis
• It has role in cell differentiation

102
 Functions of c-AMP

• It regulates the cell membrane

permeability, by increasing permeability
of cell membrane to H2O, Na+, K+ & Ca+2
• It modulates both transcription &
translation
• It regulates insulin secretion,
catecholamine biosynthesis & Melatonin
synthesis
103
• Histamine ↑ cAMP production in
parietal cells which in turn ↑ gastric
secretion
• Addition of cAMP to malignant cell
line in vitro↓ ng growth rates &
restores their morphology to normal

104
Normal activation and Deactivation

105
Cholera Toxin and cAMP

106
cGMP

107
 cGMP
• Formation: Cyclic GMP is made from GTP
by the enzyme guanylate cyclase which exists
in soluble and membrane bound form. The
enzyme requires Mn++ as a cofactor.
• Guanylate cyclase is also reported to be
stimulated by Ca++.

108
• Fate: Cyclic-GMP is hydrolysed by cyclic
nucleotide phosphodiesterase.
• The cyclic nucleotide phosphodiesterase is
trimeric consisting of three subunits, α, β and
γ.

109
• γ-subunit is inhibitory and unbinding
of γ-subunit activates the
phosphodiesterase (α, β).
• The activated phosphodiesterase (α,
β) then catalyses the hydrolysis of
c-GMP to 5’-AMP.

110
 cGMP

Functions of c-GMP:

• It serves as a second messenger in response to

nitric oxide (NO) during relaxation of smooth
muscle, so it has role in smooth muscle
relaxation and vasodilatation thus lowering of
blood pressure

111
 cGMP
• Role of c-GMP in phosphorylation
of proteins: “Muscarinic action” of
acetyl choline on smooth muscles is
mediated through c-GMP dependant
phosphorylation

112
• Role of c-GMP in vasodilation:
Compounds like nitroglycerine,
sodium nitrite, etc. cause smooth
muscle relaxation and vasodilation by
increasing c-GMP level

113
 cGMP
• Role of c-GMP in action of
neurotransmitters: GABA has been
claimed to change c-GMP level in
cerebellar tissues

• Role of c-GMP in PG’s: PG-F2α has

been shown to use c-GMP as second
messenger for its action
114
 cGMP
• Role of c-GMP in insulin actions:
Insulin action in certain tissues may be
mediated through c-GMP which activate
the ‘protein kinases’, which in turn
phosphorylates some enzymes to
modulate their activities
• It has role in Regulation of sodium
channels
115
 Dinucleotides
• The bond between the nucleotides is
the “phosphodiester bond”
• This bond is formed mainly between
the 3´OH group of sugar of one
nucleotide and 5´PO4 group of sugar of
another nucleotide
• This forms a dinucleotide
116
117
 Oligonucleotide

• A short nucleic acid is referred to as an

oligonucleotide

• The definition of “short” is somewhat

arbitrary, but polymers containing 50 or
fewer nucleotides are considered as
oligonucleotides
118
 Polynucleotides
• When nucleotides are joined together by
internucleotide bonds, polynucleotides are formed
• Polynucleotides are synonymous with nucleic acids
• Well-studied biological nucleic acid molecules range
in size from 21 nucleotides to large chromosomes
(human chromosome 1 is a single molecule that
contains 247 million base pairs)

119
 Genetic Information
• Nucleic acids are required for the
storage and expression of genetic
information
• Two chemically distinct types of nucleic
acids
– DNA
– RNA

121
 Genetic Information
• DNA, the repository of genetic information is
present in:
– Chromosomes in nucleus and mitochondria
of eukaryotic organism
– Chloroplasts of plants
– Prokaryotic cells contain single
chromosome but also contain
non-chromosomal DNA as plasmids
 Replication
• The genetic information found in DNA is
copied and transmitted to daughter cells
through DNA replication

• The DNA contained in a fertilized egg

encodes the information that directs the
development of an organism
Replication

•This development may involve the production of

billions of cells

•Each cell is specialized, expressing only those

functions that are required for it to perform its
role in maintaining the organism
• Therefore, DNA must be able to

– not only replicate precisely each time a

cell divides, but

– also to have the information that it

contains be selectively expressed
• Transcription
– RNA synthesis (first stage in expression of
genetic information)

• Translation
– Protein synthesis (code contained in the
nucleotide sequence of messenger RNA is
translated), thus completing gene expression
 Central Dogma of Molecular
Biology
• The flow of information
from DNA to RNA to
protein is termed the
“central dogma” of
molecular biology, and is
descriptive of all organisms,
with the exception of some
viruses that have RNA as the
repository of their genetic
information
 Higher Organization of DNA
• In higher organisms, DNA is organized
inside the nucleus.
• Double stranded DNA is wound round
histones to form nucleosomes .
• Chromatin is a loose term employed for
a long stretch of DNA in association with
histones.
• Chromatin is then further and further
condensed to form chromosomes .
• Similarly, the DNA molecule is
folded and compressed to 10,000
fold to generate chromosomes.

129
 ORGANIZATION OF
EUKARYOTIC DNA
• A typical human cell contains 46 chromosomes,
whose total DNA is approximately one meter
long
Proteins associated in Packaging of
DNA
• DNA is associated with a large number of
proteins,
– Small basic proteins termed as histones
– Acidic and larger non-histone proteins
– Small quantity of RNA
each of which performs a specific function in
the ordered packaging of these long molecules
of DNA
 Non-Histone Protein
• The non-histone proteins include enzymes
involved in DNA replication and repair, and
the proteins involved in RNA synthesis,
processing, and transport to the cytoplasm
• The dsDNA helix in each chromosome has a
length that is thousands of times the diameter
of the cell nucleus
 Functions of Histones
• One purpose of the molecules that comprise
chromatin, particularly the histones, is to condense
the DNA
• However, it is important to note that the histones also
integrally participate in gene regulation; indeed
histones contribute importantly to all DNA-directed
molecular transactions
• DNA and histones are present in nearly equal mass

133
 Nucleosomes
• Tightly bound histones serve to order the DNA into
basic structural units, called nucloesomes, that
resemble beads on a string
• Electron microscopic studies of chromatin have
demonstrated nucleosomes as dense spherical
particles, approximately 10 nm in diameter and
connected by DNA filaments
• Nucleosomes are composed of DNA wound around
an octameric complex of histone molecules

134
 Chromatin
• Nucleosomes are further arranged into
increasingly more complex structures that
organize and condense the long DNA
molecules into chromosomes that can be
segregated during cell division

• The complex of DNA and protein found inside

the nuclei of eukaryotic cells is called
chromatin
 Histones and the formation of
nucleosomes
• There are five classes of histones,
designated H1, H2A, H2B, H3, and
H4
• These small proteins are basic and
positively charged at physiologic pH
as a result of their high content of
lysine and arginine
 Histones and the formation of
nucleosomes
• Because of their positive charge, they
form ionic bonds with negatively
charged DNA

• Histones, along with positively

++
charged ions such as Mg help
neutralize the negatively charged
DNA phosphate groups
• The structure of all four histones is
highly conserved between species
• The carboxyl terminal two-thirds
of the histone molecules are
hydrophobic, while their amino
terminal one thirds are particularly
rich in basic amino acids
• Model for the structure
of the nucleosome, in
which DNA is wrapped
around the surface of a
protein cylinder
consisting of two each of
histones H2A, H2B, H3,
and H4 that form the
histone octamer

139
• Around ~145 bp of
DNA, consisting of
1.75 superhelical
turns, are in contact
with the histone
octamer
• Histone H1 interacts
with DNA as it enters
and exits the
nucleosome
140
• Thus, 1.75 superhelical
turns of DNA are
wrapped around the
surface of the histone
octamer, protecting 145
to 150 bp of DNA and
forming the nucleosome
core particle

141
• The disk-like
nucleosome
structure has a
10-nm
diameter and a
height of 5 nm

142
• H1 is the most tissue-specific and species-specific of
the histones
• It facilitates the packing of nucleosomes into more
compact structures
• In chromatin, core particles are separated by a
roughly 30-bp region of DNA termed “linker”
• Most of the DNA is in a repeating series of these
structures, giving the so called beads-on-a-string
appearance
• The histones interact with each other in very specific
way
• H3 and H4 form a tetramer containing two
molecules of each (H3–H4)2, while H2A and H2B
form dimers (H2A–H2B)

• Under physiologic conditions, these histone

oligomers associate to form the histone octamer of
the composition (H3–H4)2–(H2A–H2B)2

145
• These four core histones are subject to at
least six types of covalent modification or
posttranslational modifications (PTMs)

• Acetylation, methylation, phosphorylation,

ADP-ribosylation, monoubiquitylation, and
sumoylation

146
• These histone modifications play an important
role in chromatin structure and function

147
 HIGHER ORDER STRUCTURES PROVIDE
FOR THE COMPACTION OF CHROMATIN
• Electron microscopy of
chromatin reveals two
higher orders of structure
beyond that of the
nucleosome:
– the 10-nm fibril
– 30-nm chromatin
fiber

148
 HIGHER ORDER STRUCTURES PROVIDE
FOR THE COMPACTION OF CHROMATIN

• The 10-nm fibril

consists of nucleosomes
arranged with their
edges separated by a
small distance (30 bp of
linker DNA) with their
flat faces parallel to the
fibril axis

149
 HIGHER ORDER STRUCTURES PROVIDE
FOR THE COMPACTION OF CHROMATIN
• The 10-nm fibril
is probably
further
supercoiled with
six or seven
nucleosomes per
turn to form the
30-nm
chromatin fiber
150
• In interphase chromosomes,
chromatin fibers appear to be
organized into 30,000 to 100,000 bp
loops or domains anchored in a
scaffolding, or supporting matrix
within the nucleus, the so-called
nuclear matrix

151
152
• It has been suggested that each looped
domain of chromatin corresponds to one
or more separate genetic functions,
containing both coding and noncoding
regions of the cognate gene or genes

153
154
• Each phase of condensation or
compaction and organization decreases
overall DNA accessibility to an extent
that the DNA sequences in metaphase
chromosomes are almost totally
transcriptionally inert

155
156
• In total, these five levels of DNA
compaction result in nearly a 104-fold
linear decrease in end-to-end DNA length

157
159
Fate of nucleosomes during DNA
replication
• In order to replicate, the highly structured
and constrained chromatin must be
relaxed
• Although the nucleosomes are displaced,
dissociation of the nucleosome core from
the DNA is incomplete, with all the
parental histones remaining loosely
associated with only one of the parental
DNA strands
 Fate of nucleosomes during DNA
replication
• Synthesis of new histones occurs
simultaneously with DNA
replication, and nucleosomes
containing the newly synthesized
histones associate with only one
of the new daughter helices
• Therefore, the parental histone
octamers are conserved
 Nucleic Acid Structure
• Nucleic acid structure can be
described in terms of hierarchical
levels of complexity (primary,
secondary, tertiary)
• The primary structure of a
nucleic acid is its covalent
structure and nucleotide sequence
164
 Nucleic Acid Structure
• Any regular, stable structure taken up
by some or all of the nucleotides in a
nucleic acid can be referred to as
secondary structure
• The complex folding of large
chromosomes within eukaryotic
chromatin and bacterial nucleoids is
generally considered tertiary
structure
Primary Structure of DNA
dA dG dT dC
P

P
 Properties of a DNA
double helix
The strands of DNA are antiparallel

The strands are complimentary

There are Hydrogen bond forces

There are base stacking interactions

There are 10 base pairs per turn

 DNA IS MADE OF TWO STRANDS OF
POLYNUCLEOTIDE
• The sister strands of the DNA molecule run in
opposite directions (antiparallel)
• They are joined by the bases
• Each base is paired with a specific partner:
A is always paired with T
G is always paired with C
Purine with Pyrimidine
• This the sister strands are complementary but not
identical
• The bases are joined by hydrogen bonds, individually
weak but collectively strong
© 2007 Paul Billiet ODWS
DNA is a Double-Helix
(a) Primary Structure of DNA
• The primary structure is the number and sequence
of different deoxyribonucleotides in its strands
joined together by phosphodiester linkages.
• Normally there are only four different types of
deoxyribonucleotides that are found in
DNAmolecule, namely,
1. adenine deoxyribonucleotide (dA),
2. thymine deoxyribonucleotide (dT),
3. guanine deoxyribonucleotide (dG),
4. cytosine deoxyribonucleotide (dC).
• Nucleotides of each of the two helical strands
are bound to each other by covalent 3’-5’
phosphodiester linkage.
• Each such bond is formed by the ester
linkages of a single phosphate residue with
the 3’ –OH (i.e. C – 3’ –OH group of the
ribose sugar) of one nucleotide with the C –
5’ –OH group of ribose of the next nucleotide.
 P

THE SUGAR-PHOSPHATE
BACKBONE P

• The nucleotides are all

orientated in the same P

direction
P
• The phosphate group joins the
3rd Carbon of one sugar to the
5th Carbon of the next in line. P

© 2007 Paul Billiet ODWS

• This kind of bonding gives rise to a linear
polydeoxyribonucleotide strand with 2 free
ends on both sides.
• That end of the strand which bears a free 5’
phosphate group without phosphodiester
linkage is called the 5’-end.
• The opposite end bears a free 3’ – hydroxyl
or 3’ phosphate group and is called the 3’
end.
Nucleotides are
linked by
phosphodiester
bonds
• The backbone of the primary structure is the
linear strand of interconnected sugar
phosphate residues
• Purine or pyrimidine connected with the
sugar residue projects laterally from the
backbone.
 Secondary Structure of DNA
• This consists of a double stranded helix
formed by the two polydeoxyribonucleotide
strands around a central axis. This type of
model was first proposed by Watson and
Crick in their paper in Nature in 1953.
Later on they were awarded the Nobel Prize
in 1962 along with Maurice Wilkins.
• DNA is a double helix. Each of its two strands is
coiled about a central axis, usually a right handed
helix.
• The two sugar phosphate backbones wind around
the outside of the bases like the banisters of a
spiral stair case and are exposed to the aqueous
solution.
• The phosphodiester bonds in the two interwoven
strands run in opposite directions. Therefore the
strands are called antiparallel. Thus the polarity
of the two strands will be 3’ – 5’ and 5’ – 3’.
 DNA IS MADE OF TWO STRANDS OF
POLYNUCLEOTIDE Hydrogen bonds
P
G C
P
P
C G
P
P
C G
P
P
A T
P
P
T A
P
P
T A
© 2007 Paul Billiet ODWS
P
• The 3’ – 5’ strand is called coding or “template
strand” and 5' – 3' strand is called coding
‘strand’ .
• The aromatic rings of bases are hydrophobic and
they are stacked in the interior, nearly
perpendicular to the long axis of the helix.
• Adenine base of one strand of DNA is
hydrogen bonded to a thymine in the opposite
strand; while the guanine is hydrogen bonded
to a cytosine.
 Chargaff’s rule
• The ratio of purine to pyrimidine bases in the
DNA molecule is always around 1 (i.e. G + A/T +
C ≈ 1).
• This is known as Chargaff’s rule.
• In DNA, the glycosidic bonds between sugar and
bases are not directly opposite each other and two
grooves of unequal width form around the double
helix.
 Primary Structure

• 3’ → 5’-Phosphodiester bonds
(Sugar-Phosphate Bond)
• Successive nucleotides of both DNA
and RNA are covalently linked by
PO4 bridges
192
 Primary Structure

• Phosphodi- ester bonds join the

3’-OH group of deoxypentose of one
nucleotide to 5’-PO4 group of an
adjacent nucleotide
194
• The resulting long,
unbranched chain has
polarity, with both
–a 5’-end (free
phosphate) and
–a 3’-end (free OH) that
are not attached to
other nucloetides
• The bases are always
written in sequence from
the 5’-end to 3’-end

• This sequence is the

genetic code of the DNA
• Main function of
these bonds is to
stabilize the
molecule

• The
deoxyribose-phosph
ate backbone is the
constant part
• Purines and
pyrimidines project
laterally from the
backbone and form
the variable part

• The variable part is

concerned with the
transport of genetic
material
 Cleavage of Phosphodiester
Bonds
• Phosphodiester linkages between nucloetides
(in DNA and RNA) can be cleaved
– hydrolytically by chemicals
– Hydrolyzed enzymatically by nucleases:
•Deoxyribonucleases for DNA
•Ribonucleases for RNA
 Cleavage of Phosphodiester
Bonds
• Endonucleases: They cleave the
nucleotide chain at positions in
the interior of the chain

• Exonucleases: They cleave the

chain by removing the individual
nucleotides from one of the two
ends
 Secondary Structure
• DNA exist as double stranded
molecule (double helix), except
few viruses that contain single
stranded molecule
• The two chains are coiled around
a common axis called axis of
symmetry
202
 Secondary Structure
• The chains are paired in
anti-parallel manner, i.e., the 5’-end
of one strand is paired with the
3’-end of the other strand
204
• In the DNA helix
– Hydrophilic deoxyribose-phosphate
backbone is on the outside of the
molecule
– The hydrophobic bases are stacked
inside

205
 Secondary Structure
• The overall structure resembles a twisted
ladder
• Grooves: The spatial relationship
between the two strands in the helix
creates
– a major (wide) groove
– a minor (narrow) groove
207
 Secondary Structure
• These grooves provide access for the
binding of regulatory proteins to their
specific recognition sequences along
the DNA
 Clinical aspect
• Certain anticancer drugs, such as
dactinomycin (actinomycin D), exert
their cytotoxic effect by intercalating
into the narrow groove of the DNA
double helix, thus interfering with
RNA and DNA synthesis

210
 Base Pairing
• The bases of one strand of DNA are
paired with the bases of the second
strand, so that
– Adenine is always paired with
thymine
– Cytosine is always paired with
guanine
212
 Base Pairing
• One polynucleotide chain of DNA
double helix is always the
complement of other
214
 Base Pairing
• Given the sequence of bases on one
chain, the sequence of bases on the
complementary chain can be
determined
216
 Chargaff’s Rules
The specific base pairing of DNA leads to
Chargaff’s Rules:
• In any sample of double-stranded DNA:
– the amount of adenine equals the amount of
thymine
– the amount of guanine equals the amount of
cytosine
– the total amount of purines equals the total
amount of pyrimidines

217
218
 Chargaff’s Rules

•The base pairs are held

together by hydrogen
bonds:
–two between A and T
–three between G and C

219
220
– These hydrogen bonds, plus the hydrophobic
interactions between the stacked bases,
stabilize the structure of the double helix
– The base composition of DNA varies from
one species to another, but is same if isolated
from different tissues of same species
– The base composition does not change with
age, nutritional status and environment
 Template & Non-Template
Strand
• The 3´-5´strand is called Template
strand while 5´-3´ strand is called
Non Template (coding strand)
• The term template strand refers to
the sequence of DNA that is copied
during the synthesis of mRNA
228
 Template & Non-Template
Strand
• The opposite strand (that is, the strand with a
base sequence directly corresponding to the
mRNA sequence) is called the Non Template
or coding strand or the mRNA-like strand
because the sequence corresponds to the
codons that are translated into protein
 Separation of the two DNA
strands in the double helix:
• The two strands of the double helix separate when
hydrogen bonds between the paired bases are
disrupted
• Disruption can occur in the laboratory if
– the pH of the DNA solution is altered so that
the nucleotide bases ionize
– if the solution is heated above 80°C
• Phosphodiester bonds are not broken by such
treatment
• Disruption of the hydrogen bonds between
paired bases and of base stacking causes

– Unwinding of the double helix to form two

single strands (complete separation of DNA
stands along the entire length)

– Part of the length (partial denaturation) of

the molecule

231
• When DNA is
heated, the
temperature at
which one half of
the helical structure
is lost is defined as
the melting
temperature (Tm)
Hyperchromicity of denaturation
• 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

233
• So single-stranded DNA has a
higher relative absorbance at this
wavelength than does
double-stranded DNA

234
• The loss of
helical structure
in DNA, called
denaturation can
be monitored by
measuring its
absorbance at 260
nm
 Factors affecting Tm
• The Tm is influenced by
– the base composition of the DNA
– the salt concentration of the solution
• The higher the content of GC base pairs,
the higher the melting point of the DNA

236
 Factors affecting Tm
• This is because GC base pairs, with three
hydrogen bonds, require more heat
energy to dissociate than AT base pairs

• An increase in salt concentration

increases and a decrease in salt
concentration decreases the Tm

237
 Renaturation or Reannealing
• Under appropriate conditions (temp. & salt
concentration), separated strands of DNA will
renature or re-associate and form the double
helix by the process called renaturation (or
reannealing)
• This reannealing process is also often referred
to as hybridization

238
• When the temperature or pH is returned
to the range in which most organisms
live, the unwound segments of the two
strands spontaneously rewind, or anneal,
to yield the intact duplex

239
 Denaturation &Renaturation
• Renaturation of a
DNA molecule is a
rapid one-step
process
• The rate of
re-association
depends upon the
concentration of the
complementary
strands 240
 Structural forms of the double
helix
• There are three major structural forms of
DNA:
– the B form, described by Watson and
Crick in 1953,
– the A form,
– the Z form
• The B form is a
right-handed
helix with ten
residues per
360° turn of the
helix, and with
the planes of the
bases
perpendicular to
the helical axis
• Chromosomal DNA is thought to
consist primarily of B-DNA
• The A form is
produced by
moderately
dehydrating the B
form

• It is also a right-handed
helix, but there are 11
base pairs per turn, and
the planes of the base
pairs are tilted 20° away
from the perpendicular
to the helical axis
• The conformation
found in
DNA–RNA hybrids
or RNA–RNA
double-stranded
regions is probably
very close to the A
form
• Z-DNA is a
left-handed helix
that contains about
12 base pairs per
turn

• The
deoxyribose–phosp
hate backbone
“zigzags,” hence,
the name
• Stretches of Z-DNA can occur
naturally in regions of DNA that
have a sequence of alternating
purines and pyrimidines, for
example, poly GC
• Transitions between the B and Z
helical forms of DNA may play a role
in regulating gene expression
 Linear and circular DNA
molecules
• Each chromosome in the nucleus of a
eukaryote contains one long, linear
molecule of dsDNA, which is bound to a
complex mixture of proteins to form
chromatin
• Eukaryotes have closed, circular DNA
molecules in their mitochondria
• A prokaryotic organism typically contains a
single, double-stranded, supercoiled, circular
chromosome
• Each prokaryotic chromosome is associated
with non-histone proteins that can condense
the DNA to form a nucleoid
250
• In addition, most species of bacteria also
contain small, circular, extra
chromosomal DNA molecules called
plasmids
• Plasmid DNA carries genetic information,
and undergoes replication that may or
may not be synchronized to chromosomal
division
• Plasmids may carry genes that convey
antibiotic resistance to the host
bacterium, and may facilitate the transfer
of genetic information from one
bacterium to another
• The use of plasmids as vectors in
recombinant DNA technology will be
discussed in detail
 Certain DNA Sequences Adopt Unusual
Structures
• A rather common type of DNA sequence is a
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

253
254
 Certain DNA Sequences Adopt Unusual
Structures
• The term is applied to regions of DNA with
inverted repeats of base sequence having
two fold symmetry over two strands of DNA

255
 Hairpin DNA Structure
• Such sequences are
self complementary
within each strand
and therefore have
the potential to
form hairpin or
cruciform
(cross-shaped)
structures
256
Cruciform

257
 Mirror Repeat
• When the inverted repeat occurs within
each individual strand of the DNA, the
sequence is called a mirror repeat
• Mirror repeats do not have
complementary sequences within the
same strand and cannot form hairpin or
cruciform structures

258
 Mirror Repeat
• Sequences of these types are found in
virtually every large DNA molecule and
can encompass a few base pairs or
thousands

259
 RNA
• The genetic master plan is contained in the
nucleotide sequence of DNA

• It is through the ribonucleic acid (RNA)―the

"working copies" of the (DNA) ― that the
master plan is expressed

260
261
 RNA
• RNA is a polymer of ribonucleotides
of Adenine, Uracil, Guanine and
Cytosine, joined together by
3´-5´phosphodiester bonds
• RNA does not contain thymine except
in rare cases
• The pentose sugar of RNA is
D-ribose 262
 RNA
• Location: RNA is found in the
nucleolus, ribosomes, mitochondria,
and cytoplasm

• There is also a wide variety of

special-function RNAs, including
some (called ribozymes) that have
enzymatic activity
263
 Primary Structure of RNA
• It is defined as the
number and sequence of
ribonucleotides in the
RNA chain

• The sequence is
complementary to the
template strand of the
gene from which it was
transcribed

264
 Primary Structure of RNA
• The ribonucleotides are held together by 3´- 5´
phosphodiester bonds

• 3´-OH group of one nucleotide is bound to

5´-PO4 of the other nucleotide and forms a
linear strand

265
266
 Primary Structure of RNA

• Phosphodiester bonds are present

between ribosyl moieties

• The ribosyl moieties are attached to

the nucleobases by N-glycosidic
bonds
267
 Secondary Structure of RNA
• RNA has no simple, regular
secondary structure that serves as
a reference point, as does the
double helix for DNA

268
• The three-dimensional structures of
many RNAs, like those of proteins,
are complex and unique
• Secondary structure involves coil
formation of the polyribonucleotide
chain

269
 Secondary Structure of RNA
• The coiled structures are stabilized by
– Hydrophobic interactions between
purine and pyrimidine bases
– Intra-chain hydrogen bonds between
G-C and A-U
– Weak interactions, especially
base-stacking interactions
270
 Secondary Structure of RNA
• Where
complementary
sequences are
present, the
predominant
double-stranded
structure is
A-form, a
right-handed
double helix 271
 Secondary Structure of RNA
• Z-form helices have
been made in the
laboratory (under very
high-salt or
high-temperature
conditions)

• The B form of RNA

has not been observed
272
 Tertiary Structure of RNA

• This involves the

folding of the
molecule into
three dimensional
structure

273
 Tertiary Structure of RNA

• There is
cross-linking at
various sites
stabilized by
hydrophobic and
hydrogen bonds
producing a
compactly coiled
globular structure

274
 Differences between DNA and
RNA
Although sharing many features with
DNA, RNA possesses several specific
differences:
1. Size: They are considerably smaller than
DNA
2. Sugar: 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
275
 Differences between DNA and
RNA
1. Pyrimidine: The pyrimidine
components of RNA differ from
those of DNA
• Instead of thymine, RNA contains the
ribonucleotide of uracil
• Thymine is present in the rare case of
tRNA
276
 Differences between DNA and
RNA
4. Single Strand: 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 is capable of folding
back on itself like a hairpin and thus
acquiring double-stranded characteristics:
G pairing with C, and A with U
277
 Differences between DNA and
RNA
5. G≠C: 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
6. Hydrolysis: 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
278
 Differences between DNA and
RNA
7. Location: In addition to nucleus,
RNA is found in cytoplasm
8. Reverse Transcription: DNA forms
RNA by transcription but RNA
cannot form DNA
• In experiments, reverse transcriptase
can be used for this purpose
279
 Types of RNA
• Important classes of RNA
– Messenger RNA (mRNA)
– Transfer RNA (tRNA)
– Ribosomal RNA (rRNA)
– Small Nuclear RNA (snRNA)

– Other RNAs 280

 Messenger RNA (mRNA)
• This class is the most
heterogeneous in
– abundance
– size (500-6000
nucleotides)
– base sequence
– stability
• mRNA comprise about
2–5% of total cellular
RNA
281
 Messenger RNA (mRNA)
• mRNA molecules are
formed with the help of
DNA template strand
(3´-5´) during the process
called transcription
• In addition to the protein
coding regions that can be
translated, there are
untranslated regions at its
5´ and 3´ ends
282
 Function of mRNA
• The members of this class function as
messengers to convey the information in a
gene to the protein synthesizing machinery

283
 Function of mRNA
• The mRNA carries genetic information from
the nuclear DNA to the cytosol, where it is
used as a template for protein synthesis

284
 Polycistronic and Monocistronic
mRNA
• Polycistronic: If the mRNA carries
information from more than one gene (i.e. it
carries the code for more than one polypeptide)
it is called polycistronic which is
characteristic of prokaryotes

285
 Polycistronic and Monocistronic
mRNA
• Prokaryotes can have both polycistronic and
monocistronic mRNAs
• Monocistronic: If the mRNA carries
information from just one gene (i.e. it carries
the code for only one polypeptide) it is called
monocistronic, a characteristic of eukaryotes

286
 Special structural characteristics of
eukaryotic messenger RNA
• A Cap: There is
a “cap” on the
5´ end
consisting of a
molecule of
7-methylguanos
ine attached
"backward"
(5’-5’) through
a triphosphate
linkage 287
 Special structural characteristics of
eukaryotic messenger RNA
• The cap is involved in
– The recognition of mRNA by the translation
machinery
– Stabilizing mRNA against 5´ exonucleases

288
 Special structural characteristics of
eukaryotic messenger RNA
• Poly-A tail: It contains a long sequence
of adenine nucleotides (20-250) on the
3´-OH end of the RNA chain and is called
“poly-A tail”

289
• It is thought to play role
– In stability of mRNA against 3´
exonucleases
– Facilitates translation

• Both the mRNA "cap" and "poly(A) tail" are

added post-transcriptionally to mRNA
precursor molecules (pre mRNA)

290
 Transfer RNA (tRNA)

• t RNA is the smallest of the three

major species of RNA (4S)

• “S” is the Svedberg unit, which is

related to the molecular weight and
shape of the compound

291
 Transfer RNA (tRNA)

• They are single stranded globular

molecules

• They remain largely in cytoplasm

• They are generated by nuclear

processing of a precursor molecule
292
 Transfer RNA (tRNA)
• tRNAs compose roughly 20% of total
cellular RNA
• There are at least 20 species of tRNA
molecules in every cell
• Although each specific tRNA differs
from the others in its sequence of
nucleotides, the tRNA molecules as a
class have many features in common
293
 Primary structure
• tRNA molecules consist of 74-95 nucleotides in a
particular sequence
• The tRNA molecules contain not only the usual bases
like adenine, guanine, cytosine, uracil but also
contain unusual bases like
– Dihydrouracil
– Pseudouridine
– Thymine

294
 Secondary Structure
• Each single stranded
tRNA is folded
extensively
• Extensive intra chain
base pairing which
leads to a
characteristic
CLOVER-LEAF
structure

295
296
 Secondary Structure

• These folds are

stabilized by
hydrogen bonds
between
complementary
bases of the
same strand
297
 Arms or loops of tRNA
• All tRNA molecules
contain 4 main arms
or loops
1-Acceptor arm: This
is made up of
unpaired sequences of
cytosine-cytosine-ade
nine (CCA) at the
3´end

298
 Arms or loops of tRNA
• The 3´OH group of adenine binds with
the carboxylic group of a specific amino
acid and carries it to ribosomes for
protein synthesis

299
 Arms or loops of tRNA
2-Anticodon
arm: It is in
the form of a
loop and
carries
specific
sequences of
three bases
which
constitute the
anticodon 300
 Arms or loops of tRNA

• The bases of
anticodon are bonded
with three
complemntary bases
of codon of mRNA

301
 Arms or loops of tRNA
3-D arm: It contains
the base
dihydrouridine
4-TΨC arm: It
contains thymine,
pseudouridine and
cytosine
• The extra arm and
the TΨC arms help
define a specific
tRNA
302
 Function of tRNA
• The t RNA molecules serve as adapters
for the translation of information in the
sequence of nucleotides of the mRNA
into specific amino acids
• There is at least one (and often several)
specific type of tRNA molecule for each
of the amino acids commonly found in
proteins
303
 Function of tRNA
• Each t RNA carries its specific amino
acid to the site of protein synthesis

• There it recognizes the genetic code

word on mRNA and this specifies the
addition of its amino acids to the
growing peptide chain

304
 Ribosomal RNA
• Ribosomal RNA (r RNA) is found in
association with several proteins as a
component of ribososmes - -- a
cytoplasmic nucleoprotein structure that
acts as the machinery for the synthesis of
proteins from the mRNA template
• rRNAs make up 80% of the total RNA in
the cell
305
• The bases in rRNA are mainly adenine,
guanine, cytosine and uracil and a few
pseudouridine

• Some RNAs function as catalysts, for

example, rRNA in protein synthesis

• RNA with catalytic activity is termed a

“ribozyme”
 Small nuclear RNA (SnRNA)
• Small nuclear RNA (SnRNA) are
large number of small stable RNA
species found in eukaryotic cells
• Most of them are complexed with
proteins to form ribonucleoproteins
• They are distributed in the nucleus, in
the cytoplasm or in both
307
 Small nuclear RNA (SnRNA)
• The RNAs in the small nuclear
ribonucleoproteins (snRNPs or
snurps) are involved in the splicing
and modification reactions that
occur during the maturation of RNA
precursors

308
309
 Heterogeneous nuclear RNA
• Heterogeneous nuclear RNA (hnRNA)
is a diverse group of long primary
transcripts formed in the eukaryotic
nucleus, many of which will be processed
to mRNA molecules by splicing

• The are synonyms of pre mRNA

310
 Micro RNA
• miRNAs are typically 21–25 nucleotides
in length and are generated by nucleolytic
processing of the products of distinct
genes

• miRNA precursors are single stranded but

have extensive intramolecular secondary
structure
311
 Micro RNA
• The small processed mature miRNAs
typically hybridize, via the formation
of RNA-RNA duplexes within the
3'-untranslated regions of specific
target mRNAs, leading via poorly
understood mechanisms to translation
arrest
312
 Small Interfering RNAs
• siRNAs are derived by the specific nucleolytic
cleavage of larger, double-stranded RNAs to
again form small 21–25 nucleotide-long
products

• These short siRNAs usually form RNA-RNA

hybrids with their distinct targets potentially
anywhere within the length of the mRNA
where the complementary sequence exists
313
• Formation of such RNA-RNA duplexes
between siRNA and mRNA results in reduced
specific protein production because the
siRNA-mRNA complexes are degraded by
dedicated nucleolytic machinery

• Both miRNAs and siRNAs represent exciting

new potential targets for therapeutic drug
development
314
 Denaturation of RNA
• Duplexes of two RNA strands can also be
denatured
• RNA duplexes are more stable than DNA
duplexes
• At neutral pH, denaturation of a double helical
RNA often requires temperatures 20° C or more,
higher than those required for denaturation of a
DNA molecule with a comparable sequence
315
 Compounds contributing in Purine
Nucleotides
• The atoms of the purine ring are contributed by a
number of compounds including
– amino acids
• aspartic acid
• glycine
• glutamine
– CO2
– N10-Formyl-tetrahydrofolate

316
317
 Compounds contributing to Pyrimidine
Nucleotides
• The sources of the atoms in
the pyrimidine ring are
– glutamine,
– CO2
– aspartic acid
• Glutamine and aspartic acid
are thus required for both
purine and pyrimidine
synthesis
318
319
 Synthetic analogs of purines &
pyrimidines
• Chemically synthesized analogs of
purines, pyrimidines and their
nucleosides and nucleotides have
numerous applications in clinical
medicine especially for cancer
chemotherapy

320
 Basis of Chemotherapy
• The growth of cancer cells is not
controlled in the same way as cell
growth in most normal tissues
• Cancer cells have greater
requirements for nucleotides as
precursors of DNA and RNA, and
consequently are generally more
sensitive than normal cells to
inhibitors of nucleotide biosynthesis
321
 Basis of Chemotherapy
• In synthetic analogs, the heterocyclic ring or
the sugar moiety is altered in such a way that
toxic effects are produced when this analog
gets incorporated into the cellular constituents
of the body
• The toxic effects of chemotherapeutic drugs
include
– Inhibition of enzymes essential for nucleic
acid synthesis
– They get incorporated into nucleic acids
which results in disruption of base pairing 322
Inhibitors of human purine synthesis
• Inhibitors of human purine synthesis are
extremely toxic to tissues, especially to
– developing structures such as in a fetus
– cell types that normally replicate rapidly,
including those of
• bone marrow
• skin
• gastrointestinal (GI) tract
• immune system
• hair follicles

323
Inhibitors of human purine synthesis
• As a result, individuals taking such
anti-cancer drugs can experience adverse
effects, including
– anemia
– scaly skin
– GI tract disturbance
– immunodeficiencies
– baldness 324
 Synthetic analogs of Glutamine
• The first set of agents includes compounds that
inhibit glutamine amidotransferases
• Glutamine is a nitrogen donor in at least half a dozen
separate reactions in nucleotide biosynthesis
• The binding sites for glutamine and the mechanism
by which NH4+ is extracted are quite similar in many
of these enzymes
• Most are strongly inhibited by glutamine analogs
such as azaserine and acivicin

325
Synthetic inhibitors of purine synthesis

• Some synthetic inhibitors of purine

synthesis (for example, the
sulfonamides), are designed to inhibit the
growth of rapidly dividing
microorganisms without interfering with
human cell functions

326
327
PABA Analogs

328
 Methotrexate
• Other purine synthesis inhibitors, such as structural
analogs of folic acid (for example, methotrexate),
are used pharmacologically to control the spread of
cancer by interfering with the synthesis of nucleotides
and, therefore, of DNA and RNA
• Methotrexate, is an inhibitor of dihydrofolate
reductase
• This folate analog acts as a competitive inhibitor; the
enzyme binds methotrexate with about 100 times
higher affinity than dihydrofolate
329
Folic Acid Analogs

330
 Other Folic acid Analogs
• Another important folate analog,
aminopterin is identical to
methotrexate except that it lacks the
methyl group

• Aminopterin acts similarly as

Methotrexate

331
 Other Folic acid Analogs
• Trimethoprim another folate analog, has
potent antibacterial activity because of its
selective inhibition of bacterial
dihydrofolate reductase
• Trimethoprim, an antibiotic, binds to
bacterial dihydrofolate reductase nearly
100,000 times better than to the
mammalian enzyme
• It is used to treat certain urinary and
middle-ear bacterial infections 332
• Mycophenolic acid is a potent, reversible,
uncompetitive inhibitor of inosine
monophosphate dehydrogenase that is being
used successfully in preventing graft rejection
• It blocks the de novo formation of guanosine
monophosphate (GMP), thus depriving rapidly
proliferating cells, including T and B cells, of a
key component of nucleic acids
• 6-mercaptopurine inhibits the conversion of
IMP to GMP and AMP
333
Mycophenolic acid

334
 Caffeine
• Caffeine is a trimethyl derivative of
xanthine

• It inhibits phosphodiesterase, causing

prolonged action of cyclic AMP

• This is how caffeine prolongs the effect

of epinephrine
335
 Salvage pathway for purines
• Purines
• that result from the normal turnover of cellular
nucleic acids, or
• that are obtained from the diet and not degraded,
can be reconverted into nucleoside triphosphates and
used by the body in a pathway much simpler than the
de novo synthesis of purine nucleotides
• This is referred to as the "salvage pathway" for
purines and it requires far less energy than de novo
purine synthesis
 Conversion of purine bases to
nucleotides
•Two enzymes are involved:
• adenine phospho ribosyltransferase
(APRT)
 Conversion of purine bases to
nucleotides
• hypoxanthine-guaninephospho ribosyl
transferase (HGPRT)
 Conversion of purine bases to
nucleotides

• Both enzymes use PRPP as the

source of the ribose 5-phosphate
group

• The release of pyrophosphate

makes these reactions irreversible
 Lesch-Nyhan syndrome
• This syndrome
is an X-linked
recessive
disorder
associated with
a virtually
complete
deficiency of
HGPRT
• This deficiency
results in an
inability to salvage
hypoxanthine or
guanine, from
which excessive
amounts of uric
acid, the end
product of purine
degradation, are
produced
• Hypoxanthine
and guanine
arise
constantly
from the
breakdown of
nucleic acids
In addition, the lack of this salvage pathway causes
increased PRPP levels and decreased IMP and GMP
level
And glutamine:phosphoribosylpyrophosphate
amidotransferase (the committed step in purine
synthesis) has excess substrate and decreased inhibitors
available, and de novo purine synthesis is increased
• The combination of decreased purine reutilization and
increased purine synthesis results in the production of large
amounts of uric acid, making the Lesch-Nyhan syndrome a
severe, heritable form of hyperuricemia
• In patients with Lesch-Nyhan syndrome, the hyperuricemia
frequently results
• in the formation of uric acid stones in the kidneys
(urolithiasis) and
• the deposition of urate crystals in the joints (gouty
arthritis) and soft tissues
• The brain is especially dependent on the salvage
pathways, and this may account for the central
nervous system damage in children with
Lesch-Nyhan Syndrome
• Characteristic neurologic features of
the disorder include
– involuntary movements due to motor
dysfunction
– extremely hostile and compulsive
self-destructive tendencies: they mutilate
themselves by biting off their fingers, toes,
and lips
347
• Lesch-Nyhan syndrome is seen almost exclusively in
male children (X-linked recessive)
• Children with this genetic disorder, which becomes
manifest by the age of 2 years, are sometimes poorly
coordinated and mentally retarded
• The devastating effects of Lesch-Nyhan syndrome
illustrate the importance of the salvage pathways
• This syndrome was a target of early trials in gene
therapy
 Gout
Gout is a disorder of joints, characterized by
high levels of uric acid —the end product of
purine catabolism—in the blood
(hyperuricemia), as a result of either
– over-production
– underexcretion of uric acid
350
• Hyperuricemia can lead to deposition of crystals of
monosodium urate in the joints, and an
inflammatory response to the crystals, causing first
acute and then progressing to chronic gouty arthritis
• So joints become inflammed, painful and arthritic
• Hyperuricemia is typically asymptomatic and does
not always lead to gout, but gout is usually preceded
by hyperuricemia
• Nodular masses of
monosodium urate crystals
(tophi) may be deposited in
the soft tissues, resulting in
chronic tophaceous gout

• The deposition of
needle-shaped monosodium
urate crystals initiates an
inflammatory process
involving the infiltration of
granulocytes that
phagocytize the urate
crystals
• This process
generates oxygen
metabolites that
damage tissue,
resulting in the
release of lysosomal
enzymes that evoke
an inflammatory
response
• In addition, lactate production in the
synovial tissues increases, resulting
in a decrease in pH that fosters
further deposition of urate crystals
• Formation of uric acid stones in the
kidney (urolithiasis) may also be seen
• A definitive
diagnosis requires
aspiration and
examination of
synovial fluid using
polarized light
microscopy to
confirm the presence
of needle-shaped
monosodium urate
crystals
 Underexcretion of uric acid
• In most patients, gout is caused by the
underexcretion of uric acid due to defective renal
secretion
• Underexcretion can be
• Primary
• Secondary
Primary Underexcretion
• It is due to unidentified inherent excretory defects
 Secondary Underexcretion
• It is due to known disease processes that
affect how the kidney handles urate, for
example lactic acidosis (lactate and
urate compete for the same
transporter)
• Environmental factors are also important
such as the use of drugs, e.g. thiazide
diuretics
• Exposure to lead (saturine gout)
 Overproduction of Uric acid

• A less common cause of gout is

hyperuricemia from the
overproduction of uric acid

• Primary Overproduction: It is
idiopatic
 Overproduction of Uric acid
• However, several mutations have been
identified in the gene for X-linked PRPP
synthetase that resulting in the enzyme
having either
• an increased Vmax for the production of
PRPP,
• a lower Km for ribose 5-phosphate
• or a decreased sensitivity to its purine
nucleotide inhibitors
• In each case, increased availability of
PRPP increase purine production,
resulting in elevated levels of plasma
uric acid
• Lesch-Nyhan Syndrome also causes
hyperuricemia as a result of
•Decreased salvage of hypoxanthine
and guanine
 Secondary hyperuricemia
• This form of gout is due to increased availability of
purines
• It is caused by a variety of disorders and lifestyles, for
example,
• those undergoing chemotherapy (so have a high
rate of cell turnover)
• those with myeloproliferative disorders
• and those who consume excessive amounts of
alcohol or purine-rich foods
• A diet rich in meat and seafood
(particularly shellfish) is associated with
increased risk of gout
• In addition, a diet rich in low-fat dairy
products was shown to be associated with
a decreased risk
• Gout can also be an adverse effect of
seemingly unrelated metabolic diseases,
such as
 Von Gierke Disease
• Purine overproduction and hyperuricemia
in von Gierke disease
(glucose-6-phosphatase deficiency)
occurs secondary to enhanced generation
of the PRPP precursor ribose 5-phosphate

• An associated lactic acidosis elevates the

renal threshold for urate, elevating total
body urates
 Fructose intolerance
•Aldolase B is deficient

•Fructose 1-phosphate accumulates, and ATP

and inorganic phosphate levels fall significantly,
with adenine being converted to uric acid,
causing hyperuricemia
 Treatment for gout
• Gout is effectively treated by a
combination of nutritional and drug
therapies

• Foods especially rich in nucleotides

and nucleic acids, such as liver or
glandular products, are withheld from
the diet
•Acute attacks are treated with
• Colchicine, which prevents formation
of microtubules thus decrease the
movement of neutrophils into the
affected area
• Like the other anti inflammatory drugs
it has no effect on uric acid levels
• Steroidal drugs such as prednisone
 Long term Therapeutic
Strategies
• Most therapeutic strategies for gout
involve lowering the uric acid level
below the saturation point, thus
preventing the deposition of urate crystals

• Uricosuric agents, such as probenecid

or sulfinpyrazone are used in most
patients with gout because most are
"underexcretors" of uric acid
 Treatment for gout
• Allopurinol – a structural analog of
hypoxanthine and an inhibitor of uric
acid synthesis - is more toxic, and is
reserved for those patients who are
“overproducers” of uric acid
• Allopurinol is converted in the body
to oxypurinol, which inhibits
xanthine oxidase, the enzyme that
• Oxypurinol inactivates the reduced
form of the enzyme by remaining
tightly bound in its active site

• This results in an accumulation of

hypoxanthine and xanthine –
compounds more soluble than uric
acid and less likely to form
crystalline deposits, therefore, less
• In patients with normal levels of HGPRT,
the hypoxanthine can be salvaged, thus
reducing the levels of PRPP and,
therefore, denovo purine synthesis
• Febuxostat, a non-purine inhibitor of
XO, is now available
• Uric acid levels in the blood normally are
close to the saturation point
 University Questions
• Compare and contrast the structural and other features of
DNA and RNA.
• What is the structure of DNA?
• Draw the structure of ADENOSINE TRIPHOSPHATE
(ATP). Enumerate four functions of ATP.
• (a) Define with one example
i. NUCLEOSIDE
ii. NUCLEOTIDE
(b) Give four functions of NUCLEOTIDES in the human
body.
371
 University Questions
• Name the NITROGENOUS BASES found in
RNA. What are the major classes of RNA?
Write down characteristic features of any two
classes.
• What are the characteristic structural features
and functions of mRNA and tRNA?

372