N ucl e ic a c i d s

DNA- Deoxy ribonucleic acid

RNA- Ribonucleic acid
Nucleoproteins
DNA + Proteins (Histones) – Chromatin
DNA contains genetic information and it can be
transmitted by replication
Chromatin is the chromosomal material
extracted from nuclei of cells
Chromatin consists of very long double
stranded DNA molecules and nearly equal
mass of small basic proteins- Histones and
a smaller amount of non histone proteins & a
small quantity of RNA
Arranged in special manner -Beads on a string
The double stranded DNA helix in each
chromosome has a length that is thousands of
times the diameter of the cell nucleus
The purpose of histones is to condense the DNA
Electron microscopic studies of chromatin have
demonstrated dense spherical particles
called Nucleosomes
Nucleosomes are approximately 10 nm in
diameter and connected by DNA filaments
Nucleosomes are composed of DNA wound
around a collection of histones .
 Histones
Histones are most abundant chromatin proteins
They are a small family of closely related basic
proteins
Histones are of many types
H 1, H 2A , H 2B, H 3 and H 4
H 1 histones are least tightly bound to chromatin
and are easily removed by a salt solution after
which chromatin becomes soluble
The isolated core nucleosome contain four
classes of histones H2A, H2B, H 3 and H 4
The structure of slightly Lysine- rich
histones H2A and H2B appear to have been
significantly conserved between species.
The structure of Arginine rich histones H 3 and
H 4 have been highly conserved between
species.
This severe conservation implies that the
function of histones is identical in all
eukaryotes and entire molecule is involved
quite specifically in carrying out this
function.
The C- terminal 2/3 of molecules have a usual
amino acid composition while their amino
terminal 1/3 is rich in Basic Amino acids.
These four core histones are subjected to
five types of covalent modifications -
Acetylation, Methylation, Phosphorylation,
ADP-ribosylation and Covalent linkage
(H2A only) to ubiquitin, the nulear protein
The histone modifications likely play some
role in chromatin structure and function.
Possible roles of modified Histones
1 Acetylation of histones H3 and H4 is associated
with the activation or inactivation of gene
transcription
2. Acetylation of core histones is associated with
chromosomal assembly during DNA replication
3. Phosphorylation of histone H1 is associated with
condensation of chromosomes during the
replication cycle
4. ADP-ribosylation of histones is associated with
DNA repair
5. Methylation of histones is correlated with
activation and repression of gene transcription.
6. Monoubiquitylation is associated with gene
activation , repression, and heterochromatic gene
silencing.

7. Sumoylation of histones (SUMO; small ubiquitin-

related modifier) is associated with transcription
repression
Histones interact with each other in a very
special way
H3 and H4 forms a tetramer containing 2
molecules of each - (H3/H4) 2

H2A and H2B form a dimer (H2A / H2B ) &

higher oligomeric complexes
(H2A/H2B) n
Under physiological conditions two of these
tetramers associate to form the histone octamer
When the histone octamer is mixed with
purified double stranded DNA the same x-ray
pattern is formed as observed in freshly
isolated chromatin.
Nucleosomes can be reconstructed
The reconstitution of nucleosomes from
DNA & histones H2A, H2B, H3 and H4 is
independent of organismal and cellular
origin of the various components
The histone H1 and the non histone proteins
are not necessary for the reconstitution of
the nucleosome core.
In nucleosomes , the DNA is super coiled in
a left handed helix over surface of disc
shaped histone octamer consisting of central
H3 and H4 tetramers & two H2A- H2B
dimers.
 Components of Nucleic Acids

1. Nitrogenous base
2. Sugar Nucleoside Nucleotide
3. Phosphoric acid
Ribo Deoxy Ribo Deoxy Ribo
Ribo
 Nitrogenous bases
1. Purines
Adenine A
Guanine G

2. Pyrimidine
Cytosine C
Uracil U
Thymine T
 Sugar
 Ribose
Deoxyribose

Purines and pyrimidines absorb maximum

light at 260nm- nucleotides
 Mitochondrial DNA

1% of cellular DNA is in mitochondria

It is circular, double –stranded & composed
of heavy (H) and light (L) chains or
strands
It contains 16,569 base pairs
a) Encodes 13 protein subunits of the
respiratory chain (total 67)
Encodes
1. 7 subunits of NADH dehydrogenase
(complex I)
2. Cytochrome b of complex III
3. 3 Sub units of cytochrome oxidase (c iv)
4. 2 subunits of ATP synthase
b) Encodes large(16S) and small (12S)
mitochondrial ribosomal RNAs
c) Encodes 22 mitochondrial t RN A
molecules
Genetic code differs slightly from standard
code
UGA (standard stop codon) is read as
tryptophan
AGA and AGG (st. codon for Argnine) are
read as stop codon.
Contains very few un translated sequences
High mutation rate (5-10 times that of
nuclear DNA)
Comparisons of mit DNA sequences provides
evidence about evolutionary origins of
primates and other species
All mitochondria are contributed by the ovum
during zygote formation so mit. DNA is
transmitted by maternal inheritance
In case of diseases transmitted through
mit DNA, an affected mother would pass
the disease to all of her children but only
her daughters would transmit the trait
A number of diseases have now been shown
to be due to mutations of mit DNA
o A variety of myopathies
o Neurologic disorders
o Some cases of diabetes mellitus
 DNA Super coiling
Super coiling means the coiling of a coil
DNA is coiled in the form of a double helix in
which both strands of the DNA coil around
an axis
The further coiling of that axis upon itself
produces DNA super coiling
DNA super coiling is generally a manifestation
of structured strain
When there is no net bending of the DNA axis
upon it self , the DNA is said to be in a
relaxed state.
 DNA compaction involves some form of super
coiling
Replication and transcription of DNA also
affect and are affected by super coiling
Super coiling is an intrinsic property of DNA
tertiary structure
It occurs in all cellular DNAs and is highly
regulated by each cell.
 Chromosomes
DNA is organized in the form of chromosomes
Total number of chromosomes in human beings is
46 arranged in 23 pairs
At metaphase of cell division chromosomes
possess a twofold symmetry with identical
duplicated sister chromatids connected with a
centromere
The relative position of centromere is
characteristic for a given chromosome
The cetromere is an Adenine Thymine (A-T)
rich region ranging in size from 102 (yeast)
to 106(mammals) base pairs.
This complex called the kinetochore provides
the anchor for the mitotic spindle so it is an
essential structure for chromosomal
segregation during mitosis
 Human karyotype

Total chromosomes 46

Arranged in Pairs 23

Autosomes 22
Sex chromosome 01
The ends of each chromosome contain
structures called telomeres
Telomeres help to stabilize chromosomes
Telomeres consists of short , repeat TG- rich
sequences
Human telomeres have a variable number of
repeats of sequences 5/- TTAGGG-3/
Telomerase –a multi sub unit RNA containing
complex related to viral RNA dependent
DNA polymerase (reverse transcriptase) is the
enzyme responsible for telomeres synthesis
and for maintaining the length of telomere
Telomere shortening is associated with
(a) Malignant transformation
(b) Aging
Now a days telomerase has become an attractive
target for cancer chemotherapy and drug
development
Each sister chromatid contains one double
stranded DNA molecule
During inter phase , the packing of the DNA
molecule is less dense than it is in the
condensed chromosomes during metaphase
Metaphase chromosomes are transcriptionaly
inactive
The human haploid genome consists of about
3x109 base pairs and about 1.7x 107
nucleosomes
Each of the 23 chromatids in the human haploid
genome contain on average 1.3x108
nucleotides in one double stranded DNA
molecule
The length of each DNA molecule must be
compressed about 8000 folds to generate the
structure of a condensed metaphase
chromosome
In metaphase chromosomes, the 30nm
chromatin fibers are also folded into a series
of looped domains, the proximal portions
of which are anchored to a non histone
proteinaceous scaffolding within the
nucleus.
The packing ratio of each of the orders of DNA
structures is different and is as follows
Chromatin form Packing Ratio
 Naked double- helical DNA ~1.0
10nm fibril of nucleosomes 7-10
25-30nm chromatin fiber 40-60
of super helical nucleosome
Condensed metaphase 8000
chromosome of loops
The packaging of nucleoproteins within
chromatids is not random and is dependent
upon specie specific characteristics of
DNA molecules
With in a single specie, the pattern of staining
of the entire chromosome complement is
highly reproducible from individual to
individual but it differs significantly from
other species even those closely related.
Genetic material can be altered and rearranged
 An alteration in the sequence of purine and
pyrimidine bases in a gene may result in an
altered gene product
This alteration can be due to a removal or an
insertion of one or more bases
Such alterations in the genetic material result in
a mutation
Chromosomal recombination is one way of
rearranging genetic material.
Genetic information can be exchanged between
similar or homologous chromosomes
This exchange also known as recombination
occurs primarily during meiosis in
mammalian cells
It requires alignment of homologous metaphase
chromosomes & this alignment always occurs
with great exactness
 After alignment process of crossing over occurs
This usually results in an equal and reciprocal
exchange of genetic information between
homologous chromosomes
If homologous chromosomes possess different
alleles of the same genes , the crossing over
may produce noticeable and heritable
genetic linkage differences
In rare cases where alignment of homologous
chromosomes is not exact , the crossing over
may result in an unequal exchange of
information
One chromosome may receive less genetic
material and other partner receives more
genetic material leading to deletion in one
and insertion in other
Unequal crossing over does occur in human
beings e.g resulting in formation of 2 types
of Hb lepore and anti lepore
Genes
Portions of DNA which can direct &
control certain peptide formation
Structural genes
These are responsible for the formation of
a certain peptide by ribosomes
The information is sent to ribosomes Via
mRNA
Control Genes
These regulate the action of structural genes
Two types
1. Operator genes
2. Regulator genes
Operator genes
Present on chromosomes adjacent to
structural genes and function to initiate
synthesis by the structural genes
Regulator genes
It controls the function of operator gene by
inducing the synthesis of repressor
substances
Operon
A cluster of structural genes along with a
single operator gene which regulates their
activity
 Autosomes and Sex chromosomes

Autosomes Ordinary paired chromosomes

Sex chromosomes Determine the sex of an
individual
22 pairs Autosomes
1 pair Sex chromosome XY
XX
 Expressivity
It is the extent to which a heritable trait is
manifested by an individual carrying the
principal gene controlling it.
Expressivity of a gene may vary from person to
person
For example some persons carrying the gene for
blue sclera may have a very pale blue sclera
while in others it may be very dark blue
 
The expressivity of a certain gene may need the
presence of another factor e.g the human
baldness (alopecia) needs the presence of
testosterone in order to express itself
Thus a woman may have gene for baldness but
will not ordinarily become bald but if she is
given testosterone or start producing in her
own body due to some pathology then she
becomes bald.
Allele
One of two or more contrasting genes
situated at the same locus in homologous
chromosomes which determine alternative
characters in inheritance (short or tall
smooth or rough etc)
One of two or more contrasting characters
transmitted by alternative genes
Heterozygous
Possessing a non- identical pair of alleles
with regard to a given character e.g in
sickle cell trait only one of the two
homologous chromosomes carries the gene
and this person is said to be heterozygous
with regard to this gene
Homozygous
Possessing an identical pair of alleles with
regard to a given character e.g in sickle
cell disease both the homologous
chromosomes carry the genes for Hbs,
such a person suffers much more than a
patient of sickle cell trait.
Genome
All the genetic information encoded in a cell or
virus
Dominant gene
It is capable for expressing when carried by only
one of a set of homologous chromosomes e.g
Chondro dystrophic dwarfism
Retinoblastoma
Recessive gene
It is incapable of expression unless carried by
both members of a set of homologous
chromosomes.
The recessive genes include those of
Albinism
Amaurotic idiocy (infantile)
Icthyosis Congentia
Total Colour Blindness
Hemophilia
Out of these , the gene for Hemophilia is a sex
linked gene which is carried by X chromosome
while all others are carried by autosomes.
The human male has only one X chromosome,
therefore if a male carries the hemophilia gene
in his only one X chromosome he will express
this gene i.e he will suffer from Hemophilia
On the other hand the human female has two X
chromosomes, therefore she will not suffer from
Hemophilia if she is heterozygous with regard
to this gene although she can transmit this
gene to her off springs.
In other words she is a carrier of the gene but
not a patient of Hemophilia.
If a women comes to have the Hemophilia gene in
both of her X chromosomes then she will
suffer from Hemophilia it is obvious such a
woman is both a carrier and a patient of
Hemophilia
Genotype
The fundamental hereditary constitution of an
individual is called genotype e.g
Genotype of blood group
O is OO
A can be AA
AO
Phenotype
The outward, visible expression of the
hereditary constitution of an organism e.g
an individual having only one gene for
Hbs will show Sickle Cell Trait which is
his phenotype.
A person suffering from klinefelter’s
syndrome is phenotypically a male , his
genotype is XXY.
 Digestion of Nucleo proteins
Tissue rich in nuclei- liver, kidney
Nucleoproteins
Proteolytic enzymes
Protien (50%) Histones
Protamines
Nucleic acids (polynucleotides)
Nucleic acids (polynucleotides)
Nucleases in pancreatic juice
phosphodiesterases in Succus
entericus.

Mononucleotides
H2O Nucleotidases in Succus entericus

H3PO4

Nucleosides
 Nucleosides
H3PO4 Nucleosidases or
Nucleoside phosphorylases
Sugar PO4 in Succus entericus.

Nitrogenous base

Absorbed into blood stream

Stages

1. Hydrolysis of nucleic acids to

mononucleotides
2. Hydrolysis of mononuleotides to nucleosides
3. Liberation of free nitrogenous base and
phosphorylation of sugar
 Fate of sugar
Enter body pool
Fate of nitrogenous base
(i) Catabolized in the body
Guanine Thymine Uracil
Cytosine Adenine
(ii) Participate in the synthesis of nucleic acids-
Adenine
(iii) Adenine is also converted into Guanine
 Biosynthesis of Purines
They are synthesized as their
mononucleotides from carbohydrate and
protein metabolic products.
Substances required
1. 5-phosphoribosyl 1- pyrophosphate
2. Glutamine- Amide form of glutamic acid
3. Glycine- A non- essential amino acid
4. Activated derivative of tetra hydrofolate
(N-formyl tetra hydrofolate).
Derived from many sources as glycine,
serine, methionine and betaine
5.CO2- Derived from combustion of food
stuffs.
6.Aspartic acid- A non- essential amino acid
7. ATP
 Purine Biosynthesis

Three processes are involved

1. Synthesis de novo
2. Phospho ribosylation of Purines
3. Phosphorylation of Purine Nucleosides
 Synthesis de novo
1.Transfer of pyrophosphate from ATP to C-1
of D- ribose 5-PO4 (I) 5 phospho ribosyl
1-pyrophosphate (PRPP) (II)
PRPP is also an intermediate in NAD+,
NADP+, Pyrimidine biosynthesis and in
Purine salvage pathway
2. Displacement of pyrophosphate from
PRPP by amide nitrogen of glutamine
forms 5-phospho-β- D-ribosyl amine (III)
This reaction involves inversion at C-I and
forms what will become β-N-glycosidic
bond
3. Condensation of (III) with glycine forms
glycinamide ribosyl-5-PO4 (iv)
This reaction adds C-4, 5 and N-7
4. Transfer to (iv) of a formyl (C-I) group
from N5, N10 methenyl- tetro hydrofolate
forms formyl glycinamide ribosyl-5-PO4 (v)
adds C-8
5. Transamidation of (v) by the amide nitrogen
of second glutamine forms formyl
glycinamidine ribosyl-5-PO4 (vi) adds N-3
6. Elimination of water accompanied by ring
closure forms amino imidazole ribosyl-5-
PO4 (vii)
7. Addition of CO2 to (vii) requires neither
ATP nor biotin and forms amino imidazole
carboxylate ribosyl-5 PO4 (Viii) adds C-6
8. Condensation of aspartic acid with (Viii)
forms amino imidazole succinyl
carboxamide ribosyl- 5- PO4 (ix) adds N-I
9. Loss of succinyl group of (ix) as fumarate
forms amino imidazole carboxamide
ribosyl-5-PO4 (x)
10. Formylation of (x) by N10- formyl tetra
hydrofolate forms formimido imidazole
carboxamide ribosyl-5 PO4 (xi) adds C-2
11. Ring closure of (xi) forms the first purine
nucleotide – Inosine monophosphate IMP
(xii) . Oxidation and amination of IMP
forms AMP and GMP
12. Addition of aspartate to IMP forms
adenosyl succinate. Adenoyl succinate
synthatase provides a potential focus for
regulation of adenine nucleotide biosynthesis
13. Release of fumarate forms adenosine 5- mono
phosphate(AMP). It is catalyzed by adenylo
succinase which also catalyze reaction no.9
14. Oxidation of IMP by NAD+ is catalyzed by
IMP dehydrogenase and forms Xanthosine
mono phosphate (XMP)
15. Transamination by amide nitrogen of
glutamine converts XMP into GMP-
Guanosine mono phosphate.
 Conversion of mononucleotides to
Nucleoside di and tri PO4
ATP converts mononucleotides to their
Nucleoside Di and Tri PO4
Nucleoside mono phosphate or mononucleotide
ATP
Kinase
ADP
Nucleoside di phosphate
 Nucleoside Di phosphate
ATP
Kinase
ADP
Nucleoside Tri phosphate
 Energetics
5 High energy phosphate bonds are utilized
in the formation of IMP
1 High energy phosphate bond is utilized in
the conversion of IMP to AMP
2 High energy phosphate bonds are utilized
in the conversion of IMP to GMP
 Purine anti-metabolites
Inhibits Purine biosynthesis
1. Azaserine 5
2. De oxy norleucine (DON) 2
3. 6-Mercaptopurine 13&14
4. Folic acid anagonist 4 &10
Amino pterine
5. Adenine arabinoside
 Biomedical importance
By inhibiting purine biosynthesis they
prevent nucleic acid formation needed
for cellular proliferation and viral
multiplication.
So these compounds are used as Anti cancer
and Anti viral agents.
 
 Salvage reactions
Conversion of purines , purine ribonucleosides
and purine deoxy ribonuclosides to mono
nucleotides involves salvage reactions.
They require less energy
I Phosphorylation of free purine(PU) by PRPP
forming purine 5 mononucleotide (Pu-RP)

PU+PP-RP PU-RP + PPi

Examples
1.Adenine phospho ribosyl transferase
Adenine AMP
2. Hypoxanthine- Guanine phospho ribosyl
transferase
Hypoxanthine IMP
Guanine GMP
II Direct phospho rylation of a purine
ribo nucleoside( PUR by ATP)
PUR+ATP PUR-P+ADP
Examples
1. Adenosine AMP
Adenosine kinase
2. De oxy adenosine d AMP
Adenosine kinase
3. De oxy Cytidine d CMP

4. De oxy Guanosine d GMP

5. De oxy Adenosine d AMP

De oxy Cytidine kinase (iii)(iv)(v)

 Regulation of Nucleotide Biosythesis
1. PRPP pool size regulates purine nucleotide
biosynthesis
PRPP synthetase I is sensitive to phosphate
concentration and purine ribonucleotides that
act as its allosteric regulators
2. AMP and GMP feed back -regulate PRPP
glutamyl amido transferase II
Regulation of purine synthesis by PRPP
synthetase is more important as compared
with 2nd
3. AMP and GMP feed back regulate their
formation from IMP
(i) AMP Adenylo succinate synthetase

(ii) GMP IMP dehydrogenase

(iii)Cross regulation

(iv) AMPand GMP inhibits Hypoxanthine

-Guanine phospho ribosyl transferase
 Biosynthesis of Pyrimidines
Synthesized as mononucleotides
Synthesized from products of protein and
carbohydrate metabolism
These substances are similar to those used in
purine synthesis
They include
I. Glutamine
II. Aspartic acid
III. ATP
IV. PRPP
V. CO2

VI. Tetra hydrofolate

Steps
1. Pyrimidine biosynthesis begins with formation
of Carbamoyl PO4. CO2, Glutamine and ATP
are utilized. This reaction is catalyzed by
cytosolic Carbamoyl PO4 synthetase. This
enzyme is distinct from mitochondrial
enzyme
2. Condensation of Carbamoyl PO4 with Aspartate
to form Carbamoyl aspartate. This reaction is
catalyzed by Aspartate transcarbamoylase
3. Ring closure via loss of water. It is
catalyzed by Dihydro orotase and forms
Dihydro orotic acid
4. Abstraction of hydrogens from C3 and C6
by NAD+ introduces a double bond
forming Orotic acid -a reaction catalyzed
by mitochondrial Dihydroorotic
dehydrogenase
All other enzymes of pyrimidine synthesis
are in cytosol
5. Transfer of Ribose PO4 from PRPP forming
Orotidine mono PO4 (OMP) is catalyzed
by Orotate phosphoribosyl transferase
6. De carboxylation of Orotidylate forms
Uridine mono PO4 (UMP) the first true
pyrimidine ribo nucleotide
 ATP ADP
7. UMP UDP
Kinase

ATP ADP
8. UDP UTP
Kinase
9. UTP is aminated to CTP by Glutamine and
ATP
10. Reduction of ribo nucleoside di PO 4
(NDPs) to their corresponding d NDPs de
oxy ribo nucleosides di PO4
11. d UMP is substrate for TMP so d UDP is
de phosphorylated to d UMP
12. Methylation of d UMP at C-5 by N5 N10
methylene tetra hydro folate forms TMP-
Thymidine mono PO4
Enz. Thymidine synthetase
 Salvage pathway
By salvage reactions 2 pyrimidine nucleosides
and 2 deoxy pyrimidine nucleosides are
converted into their respective nucleotides
1. Uridine and Cytidine
2. Thymidine and Deoxycytidine
 
Regulation of pyrimidine nucleotide biosynthesis
1.  Regulation at gene expression level
2. Regulation of enzyme activity

First 2 enzymes of pyrimidine nucleotide

biosynthesis are sensitive to allosteric
regulation
First 3 and last 2 enzymes of the pathway are
regulated at the genetic level by co-ordinate
repression and de repression
Carbamoyl PO4 synthetase- reaction (I) is
inhibited by UTP and purine nucleotides
but activated by PRPP
Aspartate transcarbamoylase- reaction (2) is
inhibited by CTP and activated by ATP
 Pyrimidine anti metabolites
1. Pyrimidine derivatives containing fluorine ,
bromine and iodine act as powerful anti-
metabolites
I. 5- Fluorouracil
II. 5- Bromouracil
III. 5- Iodouracil
 
2. Folic acid analogs
i. Methotrexate
ii. Aminopterine
3. Cytosine arabinoside

Biomedical importance
 They interfere in the formation of
pyrimidines and act as Anti viral and Anti
cancer agents.
 Formation of dNDPs
 Reduction of NDPs forms dNDPs
Reduction at 2-C of purine and pyrimidine
ribonucloside di PO4 catalyzed by
ribonucleotide reductase complex forms
2- deoxy ribonucloside di phosphate
The enzyme is active only when cells are
actively synthesizing DNA preparatory to
cell division
Reduction requires
i. Thioredoxin- a protein cofactor
ii. Thioredoxin reductase- a flavo protein
iii. NADPH
The immediate reductant of NDPs is reduced
Thioredoxin produced by NADPH-
Thioredoxin reductase catalyzed reaction
 Synthetic pyrimidine and purine analogs
Chemically synthesized analogs of purines and
pyrimidines, their nucleosides and nucleotides
have numerous applications in clinical
medicine and research
Their effect reflects one of two processes
i. Inhibition by the drug of specific enzymes
essential for nucleic acid synthesis
 Incorporation of metabolites of drugs into
ii.
nucleic acids where they effect the base
pairing essential for accurate transfer of
information
Examples
1. 5- Floro or 5-iodo derivative of uracil or
deoxyuridine.
It serves as thymine or thymidine derivative
respectively
2. 6 Thioguanine and 6- mecapto purine
In these thiol group replaces hydroxyl group
at 6-positon

3. 5 or 6 – Azauridine, 5or 6 Azacytidine

and 8- Azaguanine .
In these nitrogen atom replaces a hetrocylic
ring carbon atom
4. Purine analog 4-hydroxy pyrazolo
pyrimidine (allopurinol).
It is used in T/M of hyperuricemia and gout,
inhibits de novo purine biosynthesis and
xanthine oxidase activity
5. The nucleoside cytabine (arabinosyl
cytosine).
In it arabinose replaces ribose
It is used in chemotherapy of cancer and viral
infections
6. Azathioprine
It is catabolized to 6- mercapto purine, is used
in organ transplantation to suppress events
involved in immunological rejection
7. Nucleoside analogs with antiviral activities-
5-iododeoxy uridine .
It is effective in T/M of herpetic keratitis- an
infection of cornea by herpes virus
 Catabolism of purines
 Human beings- purines uric acid
Amphibians, birds & reptiles purines-
uric acid & guanine
Other mammals- purines allantoin
uricase
uric acid allantion
Net excretion of uric acid in
Normal individual 400-600mg/24 hrs
 Pathway
1. Adenosine is first deaminated to Inosine by
Adenosine deaminase
2. Phosphorolysis of the N- glycoside bonds of
Inosine and Guanosine catalyzed by purine
nucleoside phosphorylase, releases ribose-
1- PO4 and a purine base- Hypoxanthine
and Guanine.
3. Hypoxanthine and Guanine form Xanthine
in reactions catalyzed by Xanthine oxidase
and Guanase respectively
 Hypoxanthine Guanine
Xanthine oxidase Guanase
Xanthine
4.Xanthine is oxidized to Uric acid in a
reaction catalyzed by Xanthine oxidase
5.Uric acid is converted into Allantoin by the
action of enzyme uricase.
But this is not present in human beings and
it is only present in some other mammals.
 Inborn errors of purine metabolism
  Gout
This is a disease characterized by episodes
of acute arthritis associated with a raised
uric acid level in blood
Normal serum uric acid level
In Male 2.5-8mg/100ml
In Female 1.5-6mg/100ml
In gout it increase to 2000-4000 31000mg
 Biochemical defect
In gouty patients rate of utilization of glycine
to form purines is usually much increased
due to an inherent metabolic defect. The
cause of gout is therefore an abnormal
increase in the rate of purine synthesis
A secondary hyperlipidemia is also frequently
seen in gout leading to increased risk of
IHD- Ischemic heart disease
Another finding is that patients of gout include
bright and intelligent persons
Large amounts of mono sodium urate is
deposited in the joints and tissues- Tophi.
A frequent complication of gout is the
formation of urate stones in the urinary tract
High serum uric acid levels are also found in
other diseases in which there is an
abnormally great turn over of nucleic acids
e.g Polycythemia, Leukemia and Psoriasis
These conditions are some time
accompanied by gouty arthritis-Secondary
gout
In some cases increased serum uric acid is
due to decrease excretion by kidney .
This is seen during therapy with Thiazide
diuretics and during prolonged Starvation.
In starvation ketone bodies are responsible
for decreased renal excretion of uric acid
Alcohol also decreases renal excretion of
uric acid

Treatment
Allopurinol.
It is compititive inhibitor of xanthine oxidase
and stops conversion of xanthine into uric
acid
 Xanthinuria
In this disease large amounts of xanthine are
excreted in urine .
The uric acid content of the urine is
correspondingly decreased.
Serum uric acid level is also very low. It is
caused by failure of body to convert
xanthine into uric acid
Large amounts of xanthine in urine lead to the
formation of xanthine calculi.
They are not opaque to x-ray and can be
easily missed
Administration of anti gout drug allopurinol
also increases urinary excretion of
xanthine.
 Catabolism of pyrimidines

 The end products of pyrimidine catabolism

are CO2, NH3, β - Alanine and
β- Aminoisobutyrate
They are highly water soluble in contrast to
sparingly water soluble Uric acid-end
product of pruine catabolism
 Steps
1.Cytosine is deaminated to Uracil
2. Uracil is converted into Dihydrouracil
after receiving 2H from NADPH + H+
3. By the addition of H2O Dihydrouracil is
converted into β- Ureidopropionate (N-
carbamoyl- β- alanine)
 4. With the addition of H2O β-
Ureidopropionate is broken down into
CO2 + NH3 + β-Alanine
5. β-Alanine after receiving O2 is converted
into CO2 + NH3 + CH3Coo-
6. Thymine is converted into Dihydrothymine
after receiving 2H from NADPH+ H+
7. Dihydrothymine after receiving H2O is
converted into β- Ureidoisobutyrate
(N- carbamoyl- β- amino- isobutyrate)
8. With the addition of H2O
β- Ureidoisobutyrate is broken down into
CO2 + NH3 + β Aminoisobutyrate
9. β-Aminoisobutyrate is transaminated to
methyl malonate semi aldehyde which
then form succinyl-CoA
 l r o l e o f D N A
Biologica
DNA is the ultimate carrier of heredity in all
eukaryotes and even most prokaryotes
except certain viruses and phages
Genes are composed of DNA in which
genetic information is contained in the
from of codes.
A sequence of 3 nitrogenous bases on DNA
strand contains code for one amino acid .
 Replication of DNA
This occurs during cell division .
The double helix structure is opened up
starting from one end just as a zipper
opens.
Each single strand of DNA then builds up an
exact complementary copy of itself i.e in
which the nitrogenous bases are
complementary to its own nitrogenous bases
Adenine gives rise to Thymine and Guanine
gives rise to Cytosine.
 Two new stands are built up, each one of
which coils around its parent strand .
In newly synthesized DNA, one strand is
derived from the parent DNA but the other
strand in newly formed .
This mode of DNA replication is termed Semi
conservative replication
One of the two strands represents the original
conserved strand , while the second one has
been newly formed.
This mechanism of DNA replication is not
confined only to double stranded DNA. e.g
the single stranded DNA (e.g phage Φ x 174
which infects E.coli:) has a replicative form
that is double stranded.
Even the RNA viruses which have single
stranded RNA as the genome, duplicate
by using a replicative form that is double
stranded
 The process of DNA replication is highly
complex
1. Leading and lagging daughter strands
It has been seen that out of two strands of
DNA which result from replication one
strand is the leading strand while the other
one is lagging .
In the leading strand synthesis of the newly
formed DNA strand occurs in the 5 / to 3 /
direction in a continuous manner .
In the lagging strand a considerable part of
the newly synthesized DNA is made in the
form of discontinuous small fragments
called Okazaki fragments named after
discoverer of this process.
These fragments are also synthesized in the
5/to 3/ direction.
Synthesis is catalyzed by DNA polymerase
III complex .
These fragments are then joined by DNA
ligase to form complete daughter strand.
 2. Multiple replication forks
The unwinding of DNA must take place
before replication .
It is brought about by Helicase which
utilizes ATP as a source of energy.
The reformation of the double helix is
prevented by a special protein namely the
Single stranded DNA binding protein
which binds to the individual strands.
The unwinding of the double strands gives rise
to Replication forks which move in both
directions
These are sites at which the newly formed or
nascent DNA synthesis takes place
A large number of replication forks are needed
because the DNA is a very long molecule.
Replication starts at a specific point and
proceeds in the both directions.
3. The naturally occurring DNA is under
wound
The naturally occurring DNA is under wound.
However when replication starts the DNA
duplex ahead of the replication fork
becomes positively super coiled.
This change is expected to stop replication
however the enzymes called DNA
Topoisomerases relieve positive super
coiling and by cutting, uncoiling and
 repairing the phospho sugar back bone
produce negative super helicity – under
winding which favors replication
4. Synthesis of primers
In E. coli the primer for DNA synthesis is a
small piece of RNA & not DNA .
Two enzymes synthesize the primers
RNA polymerase on the leading strand
Primase on the lagging
strand.
The primer RNA is then removed from both
daughter strands by the 5/ exonuclease
activity of DNA polymerase-I and is
replaced with deoxy ribonucleotides by its
polymerizing activity .
In eukaryotes DNA can also act as the primer
in addition to RNA
 
5. Histone separation
At the same time when there is unwinding of
the duplex DNA in case of eukaryotes,
histones separate from the DNA strands.
These histones are not degraded.
During replication the previously present
histones become associated with the duplex
containing the leading daughter strand
while histones for the duplex containing the
daughter lagging strand are synthesized
de novo
 Enzymatic synthesis of DNA-
Replication
Factors required for in vitro synthesis of DNA
are as follows
1. Enzymes

DNA polymerases
These are isolated from bacterial & animal
sources
2. Four Deoxy ribonucleoside tri PO4
d GTP
d ATP
d CTP
d TTP
3. DNA Template
This DNA will determine type of DNA
formed
4. Mg++ ions
 Initiation
Initiation of DNA synthesis requires priming
by a short length of RNA about 10-200
nucleotide long .
This priming process involves the
nucleophilic attack by the 3/- hydroxyl
group of the RNA primer on the α-PO4 of
Deoxy ribonucleoside tri PO4 with the
splitting up of pyrophosphate .
The 3/- hydroxyl group of recently attached
Deoxy ribonucloside mono PO4 is then free
to carry out a nucleophilic attack on the
next entering Deoxy ribonucleoside tri PO 4
again at its α-PO4 moiety, with the splitting
off Pyrophosphate.
The selection of the proper
Deoxy ribonucleoside tri PO4 whose
terminal α- phosphoryl group is to be
attacked is dependent upon proper pairing
with the other strand of DNA according to
the rules proposed originally by Watson &
Crick.
when an Adenine deoxy ribonucleoside
mono phosphoryl moiety is in the
template position a Thymidine tri PO4
will enter & its α- PO4 will be attacked
by the 3/ hydroxyl group of the
deoxy ribonucleoside mono phosphoryl
most recently added to the polymer.
 Elongation
By stepwise process, the template dictates
which Deoxy ribonucleoside tri-PO4 is
complementary & by H-bonding holds it in
place while the 3/-hydroxyl group of the
growing strand attacks and incorporates the
new nucleotide into the polymer
In this way the strand of newly formed DNA
increases in length.
 Termination
After replication of whole of the DNA strand
the RNA primers are eventually removed
where as after replication of the
mitochondrial genome the small piece of
RNA remains as an integral part of the
closed circular DNA structure.
6. Joining precursor strands

This problem is mainly faced in the lagging

daughter strand synthesis.
The RNA primer is removed by the
5/exonuclease activity of DNA
polymerase-I.
The same enzyme replaces the primer with
Deoxy ribonuceotides by its polymerizing
activity.
The final joining of the fragments is brought
about by the DNA ligase.
DNA ligase can also join DNA strands that
have been nicked
7. Special properties of DNA polymerase
This enzyme is also called kornberg’s
enzyme and it has 3 enzymatic activities in
a single polypeptide chain
(i) It has Polymerase activity & can add
Deoxy ribonucleoside tri PO4 to the
3/-hydroxyl group of a DNA primer chain.
(ii) It has both 5/ as well as 3/ Exonuclease
activity and can cleave nucleotides from
either the 3/ or 5/ ends
In DNA repair system it functions as a
5/- Exonuclease-removes the damaged
sequence and acting as a Polymerase forms
the newly repaired sequence.
(iii) In normal DNA replication it removes the
RNA primer chains by its3/-Exonuclease
activity which is followed by template
directed replacement of the RNA by DNA
due to its polymerase activity
 Proteins involved in replication
Protein Function
DNA polymerase Deoxy ribonucleotide
polymerization
Helicase Processive
unwiding of DNA
Topoisomerase Relieves torsional strain
that result from helicase
induced unwinding
DNA Primase Initiates synthesis of RNA
primers

Single stranded Prevent premature DNA

binding proteins reannealing of ds

DNA
DNA ligase Seals the single strand
nick between the nascent chain
& okazaki fragments on lagging strand
 DNA polymerase complex
A number of different DNA polymerase
molecules are engaged in DNA replication
These share three important properties
Chain elongation
Processivity
Proof reading
Chain elongation accounts for the rate (in
nucleotides per second) at which
polymerization occur
Processivity is an expression of the number
of nucleotides added to the nascent chain
before the polymerase disengages from the
template
The Proof reading function identifies
copying errors and corrects them
In E coli polymerase III functions at the
replication fork
It catalyzes highest rate of chain elongation
and is the most processive
It is capable of polymerizing 0.5 Mb of
DNA during one cycle on the leading
strand
Polymerase III a large ten sub unit protein
complex is the product of the dna E gene
in Ecoli
The 2 identical β- subunits of polymerase III
encircle the DNA template in a sliding
clamp which accounts for the stability of
the complex and for high degree of
prcessivity of the enzyme .
Polymerase II is mostly concerned with
proof reading & DNA repair
Polymerase I completes chain synthesis
between okazaki fragments on the lagging
strand
Eukaryotic cells have counter parts for each
of these enzymes plus some additional ones
Comparison of Prokaryotic & Eukaryotic
DNA polymerase

E.Coli Mammalian Function

1 α Gap filling &
synthesis of
lagging strand
II ε DNA proof
reading & repair

β DNA repair

γ Mitochondrial
DNA synthesis

III δ Processive, leading

strand synthesis
In mammalian cell the polymerase is capable
of polymerizing about 100 nucleotides per
second - a rate at least 10 folds less than
the rate of bacterial DNA polymerase
This decrease is due to interference by
nucleosomes.
 Mistakes in DNA synthesis and their
correction
Rarely during DNA replication the double helix
structure shows abnormality in that the base
facing each other are not complementary
to each other .
Thus A may face C which can not form H-bond
with it
In such cases DNA polymerase-I removes the
mismatched nucleotide
It is often followed by the insertion of a
nucleotide containing the specific
complementary base.
It has been found that a disordered repair of
DNA is the basis of several clinical
conditions. e.g Xerodema pigmentosa
Ataxia telengiectasia
Fanconi’s anemia
It is interesting to note that all these
conditions are associated with a higher
incidence of cancer
 Xeroderma pigmentosa
It is an autosomal recessive genetic disease .
The clinical syndrome includes marked sensitivity
to sunlight (ultra violet light) with subsequent
formation of multiple skin cancers and
ultimate death .
The inherited defect seems to involve in the
repair of damaged DNA.
Cells cultured from patients exhibit low activity
for the photo activated Thymine diamer
cleavage process
 Ataxia- Telangiectasia
It is an autosomal recessive disease in humans
resulting in the development of cerebellar
ataxia and lympho reticular neoplasms.
There appears to exist an increased sensitivity
to damage by x-rays.
 Fanconi’s Anemia
 Autosomal recessive anemia characterized by
increase frequency of cancer &
chromosomal instability.
It has defective repair of cross linking damage.
 Damage to DNA
I Single base alteration
a. Depurination
b. Deamination of Cytosine to Uracil
c. Deamination of Adenine to Hypoxanthine
d. Alkylation of bases
e. Insertion or deletion of nucleotide
f. Base analog incorporation
 II Two base alteration
a. U V light induced Thymine- Thymine
diamer
b. Bifunctional alkylating agent cross linkages

III Chain breaks

a. By ionizing radiation
b. Radioactive disintegration of back bone
element
 IV. Cross linkages
a. Between bases in same or opposite strands
b. Between DNA & protein molecules
-Histones
 1 Mismatch repair
Mismatch repair corrects errors made when
DNA is copied e.g
i C could be inserted opposite an A
ii The polymerase could slip or stutter and
insert 2 to 5 extra unpaired bases
Repair
Specific proteins scan the newly synthesized
DNA using Adenine methylation with a
GATC sequence as the point of reference
Template strand is methylated while newly
synthesized strand is not.
This difference allows the repair enzymes to
identify the strand that contain errant
nucleotide which requires replacement.
If a mismatch or small loop is identified, a
GATC endonuclease cuts the strand
bearing the mutation at a site
corresponding to the GATC
An exonuclease then digests this strand
from the GATC site,through the mutation
thus removing the faulty DNA
This can occur from either end if the defect
is bracketed by two GATC sites.
This defect is then filled in by normal
cellular enzymes according to base
pairing rules.
 2. Base excision repair
The depurination of DNA which happens
spontaneously owing to the thermal
lability of the purine N- glycosidic bond
occurs at a rate of 5000- 10000/ cell/d at
37oc
Specific enzymes recognize a depurination
site and replace the appropriate purine
directly without interruption of the
phospho diester back bone
Cytosine, Adenine & Guanine bases in DNA
spontaneously form Uracil, Hypoxanthine
or Xanthine respectively.
Since normally these are not present in DNA
so specific N- glycosylases recognize these
abnormal bases and remove the bases from
DNA
This removal marks the site of defect
Then an Apurinic or Apyrimidinic
endonuclease excise the Abasic sugar.
The proper base is then replaced by a repair
DNA polymerase
Then a Ligase returns the DNA to its original
state
This series of events is known as Base
excision repair.
By a similar series of steps involving initially
the recognition of defect, Alkylated bases
and Analogs can be removed from DNA
and the DNA returned to its original
content
This mechanism is only suitable for
replacement of a single base but is not
effective at replacing regions of damaged
DNA
 3. Nuecleotide excision repair
This mechanism is used to replace regions of
damaged DNA up to 30 Bases in length
Common examples of DNA damage include
(i) Ultra violet light -Which induces formation
of Cyclobutane pyrimidine - pyramidine
diamer
(ii) Smoking - which causes formation of
Benzo pyrene- guanine adducts
(iii) Ionizing radiation
Cancer chemotherapeutic agents
Variety of chemicals found in environment
These can cause Base modifications, Strand
breaks, Cross linkage between bases of
apposite strands or between DNA and
proteins
These all are repaired by Nucleotide excision
repair system
This complex process involves the hydrolysis
of two phospho diester bonds on the strand
containing the defect.
A special excision nuclease ( exi nuclease )
consisting of at least 3 subunits in E coli
and 16 polypeptides in humans perform this
important task
In eukaryotic cells enzymes cut between 3rd to
5th phospho diester bond 3/ from the lesion
and on 5 / side the cut is between 21st and
25th bond.
Thus a fragment of DNA 27-29 nucleotides
long is excised
After the strand is removed it is replaced by
exact base pairing through the action of
another Polymerase (δ /ε in humans) & the
ends are joined to the existing strands by
DNA Ligase.
 4. Double strand break repair

The repair of double strand break is part of the

physiologic process of immunoglobulin
gene rearrangements.
It is also important for repairing damaged DNA
as a result of
(i) Ionizing radiation
(ii) Oxidative free radical generation.
Some chemotherapeutic agents destroy cells
by causing double strand breaks or preventing
their repair
Two proteins are initially involved in non
homologous rejoining of a double strand
break
(i) Ku - a heterodiamer of 70 & 86 KDa subunit
It binds to free DNA ends & has latent
ATP -dependent Helicase activity
The DNA- bound Ku hetrodiamer recruits an
unusual DNA- dependent protein kinase (
DNA- PK)
DNA- PK has a binding site for DNA free
ends & other for double stranded DNA
just inside these ends.
It therefore allows the approximation of the
two separated ends
The free end DNA- Ku - DNA- PK complex
activates the kinase activity in latter
DNA- PK phosphorylates Ku & the other
DNA- PK molecule on the apposing strand
in trans.
DNA PK then dissociates from the DNA & Ku
and results in activation of Ku helicase
This results in unwinding of the two ends.
The unwound , approximated DNA forms base
pairs, the extra nucleotide tails are removed
by an exonuclease, and the gaps are filled
and closed by DNA ligase.
 Enzymatic synthesis of RNA

The important factors required are as

follows:

(i) RNA- polymerase

It does not need a primer and does not possess
nuclease activity
(ii) Ribo nucleoside tri PO4

ATP
GTP
CTP
UTP
(iii) DNA
It acts as the template
 Formation of messenger RNA (mRNA)
  TRANSCRIPTION
It is the process of formation of mRNA .
DNA contains genes & is present in nucleus
so nucleus is called information center of
cell.
This information is sent in the form of mRNA
molecules to the ribosomes which are the
sites for protein synthesis in cytoplasm.
The mRNA is formed under the influence of
DNA in nearly the same way as DNA
replicates itself.
But RNA has Ribose instead of Deoxy
ribose & Uracil instead of Thymine .
RNA is a smaller molecule as compared with
DNA
 Initiation
In the formation of mRNA the first step is the
binding of DNA dependent RNA
polymerase to the DNA under the influence
of start signals on specific sites on DNA
molecule called the promoter regions.
The DNA dependent RNA polymerase is made
up of a Core molecule having 4
subunits (α,α,β,β1) and 2 Zn molecules .
The binding of RNA polymerase to the
promoter is brought about by one of the
sigma factors which are many in number
each being specific for binding the core
enzyme to specific sites (promoters) on
DNA .
The core enzyme along with sigma factors is
called the RNA polymerase holoenzyme
This leads to the opening up of the double
helix of DNA.
One of the two strands of DNA gives rise to
mRNA molecule just as in DNA
replication it gives rise to DNA strand .
This strand is called the template strand while
the other DNA strand is called the coding
strand for that gene.
The template strand for another mRNA may be
the one that is the coding strand for a
different mRNA .
Thus each strand of duplex DNA acts as
template strand for some mRNAs & coding
strand for other mRNAs
 Elongation

The initiation of mRNA formation is

followed by chain elongation resulting in a
progressive increase the length of the
mRNA molecule.
In this process the elongation complex
containing the core enzyme moves along the
DNA molecule which goes on un
winding & giving rise to phospho diester
linkages between the ribo nucleoside tri PO4
complementary to the bases of DNA .
It has been found out that one RNA
polymerase by its so called unwindase activity
opens up 17 base pairs of the template DNA
strand.
 Termination
When the molecule of mRNA has reached the
desired length ,it separates from the DNA
strand.
This is brought about by specific termination
signals on DNA molecule.
It is followed by the recoiling of the two
strands of this portion of DNA molecule
undergoing transcription thus restoring the
original state.
A protein termed rho has been isolated.
It binds to RNA polymerase and can sense
Terminating signals on DNA.
After termination of the mRNA synthesis
their core enzyme separates from the
DNA template strand.
However with the help of another sigma
factor the core enzyme gets bound to
another promoter and thus a new mRNA
synthesis starts at different place.
 Pribnow box, tata box and other boxes
It is found out that about 10 base pairs
upstream of the promoter there is an A-T
rich sequence which due to lack of G-C
pairs has low melting point .
This A-T sequence comprises 6 bases
i.e 5/ TATAAT-3/ which has been termed
pribnow box in prokaryotes .
This box guide RNA polymerase for
attachment to the promoter.
A second nucleotide sequence
5/ - TGTTGACA-3 / located up to 35 bases
upstream also help in this process.
The easy melting property of pribnow box
facilitates the unwinding of the DNA
duplex for RNA polymerase holoenzyme
to start synthesizing the mRNA on the
template strand.
This work was done in E coli, other bacteria
have different boxes.
In eukaryotes DNA regions corresponding to
pribnow box and 5/TGTTGACA-3 / sequences
are present , though at higher upstream levels
& have been designated TATA or Hogner
box & CAAT box respectively & serve the
same purpose.
 Enhancers & Silencers
These factors are capable of increasing or
decreasing the rate of transcription in
eukaryotes .
These can occur both upstream and
downstream from the promoter and may be
quite far away .
Mechanism of action is still not fully known
but Steroids, T4, C-AMP, Retinoic acid etc
act on the genome via these factors.
 Processing of RNA molecule
With the exception of prokaryote mRNA all
other RNA molecules i.e t RNA ,r RNA in
prokaryotes & m RNA, t RNA, r RNA &
5S RNA in eukaryotes undergo processing
before they become functional.
It has been shown that mRNA produced in
the nucleus is of high mol wt. as compared
to mRNA which goes to cytoplasm for
protein synthesis.
The heterogeneous nuclear RNA is abbreviated
as hn RNA & it is processed & modified to
mRNA .
The mRNA is a much smaller molecule than
DNA because only a small part of the DNA
molecule is transcribed as mRNA molecule
A sequence of three successive bases on mRNA
molecule is termed as Codon .
It directs the entry of a specific amino acid
into the peptide chain growing at the
ribosomes
Each mRNA is specific for a single peptide
chain.
The turn over rate of mRNA is very high &
it has to be synthesized continuously
t RNA& r RNA are also produced by the
same mechanism .
In animal cell there are DNA-dependent RNA
synthesizing systems.
Three enzymes DNA dependent RNA
polymerase I (A) II(B) III(C) are present.
Enzymes
I Catalyzes the synthesis of r RNA
II Catalyzes the synthesis of m RNA
III Catalyzes the synthesis of t RNA
& 5S RNA
 Introns & Exons
 Human beings possess sufficient amount of
DNA to code for 3 million pairs of genes.
However the number of proteins in man
appear to be at the most 50,000.
Thus it is obvious that large areas of DNA
molecule does not give rise to active
mRNA.
In other words these areas do not act as genes
unlike other areas which act as genes
It has been shown that long sequences of DNA
that do not contribute to genetic information
ultimately translated into the amino acid
sequences of protein (introns) occur as
interspersed within the amino acid coding
portions (exons) of many genes of
eukaryotes.
The primary mRNA molecules i.e the
transcripts of the structural genes contain
bases complementary to the exons as well as
the interspersed introns.
Before the mRNA leaves the nucleus the
RNA sequences formed under the
direction of introns are cleaved out of the
transcript & the RNA sequences made
under direction of the exons are spliced
together .
The conversion of the primary transcript into
mRNA is done by special structures
termed spliceosomes which comprise the
primary transcript, 4 small ribonucleases
and an unknown number of proteins .
During this process RNA molecule undergoes
a change in shape resembling a lariat (rope
with a loop at one end ) of an exons -
directed RNA segment.
Further changes undergone by the primary
transcript before it leaves the nucleus as m
RNA include the followings
(i) Capping of molecule with 7- methyl
guanosine attached through a tri PO4
linkage to the 5/ terminal end of mRNA
This capping helps stabilizing mRNA and
facilitates the start of translation
(ii) Addition of a poly (A) chain occurs in most
but not all cases.
The poly (A) tail has a chain of 40 to 200
adenine nucleotides attached to the 3/ end.
This tail may serve to stabilize the mRNA and
facilitate its exit from the nucleus .
After the mRNA enters the cytoplasm this poly
(A) tail is gradually shortened
 Types of Introns
There are four groups of introns

Group I.
It is self- splicing and no protein enzymes
are involved in its splicing activity.
ATP is also not needed .
However , it requires a guanine nucleoside or
nucleotide.
 Group II.
It resembles group I introns except for the
identity of the nucleophile in the first step and
formation of a lariat- like intermediate.
The intrinsic enzymatic activity of group I and
II introns was the first demonstration of the
fact, that some RNAs too can alone act as
enzymes (termed ribozymes).
 Group III.
This group includes those found in nuclear
mRNA primary transcript.
Their removal is brought about by a large
protein RNA complex called spliceosome,
and for this reason these introns are called
spliceomal introns.
 Group. IV
These introns are spliced by an endonuclease
that needs ATP.
This group is the only type of intron known to be
spliced by a protein enzyme .
After the introns have been spliced , the two
ends of the exons are joined by a
mechanism similar to the DNA ligase
reaction.
The introns remain in the nucleus and are
eventually degraded.
It should be noted that genes for histones
have no introns.
 Enzymatic functions of RNA

Certain RNA molecules have been shown to

act as enzymes e.g. small nuclear RNA
molecules probably catalyze the splicing
out of intron directed transcripts.
 Formation of t RNA
These molecules are transcribed as large
precursors.
Frequently these precursor molecules contain
the sequences of more than one t RNA .
A specific class of RNA-ase brings about
nucleolytic processing and the molecules
are reduced in size
The t RNA molecules are further modified .
This includes methylation, deamination or
reduction that probably occur within the
nucleus .
The attachment of characteristic C-C-A
terminus at the 3/ end of the molecule
takes place in the cytoplasm
Modifications in post transcriptional reactions
give rise to modified bases such as
Pseudouridine Thiouridine
Inosine Methylguanosine
Ribothiouridine Dihydrouridine
N6- isopentenyladenosine
 Formation of r RNA
The genes for r RNA are present in the
nucleolus of the cell and hundreds of
copies of these genes occur in every cell to
provide large amount of r RNA for
producing about 107 ribosomes required
for each cell replication.
In mammalian cells there are 3 types of r
RNA which are transcribed within the
nucleolus to form the ribosomal subunits
found in the cytoplasm.
The primary transcript is 45 S molecule
that undergoes extensive methylation in
the nucleolus.

It gives rise to the following Segments:

 (i) 28S segments
These segments self assemble with
ribosomal proteins to form the larger 60S
subunits
(ii) The 5.8 S r RNA
Becomes part of the larger ribosomal subunit
(iii) The 18S r RNA molecule
Associates with appropriate ribosomal proteins
to give rise to the small (40 S) ribosomal
subunit.
(iv) The 5 S r RNA is mostly produced as a
seperate transcript by RNA polymerase III
instead of RNA polymerase I
Becomes part of the larger ribosomal subunit
 Role of RNA as the genetic material
In higher animals including man the flow of
genetic information is from DNA to RNA -
Transcription
Then from RNA to protein synthesis - Translation
In many viruses the genetic material is represented
by RNA there being no DNA present in these
viruses .
The viral RNAs have been found to transmit their
genetic information by following mechanisms
1. RNA RNA protein pathway
Poliomyelitis virus represents this class of
viruses .
The virus contains an enzyme RNA
-dependent RNA – polymerase also called
Replicase, which catalyzes the synthesis of
viral RNA in the host cell.
In this way parent RNA is copied to give rise
to the progeny virus particles
2. RNA DNA RNA protein pathway

The RNA tumor viruses which comprise a

group of animal viruses which transmit the
genetic information by this mechanism
They comprise a group of animal viruses
casing leukemia and sarcoma in animals
I. Avian myeloblastosis
II. Virus causing chick leukemia
III. Rous sarcoma virus causing sarcoma in
chicks
IV. Murine & feline leukemia viruses causing
leukemia in mice & cats respectively
V. Viruses causing sarcoma in monkeys
After entry into the host cell these viruses
release their RNA & an enzyme named
RNA directed DNA polymerase also called
Reverse transcriptase.
This enzyme catalyzes the formation of DNA
from the RNA template.
DNA is complementary to the viral RNA & is
also called the pro viral DNA or pro virus
The pro viral DNA persists in the host cell in a
stable form and codes for RNA & protein
components of the virus both of which
combine to produce the viral progeny .
The discovery of reverse transcriptase is of
great interest in the area of molecular
biology & it helped in the synthesis of genes
i.e. DNA segments from purified mRNA
3. Synthesis of double stranded DNA
from mRNA

This is done is following way

I. mRNA is hybridized at its poly A tail with a

short chain of dT that acts as primer for
reverse transcriptase
II. Reverse transcriptase is used to synthesize
complementary DNA –(c DNA)
III. mRNA is degraded & the single DNA
strand is left behind
IV. DNA polymerase is used to synthesize the
double stranded DNA from the c DNA
strand.
 Synthesis of proteins by the cell
Translation

 The protein synthesis takes place in ribosomes

present in the cytoplasm.
Ribosomes in the case of mammals are
assembled in the nucleoli .
The information contained in the genes ( which
are parts of DNA molecules) is transmitted
to the cytoplasm in the form of mRNA.
The amino acids are brought to the ribosomes
in the form of complexes of amino acids
with specific t RNAs
The amino acids are then transferred to the
ribosomes where they are incorporated
into peptide chains.
This process has been termed translation of
genetic code
Protein synthesis is highly complex process
& is primarily regulated by DNA
A large number of substances take part in this
process in a highly organized &
coordinated way.

(i) Amino acids:

There are 20 amino acids which act as the
primary building blocks in protein synthesis
by combining with each other through
peptide linkage
(ii) Ribosomes:
These have a size of about 200Ao & a mol. wt of
2.5 million.
They are composed of 2/3 RNA & 1/3 protein.
The ribosomes have the property of undergoing
reversibly Dissociation & Association.
In case of E coli, the associated state of
ribosomes possess an S value of 70
On dissociation it gives rise to 2 components,
one having a value of 30S & the second
having a value of 50S
The component with 30S has only one
molecule (16S) of r RNA & about 21
proteins
The 50S component has 2 molecules of r RNA
(23S &5S) and about 34 proteins
Thus ribosome in the associated state has 3
molecule of r RNA and about 55 proteins
With one exception each ribosomal protein is
present in a single copy for ribosomes.
In higher animals
1.Associated form 80S
2.Dissociated form
2 components 40S
60 S
Ribosomes have two types of sites
1. A site Aminoacyl site
2. P site Peptidyl site
 
The A site binds the amino acid t RNA
complex which then is translocated to the
P site where lengthening of the peptide
chain takes place.
3. Messenger RNA (mRNA)
 mRNA is produced in the cell nucleus under
guidance of DNA this process is called
Transcription
The mRNA then leaves the nucleus & enters the
cytoplasm where it gets associated with
ribosomes.
mRNA has the remarkable property that its
three consecutive bases act as codons for
various amino acids.
In addition it has codons for initiation of peptide
synthesis & also for termination
Codons
Three successive nitrogenous base triplet in a
molecule of mRNA is know as a codon.
It is responsible for the incorporation of one
particular amino acid in the protein molecule.
This was first shown by experiment in which an
RNA molecule containing only uracil bases
( poly-U) was used to synthesize protein in
vitro
It was seen that a polypeptide consisting only
Phenylalanine resulted.
Thus it was concluded that the UUU is the code
for the amino acid Phenylalanine.
The mRNAs containing Adenylate (poly A) &
Cytidylate (poly C) caused the formation of
peptide chains containing Lysine & Proline
respectively.
Thus the triplets AAA & CCC coded for
Lysine & Proline respectively.
Now the triplets of other amino acids are also
found out .
There are 4 types of bases in mRNA
A, G, C & U.
These are arranged in triplet forms having as
many different sequences as possible =64
different triplets
There are only 20 amino acids that participate
in protein synthesis so most of the amino
acids have more than one codon.
  Characteristics of genetic Codon
(i) Codon Assignments
Out of 64 combinations of 3 bases , 61 have
been assigned to the coding of amino acids
Three triplets UGA,UAG,UAA do not act
as codons for amino acids but act as stop
signals for chain termination
Two triplets AUG& GUG act as codon for
methionine & valine respectively
In addition AUG & less frequently GUG also
signals initiation of peptide chain.
The codon AUG at the beginning of the
message is recognized by a special initiation
t RNA .
In bacteria this t RNA carries an N- formylated
methionine.
The formyl group is added to the methionine
after that amino acid is attached to the
initiatior t RNA by the enzyme
Transformylase which uses N- Formyl
tetrahydrofolate as the carbon donor.
In eukaryotes the initatior t RNA carries a
methionine that is not formylated .
In both prokaryote & eukaryote cells this N-
terminal methionine may be removed before
the functional protein has been completed.
(ii) Degeneracy or Redundance of the code
There are 61 codons for amino acids but there
are only 20 amino acids to be coded.
So some amino acids are coded by more than 1
codon, this is called code degeneracy.
e.g. all amino acids except methionine &
tryptophan have more than one codon.
The codons which code for the same amino acid
are called synonyms.
Most synonyms differ only in the third base of
the codon- wobble base 
(iii) Non ambiguity or specificity of the code
This means that a given codon codes for only
one amino acid

(iv) Universality
The genetic code is same for all species of
plants, animals, viruses & bacteria, except
in case of mitochondrial DNA in mammals
(v) Co- linearity of gene & product
In prokaryotes the product of a gene is the
peptide specified by the base sequence in
the gene.
In eukaryotes the product of a gene is the
peptide specified by the base sequence of
the exon regions of the gene , the intron
parts are transcribed but are spliced out
(vi) Non- over lapping & comma less
The code is read from a fixed starting point as a
continuous sequence of bases taken three at
a time. For example AUUCGCAUAGGU
will be read as AUUCGCAUAGGU with out
any punctuation e.g. comma between the
codons
It is obvious that if one or two nucleotides are
either deleted from or added to the mRNA at
some place by mistake
 It will result in altering of the reading frame
and resulting peptide may come to have
different primary structure from this point.
e.g .AAAAUCGAC
If U is deleted then codes will be
AAAACGACX
If G is inserted then codes will be
AAAAGUGCAC
 Wobble
In certain cases more than one codon of a
certain amino acid can bind to a single anti
codon because the third base of the codon is
not as critically important as the first two
bases.
This property is called wobble & the base is
called wobble base e.g. the two codons for
arginine AGA& AGG can bind to the same
anticodon UCU although anti codon for the
latter ( i.e AGG) should theoretically be
UCC.
Similarly three codons for glycine GGU, GGC
and GGA, can bind to one anti codon, CCI
although theoretically the anti codons for
these three codons should be CCA, CCG &
CCU respectively .
It is important to note that I (inosine) is not the
usual but a peculiar base appearing in this
t RNA molecule.
4. Transfer RNAs
These are single chain RNA molecules of
relatively smaller mol wt.
There is a separate t RNA for each of the 20
amino acids for which they act as the
adapter molecules.
The t RNA were first described as having a
clover- leaf shape, but now they have
been shown to possess L-shape .
Although these molecules are single stranded
but they do show base pairing in many
regions where complementary bases situated
on their molecules come in close
approximation with each other .
In addition to the usual bases present in RNA,
the t RNA molecules show the presence of
unusual bases e.g dihydrouracil, inosine,
pseudourdine etc.
Because t RNA is single stranded therefore it
possess two free ends .
One end represent 5/ end to which is usually
attached Guanine.
The other end is the 3/ end where an Amino acid
specific for the t RNA gets attached in the
first stage of protein synthesis - Stage of
activation
This 3 / end has the terminal base sequence as
Cytosine - Cytosine - Adenine or CCA in all
cases
t RNA has a small region in its molecule
which is termed the anti codon which acts
as the recognition site for the template
(mRNA).
The anti codon has sequence of 3 bases which
give the t RNA the specific property to
recognize the site of codon present over the
mRNA.
The codon also possesses a sequence of 3
bases which are complementary to anti
codon
 
5. Enzymes & proteins
Various steps of protein synthesis need
specific enzymes & specific proteins

6. High energy phosphate compounds

ATP
GTP
 Stage in protein synthesis (prokaryotes)

There are 4 stages in protein synthesis

1. Activation of amino acids
2. Initiation
3. Elongation
4. Termination
 (i) Activation of amino acids
This means formation of amino acyl-tRNA
complexes.
It needs ATP & is catalyzed by amino acyl-
tRNA synthetases which are highly specific

Amino acid + tRNA +ATP Amino acyl -

tRNA complex +AMP+PPi
The amino acid is attached through an ester
linkage to the C No 3 of the ribose part of
the 3/ terminal adenosine of the t RNA
specific for the amino acid .
Hydrolysis of pyrophosphate (PPi) drives
the reaction to the right.
 (ii) Initiation
This is start of peptide chain formation .
It is the 5/ terminus also called the cap
terminus of mRNA where protein synthesis
is initiated.
In all these cases it is the complex of formyl
methionine with its specific t RNA that is
first of all brought to the ribosomes to
initiate synthesis.
This t RNA which is called t RNAf is different
from the t RNA which inserts methionine in
internal positions in the peptide chain the
later is called t RNAm.
The formation of formyl- methionine- tRNA
complex takes place in 2stage.
1. Methionine + t RNA methionine-tRNA
Enzyme. Amino acid – t RNA synthetase
It is same for both t RNA f and t RNA m
2. Addition of formyl group to tRNA
Methionine- tRNA+N-formyl tetrahydrofolate

Formyl methionine- tRNA + tetrahydrofolated

The formyl methionine tRNA complex thus
formed is attached to the P site of the
ribosomal component of 30S and forms
what is called 30S initiation complex.
The A-site remains empty .
This step needs Mg++ ions.
GTP & 3 Protein factors termed initiation
factors 1,2& 3 and one of the 2 codons for
initiation AUG & GUG are also needed
This is followed by an association of 30S&
50S components of ribosomes in 1:1
molar ratio forming 70S initiation
complex
During this association reaction GTP is
hydrolyzed to GDP & Pi which along
with initiation factors No 1 & 2 are
released from the complex.
 (iii).Elongation
This needs 3 protein factors termed elongation
factors Tu, Ts & G.
Steps include
(i) The new (second) specific amino acid – t
RNA complex comes to empty A site of
the ribosomes as guided by the anti codon
on its t RNA
It gets attached to A-site because the
complementary codon is present on the
mRNA region facing it.
(ii) A peptide bond in created between this
second amino acyl2 –tRNA2 at A site &
methionine (present as formyl methionine –
t RNA at P site.
(iii) The t RNA of formyl methionine- t RNA
complex is split off & the P-site becomes
empty
(iv) The ribosome moves one codon towards 3 /
end. The P-site is now at codon 2& t-RNA2-
amino acyl2 – formyl methionine complex
becomes translocated on this site catalyzed
by the enzyme translocase
(v) Due to translocation of the dipeptide-tRNA 2
complex to the P-site , A-site becomes empty.
The movement of the ribosomes by one codon
at the 3/ end brings the A-site now at codon 3
The corresponding amino- acid t RNA3 becomes
attached at site A
Codon 3 attracts the third amino acyl3 t RNA3
having the complementary anti codon at site A
This amino acid now forms a peptide bond with
the second amino acid of the dipeptide-tRNA 2
at the P site .
The ribosome moves by still another codon & the
P- site again becomes free by the splitting up
of the t RNA of the amino acid No-2
The t RNA3 along with attached tri peptide at
A-site is then translocated to P-site .
Site A becomes available for forth amino
acyl-t RNA complex
All these steps are repeated again & again.
The peptide chain goes on lengthening
It is important to remember that peptide chain
lengthening occurs at A site & movement
of the ribosome by one codon vacates A
site & peptide chain get attached to P site.
Attachment of amino acyl t RNA to the A site
& translocation of the growing peptide
chain to P-site each needs hydrolysis of one
GTP to GDP &Pi
 (iv) Termination
This means stoppage of further elongation of
the peptide chain.
Three codons UAA, UAG & UGA act as the
stop signals.
These codons present on the mRNAs do not
have any t RNAs that can pair with them
Therefore they do not allow the attachment
of t RNA anticodons to the codons of
mRNA.
Special proteins termed release factors 1,2& 3
are bound to the termination codons & are
involved in the detection of these signals.
Termination is associated with the activation of
the enzyme peptidyl transferase which now
act in reverse.
When the last amino acid t RNA complex has
become attached to the ribosome, it brings
about hydrolytic cleavage of the ester bond
between the t RNA & its corresponding
amino acid.
The peptide leaves the ribosomes .
Later t RNA left behind is also split off.
The ribosome now dissociates into 30S & 50S
components and can restart another cycle of
peptide synthesis.
 Modification of peptide chain after synthesis
Post translational changes
Many proteins undergo modifications after
their molecules have been synthesized by
the above mentioned process.
Following changes can be brought about in the
newly synthesized peptide chain
(i) Formyl group of formyl methionine or even
methionine present as the first amino acid
in the peptide chain is split off
(ii) Disulfide (-S-S) bonds may be created
between adjacent cysteine residues
(iii) Proline may be hydroxylated to
hydroxyproline
(iv) Carbohydrates, prosthetic groups,
coenzymes and PO4 groups may be added to
the peptide
(v) The peptide molecule may be cleaved at
certain sites e.g pro insulin in changed to later
by loss of a peptide fragment from its
molecule.
 Interference with protein synthesis
Many agents interfere with protein synthesis at
different steps & inhibit it
(i) Streptomycin
It inhibits initiation
It also results in misreading of codons
(ii)Tetracyclines
These become attached to 30S ribosomal
component & inhibit its binding to amino acid
t RNA
(iii) Chloramphenicol
It inhibits peptidyl transferase thus termination
step does not take place.

(iv) Puromycin
Prevent the termination step
(v) Erythromycin
It binds with 50S ribosomal component.
This results in an inhibition of translocation
 Translation process (protein synthesis) in
Eukaryotes
The eukaryotes being much more complex than
prokaryotes has many difference in protein
synthesis from that occurring in
prokaryotes.

Salient difference are as follows:

 (i)AUG is the only initiating codon
(ii) The initiating amino acid is methionine
instead of formyl methionine
(iii) There are two RNA molecules that recognize
AUG codons i.e met t RNAf or met- t RNAi
The internal AUGs are recognize by
met - t RNAi
(iv) The ribosomal subunits are 40S & 60S.
When associated give rise to 80S ribosome
(v) The mRNA has a cap structure which is
important for initiation. The mRNA binding is
initiated by a cap binding protein by
interacting with the 5/ cap structure of mRNA
(vi) The number of initiation factors is much
higher, at least 10
(vii) ATP is required for initiation
(viii) The mRNA has an untranslated region at
5/ end which may be more than 700
nucleotides long.
Initiation occurs at the first AUG codon in
more than 90% cases
(ix) A single release factor recognizes all three
termination codons.