Nucleotide chemistry

• Purine and pyrimidine bases and their major

derivatives, the nucleosides and nucleotides are the
building units of nucleic acids.
• There are 2 main types of nucleic acids:-
1 Deoxyribonucleic acid (DNA)
2 Ribonucleic acid (RNA)
• The building unit of nucleic acid is the nucleotide , it
consists of ;
1 Nitrogenous base (purine or pyrimidine base)
2 Pentose sugar (ribose or deoxyribose)
3 Phosphate group.
 Nitrogenous bases (purine and
pyrimidine bases)
• They are heterocyclic ring compounds (contain
carbon & nitrogen).
• There are 2 classes according to their abundance
in cells.
Major bases :- present in large amount ,they
include :
– Purines: - adenine, guanine present both in DNA and
RNA.
– Pyrimidines :-
• Cytosine: - present both in DNA and RNA.
• Thymine: - present in DNA only.
• Uracil: - present in RNA only
 Nucleosides
• It consists of base and sugar (ribose or deoxyribose).
• Numbering of the sugar atoms employs a prime (e.g 3` or 5`)
to distinguish sugar atoms from those of the base.
 Nucleotides
• The nucleotide consists of; Base, sugar and
phosphate.
• Phosphate is attached to C-5 of the sugar.
 Nucleic acid (polynucleotide)

Nucleotide

Nucleoside Phosphate

Nitrogenous base Pentose sugar

(purine or pyrimidine) (ribose or deoxyribose)
 Functions of nucleotides
A. Both Purine and pyrimidine nucleotides enter
in structure of nucleic acids; DNA and RNA.
B. nucleotides enter in the structure of :-
1. High energy stores ATP and GTP.
2. Intracellular signals: - cAMP and cGMP act as 2nd
messenger for many hormones.
3. Enter in the structure of many coenzymes such as
NAD, NADP, FAD and FMN which act as hydrogen
carrier.
COASH, which act as acid carrier.
 1) DNA (Deoxyribonucleic acid)
❑ Site;
• In eukaryoteic cells: DNA is
found in
– Nucleus (called nuclear DNA).
– Mitochondria (called mt. DNA).
• In prokaryotic cells:
– There is a single chromosome
that contains DNA.
– And a non-chromosomal DNA
called plasmid.
❑ Structure;
1. The monomeric nucleotides
of DNA are linked
together by 3`,
5`phosphodiester bonds.
The polymer posses a polarity i.e one end with 3`-hydroxyl and the other end
with 5`-phosphate. The bases located along the DNA strand always read from the
5` end of the chain to the 3` end.
DNA is a double stranded DNA molecule: -
– The 2 strands are held
together by hydrogen
bonds between the bases
(A with T and G with C).
– They are anti-parallel i.e
one strand runs from 3`⭢
5` and the other runs from
the 5`⭢ 3`.
• Pairing rule:-
– The bases are paired; A
with T and G with C. There
are 2 hydrogen bonds
between A and T bases
(A=T) and 3 between G and
C bases (G ≡ C).
• Double-stranded DNA consists of: -
– The template strand: - Is that copied during nucleic acid
synthesis.
– The coding strand: - It is so named because it matches the
RNA transcript that encodes the protein.
 Functions of the DNA
• The genetic information stored in the nucleotide
sequence of DNA serves 2 purposes:-
1) transcription i.e formation of RNA from DNA.
2) replication i.e formation of new DNA from parent
DNA.
• Replication of DNA is semiconservative i.e each
daughter DNA molecule contains one strand from
the parent double-stranded DNA and a new
complementary strand.
 Differences between RNA and DNA

DNA RNA
Name Deoxyribonucleic acid Ribonucleic acid
No of strands Double-stranded Single-stranded
Sugar 2`-deoxyribose Ribose
Bases:
• Purine Adenine & Guanine Adenine & Guanine
• Pyrimidine Cytosine & Thymine Cytisine & Uracil

Number No of adenine = No of thymine The No of bases doesn't

No of guanine = No of cytosine necessarily be equal.
 Types of RNA
1- Messenger RNA (mRNA)
Function: - They function as messengers carry
the information in a gene to the ribosomes
where they act as templates for protein
synthesis.
Modification of messenger RNA
• Its 5' end: - Is capped by 7-methylguanosine
through a triphosphates linkage.The cap
protects the mRNA from the attack by 5'-
exonuclease.
• Its 3'end: - Is attached to a polymer of
adenylate residues from 20-250 nucleotides
in length called poly (A) tail.The tail
protects the mRNA from the attack by 3'-
exonuclease.
• Splicing The mRNA immediately synthesized
from the DNA is called heterogeneous
nuclear RNA (hn RNA), that is must be
processed to generate the mature mRNA
that act as a template for protein synthesis.
 2- Transfer RNA (tRNA)
• There are at least 20 tRNA, at
least one for each of the 20 a.a
required for protein synthesis.
• The most important arms of
transfer RNA
1) The Acceptor arm:-
– This arm carries the
appropriate amino acid at 3'-
OH group.
2) The anticodon arm:-
– It recognizes the 3-letter codon
on mRNA.
– The anticodon arm sequence is
responsible for the specificity
of tRNA.
 3- Ribosomal RNA (rRNA)
• A ribosome: - Is a cytoplasmic nucleoprotein
that acts as machinery for the synthesis of
proteins from mRNA templates.
• Eukaryotic ribosome consists of 2 subunits:-
– The 60s ribosomal subunit: - Consists of 50
polypeptides and 3 types of rRNA (5S, 5.8s and
28s rRNA).
– The 40s ribosomal subunit: - consists of 35
polypeptides and one rRNA (18s).
• Both bind to from the 80s ribosome.
 4- Small nuclear RNA(sn RNA)
• snRNAs are involved in RNA processing and
gene regulation.
• U1, U2, U4, U5 and U6 are involved in intron
removal and processing of hnRNA into mRNA.
• They are considered as nucleoprotein
enzymes called ribozymes.
 DNA organization
• Definition: - DNA organization is the process by which
the very long DNA molecules are packaged inside the
nucleus.
• Chromatin: -
– Def: - It is the chromosomal material extracted from nuclei
of cells of eukaryotic organisms.
– It consists of:-
1. Very long double stranded DNA molecules.
2. Histones: - these are small basic proteins.
3. Non-histone proteins: - like enzymes involved in DNA
replication such as topoisomerases and transcription
such as RNA polymerase.
4. Small quantity of RNA.
• Histones:-
– Def: - These are closely related basic proteins (they are positively
charged proteins rich in basic amino acids as arginine and lysine)
– There are 4 types:- H1, H2 (H2A, H2B) , H3 and H4.
– H2A, H2B , H3 and H4 form core of histone called octamers consist of
8 histone .
– While H1 histones are the one which link the octamers
– The organizational unit of chromatin is called nucleosome
• Nucleosomes:-
• Def: - These are dense spherical structures containing DNA and core of
histones (histone octamers). Nucleosomes appear like beads on a string.
• H1 histone helps in super-packing of nucleosomes in the nuclei.
 Higher ordered structures of
compact chromatin
1. The 10-nm fibril:-
– The disc like nucleosome structure
has a 10-nm diameter
2. The 30-nm fiber:-
– The 10-nm fibril is supercoiled
with 6 or 7 nucleosomes per turn
to form 30-nm chromatin fiber.
3. Formation of loops:-
– The 30-nm fibers are folded into a
series of looped domains forming
non-condensed loops
4. Chromosome:-
– It consists of 2 sister chromatids
connected at a centromere.
– The centromere is A-T rich region.
 According to density of chromatin, 2
types are known
1. Euchromatin: - Less dense and
transcriptionally active.
2. Heterochromatin: - More dense and
transcriptionally inactive there are two types of
heterochromatin.
– Constitutive heterochromatin:- It is always condensed
and thus inactive, I is found in regions near the
centromere.
– Facultative heterochromatin:- sometimes it is
condensed and in other times it is less condensed and
appear as euchromatin e.g. one of the X-
chromosomes of females is active only during
gametogenesis , then become inactive.
 Mitochondrial DNA (mt DNA)
• Features of mt DNA:-
• Human mitochondria contain 2 to 10 copies of a small
circular doubl-stranded DNA molecule.
• Mt DNA forms about 1% of the total cellular DNA.
• Mt DNA codes for (2 mt rRNA, 22 mt tRNA and 13 proteins)
that play a role in respiratory chain.
• Its genetic code differs slightly from the standard
code.
• High mutation rate (5 to 10 times of nuclear DNA).
• Completely maternally inherited.
 Replication
• Definition:- Replication is the synthesis of
DNA. It occurs during the S phase of cell cycle.
• Steps:-
1. Identification of the origins of replication (ori)
2. Unwinding (denaturation) of dsDNA to provide a
ssDNA template
3. Formation of the replication fork
4. Initiation of the DNA synthesis and elongation
5. Excision and replacement of the RNA primers
6. Reconstitution of chromatin structure
 1- Identification of the origins of
replication (ori)
• The ori is a specific sequence of bases of DNA at
which replication starts.
• 2- Unwinding (denaturation) of dsDNA to
provide a ssDNA template
• 2 types of proteins are responsible for
unwinding:-
1. DNA helicase:- Allows progressive unwinding of the
DNA.
2. Single stranded DNA-binding proteins (SSBs):- That
stabilizes the complex.
• This leads to DNA unwinding and active
replication.
 3- Formation of the replication fork
• As the 2 strands unwind and separate,
they form a "V" shape where active
synthesis occurs called the replication
fork.

• The replication fork is formed of 4

components:-
1. The DNA helicase: - That unwinds a
short segment of the parental duplex
DNA.
2. A primase: - That initiates synthesis of
an RNA molecule, needed for priming
DNA synthesis.
3. The DNA polymerase: - That initiates
nascent, daughter strand synthesis.
4. SSBs: - That binds to ssDNA and
prevents premature reannealing of
ssDNA to ds DNA.
• The replication fork moves along the
DNA molecule as synthesis occurs.
• Replication is bidirectional, moves in
both directions away from the origin
of replication.
 4- Initiation of the DNA synthesis
and elongation
• Formation of the RNA
primer: -
– DNA polymerase is unable
to synthesize DNA except,
by the presence of RNA
primer.
– RNA primer: - is a short
fragment of RNA, about
10 nucleotides in
length, synthesized by
primase.
– It has a free-OH group at 3'
end, this -OH group serves
as the acceptor of the first
nucleotide from DNA
polymerase.
 Replication exhibits polarity
• The DNA strands are anti parallel,
therefore the newly replicated
strands grow in opposite directions.
• 2 types of strands are present:
– Leading strand:- Is the strand that is
being copied in the direction towards
the replication fork, it is synthesized
continuously. It needs one RNA primer.
– Lagging strand: - Is the strand being
copied in the direction away from the
replication fork.
• It is synthesized discontinuously.
• It is synthesized in short segments of DNA
attached to an RNA primer called Okazaki
fragments.
• Many RNA primers are needed for the
lagging strand.
• This process is called semi
discontinuous DNA synthesis.
 5- Excision and replacement of
the RNA primers
– DNA polymerase III synthesizes the DNA fragment
of the lagging strand, until it becomes very close
to the next RNA primer.
– Then, DNA polymerase I excise the primer and
replace it with DNA nucleotides.
– The reaming nick is then filled by DNA ligase.
6- Reconstitution of chromatin structure:-
– Newly replicated DNA is rapidly assembled into
nucleosomes and reforms the chromatin again.
 Classes of proteins involved in
replication
Protein Function

DNA polymerases Deoxynucleotide polymerization

(chain elongation) and proofreading
function).

Helicases Processive unwinding of DNA.

DNA primase Initiates synthesis of RNA primer

Single-strand binding proteins (SSBPs) Prevent premature reannealing of dsDNA.

DNA ligase Seals the single strand nick between the

nascent chain and Okazaki fragments on
lagging strand.
 DNA synthesis occurs during the
S phase of cell cycle
• The replication of the DNA genome occurs at
a specified time during the life span of the
cell called the synthetic or S phase.
• The life span of the cell undergoes 4 phases:-
1. Gap 1 (G1):- The cell prepares for DNA synthesis.
Damage DNA detected.
2. Synthetic (S):- Replication of DNA genome.
Incomplete replication detected.
3. Gap 2 (G2):- The cell prepares for mitosis. Damage
DNA detected.
4. Mitotic (M):- The cell undergoes mitosis. Improper
spindle detected.
 DNA repair
• Definition: - It is a mechanism to repair damaged DNA.
• Causes of DNA damage:-
– Imperfect proofreading (replication error)
– Environmental: - e.g ionizing radiation.
– Physical: - e.g x-ray and ultraviolet rays.
– Chemical: - e.g alkylating agents.
 Mechanisms of DNA repair

• Abnormal regions of DNA, either from copying

errors or DNA damage are replaced by 4
mechanisms:-
1. Mismatch repair
2. Base excision repair
3. Nucleotide excision repair
4. Double strand break repair.
• The defective region in one strand can be
repaired by relying on the complementary
information stored in the unaffected strand.
 I- Mismatch repair
• Function: - It corrects errors made during DNA
replication e.g when a C is inserted opposite an A
instead of T or when the polymerase inserts 2 to 5
unpaired nucleotides.
• Mechanism:
The correct (old) strand is methylated, while the
other strand is not methylated, this allows the
repair system to identify the strand that contains
the nucleotide error.
• Important notes: -
– Hereditary nonpolyposis colorectal cancer are due to
mutations in proteins involved in mismatch repair.
 3- Nucleotide excision repair
• Function: - This mechanism is used to replace
regions of damaged DNA up to 30 bases in
length.
• Important: -
– Exposure of a cell to UV light can result in the
covalent joining of two adjacent pyrimidines usually
thymines producing a dimer.
– These thymine dimers prevent DNA from replicating the
DNA beyond the site of dimer formation.
– They are excised by UV-specific endonuclease in a
process known as nucleotide excision repair.
– xeroderma pigmentosum (XP) skin cancers,caused
by defects in nucleotide excision repair of UV
damage in humans.
 Transcription
• Definition: - It is the process of RNA synthesis.

• Differences between transcription and replication: -

1. Formation of Ribonucleotides rather than
deoxy ribonucleotides.
2. U replaces T as the complementary base for A in
RNA.
3. A primer is not involved in RNA synthesis.
4. Only a small portion of the genome is
transcribed or copied into RNA, whereas
the entire genome must be copied during
DNA replication.
5. There is no proofreading function during
transcription.
 Transcription unit
• Definition: - Is defined as that region of DNA that
includes the signals for transcription initiation,
elongation and termination.
• It consists of (the promoter region, the transcribed
region and the termination region).
• Transcription is carried out by a DNA-dependent RNA
polymerase that enhances polymerization of
ribonucleotides into RNA strand.
1) The promoter region:-
– Definition: - Ii is that part of the DNA on the transcription
unit to which the RNA polymerase binds with high affinity,
to ensure accurate initiation of transcription.
 Eukaryotic promoter
• The promoter consists of:-
1. The 5'-TATAAAAG-3'sequence (TATA box):-
– It is located 25-30 bp upstream to the transcription
start site.
– Importance: - It defines where transcription is to start
along the DNA.
2. The GC and CAAT boxes:-
– They are located about 70 to 80 nucleotides upstream
from the start site.
– Importance: - It determines how frequency the
transcription occurs.
 2) Transcribed region
• Definition: - Is the stretch of the DNA that is to be
transcribed into RNA molecules.
• The first nucleotide in the transcribed region is
designed +1 and called the start site of transcription.
• The nucleotides within the transcribed region i.e after
the starting point are called downstream.
• The nucleotides located before the starting point are
called upstream and take a negative sign e.g-1,-2.ect.
3) Termination region:-
• Definition:- It is a stretch of the DNA at the end of the
transcribed DNA.
Mammalian DNA-dependent RNA polymerase:- There are
3 types of eukaryotic RNA polymerases.
1. RNA polymerase I: - synthesize the large rRNA (5.8s, 18s
& 28s).
2. RNA polymerase II: - synthesize the mRNA and miRNA.
3. RNA polymerase III:- synthesize the tRNA, snRNA and 5s
rRNA
 Post-transcriptional modifications of RNA
• The primary transcript is called heterogeneous nuclear RNA (hnRNA) is
modified into mature m RNA by:-
1. 5' capping:-
– The 5' end of the mRNA requires a cap formed of 7- methyl-
guanosine Protects the 5ˋ end of mRNA from attack by 5ˋ exonuclease
and Facilitate the initiation of translation.
2. Poly (A) tail:-
– Up to 200 adenine nucleotides are added to the 3' end of the mRNA
molecule.
– The poly (A) tail is synthesized by poly (A) polymerase enzyme.
– Poly (A) tail protects the 3' end from the attack by the 3' exonuclease.
3. Splicing (removal of introns and ligation of exons)
– Exons: - These are the coding sequences.
– Introns: - These are non-coding sequences.
• Introns must be removed by a process called splicing.
• Spliceosome: - Is a special structure, involved removal of introns and
ligation of exons
 The genetic code
Introduction:-
There are 20 amino acids required for protein synthesis, thus their
must be at least 20 codons (one for each amino acid) that make the
genetic code.
Codon: - is the sequence of 3 nucleotide bases on mRNA that determines
the type and position of amino acid on a protein.
There are 4 different nucleotides in the mRNA and the genetic codon is
triplet thus there are (4)3= 64 codons e.g. AUG codes for methionine.
The genetic code: - is the collection of the genetic codons.
Nonsense (termination) codons: - there are 3 codons (UAA, UAG and UGA)
don't code for amino acids and are called nonsense codons or termination
codons as they terminate the translation process.
 Characteristics of the genetic code
1. Degeneracy:-
Definition:- It means that multiple codons can code for
one amino acid.
There are 61 codons and only 20 amino acids, this
means that there must be more than one codon code
for the same amino acid.
2. Unambiguous: - It means that each codon codes only
for a single amino acid.
3. Non overlapping:- It means that the reading of the
genetic codons during protein synthesis doesn't
involve any overlap of codons.
4. No punctuation:- It means that there is no punctuation
between codons and the message is read in a
continuing sequence of nucleotide triplets until a
translation stop codon is reached.
The codon-anticodon recognition
The anticodon arm of the tRNA recognizes the mRNA
codon.
The binding of tRNA anticodon to the codon follows
the rules of complementarity and antiparallel i.e the
codon is read 5ˋ3ˋwhile the anticodon is read 3ˋ5ˋ.
The anticodon arm is responsible for the specificity of
tRNA.
 Mutation
Definition: - It is a change in the nucleotide
sequence of the DNA.
Types: - There are 2 types of mutations.
1- Point mutation (single base changes).
2- Frame shift mutation.
1. Point mutation (single base changes):-
There is a substitution of one base by another.
There are 2 types
A. Transition mutation:- occurs when one pyrimidine is
changed to other pyrimidine or when one purine is
changed to purine.
T C
A G
B. Transversion mutation: - Occurs when a purine
is changed to either of the 2 pyrimidines or
when a pyrimidine is changed to either of the 2
purines.

Effects of point mutation:-

1. Silent mutation: - It means that the codon
containing the changed base still code for the
same amino acid.
2. Missense mutation: - it occurs when the codon
containing the changed base code for a different
amino acid.
The effect of the mistaken amino acid Protein
function may be;
1) Acceptable: - When the resulting protein is not
distinguishable from the normal one e.g Hb Hikari.
2) Partially acceptable: - The function of the
produced protein is partially affected e.g Hb S.
3) Unacceptable: - The produced protein is non-
functioning e.g Hb M.
3. Nonsense mutation: - Occurs when the codon
containing the changed base become a termination
codon. This may lead to premature termination of
translation and short protein.
2. Frameshift mutation
It may be produced either due to deletion or insertion
of nucleotide (s) from the coding strand of a gene.
Effects:
I. If there is deletion or insertion of a single
nucleotide, this may lead to:-
Garbled translation of the mRNA distal to the single
nucleotide deletion.
Nonsense codons: - May result from deletion,
leading to premature termination of translation.
II. If there is deletion or insertion of 3 nucleotides or
multiple of 3 from the coding region, this result in a
protein with missed amino acid or added amino acid.