Nucleic acids

BY: Endeshaw C (MSc, Medical Biochemistry)

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 Nucleic Acids
• Nucleic acids are molecules that store information for
cellular growth and reproduction.
• The nucleic acids includes deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA).
• DNA and RNA are large molecules consist of long chains
of monomers called nucleotides.
• The nucleotides consist of a
– Pentose sugar
– Nitrogen base
– Phosphate group

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Nitrogen Bases
– The nitrogen bases in DNA and RNA are
• Pyrimidines (single ring) -C, T, and U and
• Purines (double ring) - A and G.

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 Pentose Sugars
• The pentose (five-carbon) sugar in
– RNA is ribose
– DNA is deoxyribose with no O atom on carbon 2’

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 Phosphoric acid (H3 PO4)

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 Nucleoside and Nucleotides
• Nitrogenous bases with pentose sugar= nucleoside
• A nucleotide is a nucleoside that forms a phosphate ester with the
C5’ –OH group of a sugar
• In the nucleotide structure the nitrogenous base is attached to the
first carbon atom of the pentose sugar via an N- Glycosidic linkage
• The phosphate group is attached to the 5th carbon via ester linkage
• It is named using the name of the nucleoside followed by 5’-
monophosphate.

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A. Nucleotides present in DNA (deoxyribonucleoside monophosphate)
1.Deoxy adenosine mono phosphate /dAMP (deoxy adenylic acid)
2.Deoxy guanosine mono phosphate/dGMP (deoxy guanylic acid)
3.Deoxy cytidine monophosphate/dCMP (deoxy cytidylic aicd)
4.Deoxy thymidine monophospahate/dTMP (deoxy thymidylic
acid)
B. Nucleotides present in RNA (ribonucleoside monophosphate)
1. Adenosine mono phosphate /AMP (Adenylic acid)
2. Guanosine mono phosphate/GMP(Guanylic acid)
3. Cytidine monophosphate/CMP (Cytidylic aicd)
4. Uridine monophospahate /UMP (Uridylic acid)

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Important Nucleoside monophosphates
1. 3’-5’cyclic AMP (cAMP)
– a 2nd messenger for hormones, such as glucagon, adrenaline
– 2nd messenger is a molecule that is produced intra-cellularly
upon an extracellular hormonal stimulation, and mediate the
hormonal action within the cell
2. Cyclic GMP (cGMP)
– also serve as a second messenger for ANP & NO

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cAMP structure 8
Primary Structure of Nucleic Acids
• The nucleotides undergo polymerization to form long
nucleic acid polymer
• In the primary structure of nucleic acids, nucleotides
are joined by phosphodiester bonds.
• The 3’-OH group of the sugar in one nucleotide forms
an ester bond to the phosphate group on the 5’-carbon
of the sugar of the next nucleotide.
• A nucleic acid has a free 5’-phosphate group at one end and a
free 3’-OH group at the other end.
• It is read from the free 5’-end using the letters of the bases.
• This example reads
—A—C—G—T—.
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ENDESHAW C.
 Primary Structure of Nucleic Acids
P
G

P
C

P
C

P
A

P
T

P
T
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Important Nucleoside triphosphates
1. ATP –high energy molecule (energy currency of the cell)
2. GTP- High energy molecule
3. CTP- High energy molecule
4. UTP- High energy molecule

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ATP ENDESHAW C. 11
DEOXYRIBONUCLEIC ACID (DNA)
• The structure of the poly deoxy ribonucleotide consists
of hundreds or thousands of deoxyribonucleotides
linked by 3’-5’- phosphodiester linkages
• This polymer has a 3’ end with a free sugar –OH group
and a 5’ end with a free phosphoric acid group.

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 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA)
✓ The double helical model of DNA
– was proposed by James Watson and Francis Crick
– represents the secondary structure of DNA
✓ Double helical model of DNA has the following
characteristics
– DNA structure consists of two poly deoxy ribonucleotide strands
which wind around each other in a right handed manner
around an imaginary line to form a right handed double helix
– The two strands are joined by pairing between the nitrogenous
bases of opposing strands=H-bond

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 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA)
• The two strands are running in
opposite direction, one in 5’→3’
direction and other in 3’→5’ direction
(anti parallel)
• This double helical structure of DNA
resembles a spiral stair case
– The steps of the stair case are represented
by the base pairs
– the hand rails are represented by the
sugar phosphate backbones.

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 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA)
Hydrogen bonds
❑ Base pairing rule
P
• The bases of the two strands are paired G C
by strict base pairing rule P
P
• The bases along one strand complement C G
the bases along the other P
P
• A purine always pairs with a pyrimidine C G
P
hence;
P
– Adenine (A) always pairs with thymine (T). A T
– Guanine (G) always pairs with cytosine (C). P
P
• Two H-bonds are formed b/n A & T T A
• Three H-bonds are formed b/n G & C P
P
T A
P
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 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA)
❑ Complementary nature
• Due to the complementarity in base pairing, the two anti-parallel
strands of the DNA are also complimentary but not identical.
1. When there is A in one strand, there will be T in the
corresponding position of the other strand.
2. When there is G in one strand the corresponding position of
the other strand will be occupied by C
3. There will be equal number of nucleotides in both strands.
4. The number of A nucleotides in one strand will be equal to the
number of T nucleotides in the opposing strand and also in the
entire DNA.
5. Similarly, the number of G nucleotides will be same as the
number of C-nucleotides
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 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA)

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ENDESHAW C. nature of DNA strand 17
 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA)
❑Major forces stabilizing DNA Structure
• The forces stabilizing the DNA double helical structure can be
divided in to covalent bonds and non-covalent bonds.
1. Covalent bonds :
– The nucleotides in each strand are joined by 3’-5’Phosphodiester linkages
– Formed between the 5th carbon of one nucleotide with the 3rd carbon of
another nucleotide via a phosphoric acid group.
– It is considered as the backbone of DNA
2. Non-covalent bonds.
– H-Bonds –formed between the nitrogenous bases of opposing strands
– Van der Waals forces –formed between uncharged atoms of both strands
– Hydrophobic interactions – the nitrogenous bases are uncharged &
hydrophobic, while phosphate groups of each strand are negatively
charged & hydrophilic. Hence the bases become stacked inside the double
helical structure & the phosphate group tends to be in the periphery
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 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA
❑Helix parameters
• The double helical structure of DNA has 2.0 nm width
• The height of one turn (pitch of the helix) is 3.4 nm
• There are 10.4 base pairs in each turn (n=10.4).
• The distance contributed by one base pair is
3.4/10.4=0.34nm
• There are two grooves that run along the length of the
DNA molecule
1. Major groove-is the space between two adjacent turns in the
DNA molecule. It has 1.2nm width
2. Minor grove –is the space between the two strands of the
helix. It has a width of 0.6nm.

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 WATSON & CRICK DNA STRUCTURE
(DOUBLE- HELICAL MODEL OF DNA

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 Functions of DNA
• DNA is the genetic material of living systems.
– It contains all the information required for the formation of an individual or
organism
– The genetic information in DNA is converted to characteristic features of
living organisms like color of the skin and eye, height etc.
• DNA is transmitted from parent to offspring and hence DNA flows
from one generation to other in a given species.
• The amount of DNA per cell is proportional to the complexity of
organism and hence to the amount of genetic information.
• The amount of DNA in any given species or cell is constant and is
not affected by nutritional or metabolic states.

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 Ribonucleic acids (RNA)
• RNA is the polymer of ribonucleotides i.e. polyribonucleotide
• RNA are found in nucleus & cytoplasm of eukaryotic cells.
• They are also present in prokaryotes.
• General function
– involved in the transfer & expression of genetic information.
– RNA also function as genetic material for viruses.
– They act as primers for DNA formation.
– Some RNA act as enzymes as well as coenzymes.

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• Based on their specific functions, RNA are classified 3
types of RNAs in all prokaryotic & eukaryotic cells i.e.
1. Messenger RNA (mRNA)
2. Transfer RNA (tRNA)
3. Ribosomal RNA (rRNA)
– They differ from each other by
• Size
• Function
• Stability

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1. Messenger RNA
• It accounts for 1-5% of total cellular RNA.
• Varying sizes but its molecular weight ranges from 30-50 KDa
• Majority of mRNA has primary structure & are single-stranded
linear molecules
• Eukaryotic mRNA molecules are
– capped at 5’ end- the cap is methylated GTP (m7 GTP)- a modified base
– Polyadenylated tail at 3’ end- poly A tail has 20-200 adenine nucleotides
• Capping & poly A tail protects mRNA from nuclease attack/damage

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1. Messenger RNA…
• In mRNA, 3 adjacent bases are called a codon which code for
amino acids during protein synthesis
– each codon specifies one amino acid
• There is a coding region in the center which carries codons
specifying amino acids to be incorporated in the protein structure
• A region of mRNA that carry information for the synthesis of one
polypeptide chain is called cistron
1. Monocistronic mRNA
– mRNA that codes for only one protein (polypeptide)
– Eukaryotic mRNA
2. Polycistronic mRNA
– mRNA that codes for more than one protein (polypeptide)
– Prokaryotic mRNA

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 Functions of mRNA
1. mRNA is direct carrier of genetic information from
nucleus to cytoplasm
– transport of genetic information from gene (DNA) to the site
of protein synthesis in the cytoplasm
– Genetic information is present in mRNA in the form of
genetic code
2. mRNA molecule acts as template for protein synthesis
– Usually a molecule of mRNA contains information required
for the formation of one protein molecule➔ Monocistronic
– Some times single mRNA may contain information for the
formation of more than one protein ➔ polycistronic

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 2. Transfer RNA (tRNA)
• It is also called soluble RNA/adapter molecule
• It constitutes 10-20 % of the total cellular RNAs.
• It is small molecule containing only 75 nucleotides
• The molecular weight is about 25KDa
• There is at least one tRNA molecule to each of 20 amino acids
required for protein synthesis
• The primary structure of tRNA undergo extensive folding to form
a clover leaf shaped secondary structure
– this is achieved by the base pairing between self
complimentary regions in the molecule.
• Prokaryotic tRNAs are more stable compared to eukaryotic tRNA

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• Structurally, all tRNA have 4 arms.
1. Dihydrouridylic/DHU arm: D-loop
– containing an unusual base called dihydrouracil
– this arm binds with active site of the enzyme aminoacyl tRNA
synthetase
2. Ribothymidine/pseudouridine/TψC arm=T-loop
– containing a modified bases pseudouridine and ribothymidine
– binds to ribosome during protein synthesis
3. Anti-codon arm
– contain the anti-codon
– anti-codon are three adjacent bases in tRNA that form
complementary base pairing with the codon on mRNA during
translation
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4. Amino acid arm
– carries amino acid
– All tRNA molecules have a common CCA sequence at 3’ end
– The 3’ end is called amino acyl attachment site
– The amino acid binds to–OH group present in the 3’ end by
an ester linkage (between the –OH group of tRNA and the
–COOH group of the amino acid) to form the amino acyl
tRNA (activated amino acid).
5. Variable arm
– It is a short loop that contains sequence varying among
different tRNA molecules
– It is present between pseudo-uridine arm & anti-codon arm.

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Functions of tRNA
• tRNA is involved in the transfer (transport) of
amino acids to the site of protein synthesis
– It is the carrier of amino acids
• It functions as an adapter molecule that translate
the genetic information present as nucleotide
sequence in the mRNA molecule to the amino acid
sequence in the polypeptide synthesized.

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3. Ribosomal RNA (rRNA)
• rRNA accounts for 80% of total cellular RNA.
• It is present in ribosomes in combination with certain proteins to
form ribonucleoprotein.
• rRNAs are classified into different types based on their
sedimentation coefficient(S) –Svedberg Unit (S)
• Prokaryotes have 70 S ribosome containing 50S large ribosomal
unit and 30S small ribosomal unit
– there are 3 types of rRNA molecules in prokaryotes
• Eukaryotes have 80S ribosome containing large (60S) & small
(40S) ribosomal subunit
– There are four types of rRNAs in eukaryote s

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rRNA Structure
• rRNA molecules have secondary structure.
– Intra strand base pairing b/n complementary base generates
double helical segments or loops, known as domains.
– The three dimensional tertiary structure of rRNA is highly
complex

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Functions of rRNAs
1. It is a structural component of ribosome together
with proteins
– thus it is required for the formation of ribosomes
2. It interacts with tRNA during translation
3. It provides peptidyl transferase enzymatic activity
(ribozymes)

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✓ Complete the chart by reading each term or phrase and placing a check in the appropriate column.

DNA RNA Both

Deoxyribose
Ribose
Double stranded
Single stranded
Nucleotides
Found in nucleus only
Found in and out of nucleus
Cytosine
Guanine
Adenine
Thymine
Uracil
Double helix
Replication
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 CENTRAL DOGMA
• The genetic information is stored in the DNA - (4-letter
language of N-bases -A, T,C& G).
• A fragment of DNA molecule that determines a character
or a property is called a gene
• Human DNA contains about 100,000 genes.
• A gene expresses a character by the synthesis of a protein.
– these proteins will carryout the manifestation of that character
• The process of genetic information transfer and expression
is called central dogma of molecular genetics
• Involves three processes
1. Replication – synthesis of DNA from DNA
2. Transcription–synthesis of RNA using DNA as a template
3. Translation– synthesis protein using RNA as a template
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CENTRAL DOGMA

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If the cell is a school
• The Nucleus is the school
office
• The Nucleolus is the
principal’s office
• The DNA is the principal
RNA splicing

• Ribosomes are the cafeteria

ladies
• mRNA is the email from the
principal to the cafeteria lady

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Replication
• The process of synthesis of a new DNA molecule
using a parent DNA molecule as a template is
called DNA replication.
• It takes place in the nucleus of the cell.
• During replication, a new strand is synthesized on
each of the parental strands.-semiconservative
– The daughter DNA molecules will have one parental
strand & one newly synthesized strand
• DNA replication & chromosome duplication are
parallel processes-takes place just before cell division
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Enzymes of DNA replication
• Replication is carried out by the combined action of a number of enzymes
and other protein factors.
1. DNA polymerase
– It is the chief enzyme involved in the polymerization of deoxy-ribonucleotides
leading to the synthesis of new strand
– In higher organisms, there are 5 different types of DNA polymerases–alpha, beta,
gamma, delta, epsilon.
2. RNA polymerase or RNA primase
– It is involved in the synthesis of a short RNA fragment called RNA primer
3. DNA topoisomerase: uncoiling of DNA
4. DNA helicase
– separation of the two parental DNA strand during replication.(utilizes the energy
of ATP hydrolysis to break the H- bonds between bases)
5. DNA ligases
– joining of short DNA fragments to form a long continuous strand
6. Single strand binding proteins (SSBPs)
– are factors that prevent the re-association of the separated strands of DNA

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Phases of DNA replication
• The process of DNA replication has three phases;
initiation, elongation, and termination
1. Initiation
• Involves the formation of the replisome
– The association of DNA and the various enzymes involved in
replication is called Replisome
• The sequence of events in initiation are
✓ DNA-topoisomerase recognizes autonomous replicating sequences
(ARS) and starts the uncoiling of DNA.
✓ DNA helicase separates the two strands of the uncoiled DNA
✓ SSBP binds to the single strands and stabilizes them.
✓ RNA polymerase (RNA primase) synthesizes a short fragment of
RNA at the 3’ end of the 3’to 5’ strand
✓ DNA polymerase binds to the DNA
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The Tangle Problem

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 45
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2. Elongation
• On the 3’ to 5’ parental strand-
– DNA polymerase catalyzes the sequential addition of deoxyribonucleotides
to the RNA primer leading to its extension-leading strand
• On the 5’to 3’ parental strand
– the synthesis of new strand takes place by formation of short fragments
called Okasaki fragments-lagging strand
• DNA polymerase
– removes the RNA primer present at the 5’ end of the leading strand and the
Okasaki fragments by its exonuclease activity
– also fills the spaces by the addition of deoxy ribonucleotides.
• DNA ligases → joins the fragments to form two continuous strands.

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3. Termination
• The process of elongation stops when parental DNA is
completely replicated & two daughter DNA molecules
are formed,
• Each daughter DNA are exact copy of the parental DNA
molecule
– one strand is parental and one is newly synthesized
• The proof reading activity of DNA polymerase increases
the fidelity of replication.

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The characteristic of DNA replication
1. DNA replication is semi-conservative
– the daughter DNA molecules will have one parental strand and
one newly synthesized strand
2. The synthesis of the new strand takes place only in
the 5’ to 3’ direction.
3. Semi-discontinuous replication
– In 3’-5’ parental strand replication takes place continuously
– In 5’-3’ strand replication occurs discontinuously
by the formation of Okasaki fragments
4. Bidirectional replication
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Transcription
• Transcription refers to the synthesis of RNA molecules
using DNA as a template-
– DNA based RNA synthesis
– It takes place in the nucleus.
• RNA synthesis takes place by polymerization of
ribonucleotides by RNA polymerase.
• Three different types of RNA polymerases.
– RNA polymerase I- rRNA synthesis.
– RNA polymerase II- mRNA synthesis
– RNA polymerase III - tRNA synthesis

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• The process of transcription can have four phases
1. Initiation
2. Elongation
3. Termination
4. Post transcriptional processing

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1. Initiation: initiation factors/sigma factor
• RNA polymerase identifies the promoter site in gene
– eg. Hogness box (TATAA box) and CAT box (CAATA).
• The DNA molecule partially unwinds with the help of so
many protein factors called initiation factors (IFs)
• RNA polymerase catalyzes the addition of the first
nucleotide according to the complimentary base pairing
rule.
• The enzyme gradually moves one nucleotide ahead and
catalyzes the addition of the second nucleotide.

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2. Elongation: RNA polymerase
– As the RNA polymerase moves along the DNA molecule
– RNA molecule will unwind the downstream and wind the
upstream so that we feel the movement of the transcription
bubble in the direction of the movement of RNA polymerase
– More and more ribonucleotides become incorporated to
RNA structure
3.Termination: “rho” factor
– Termination takes place when RNA polymerase recognizes
certain sequences called termination signals.
– When the termination signal is reached certain proteins called
“rho” will bind to the RNA molecule.
– Once Rho is bound RNA polymerase, it cannot proceed further.
– The enzyme dissociates from DNA& the nascent
RNA molecule is released.
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4. Post transcriptional modification/ processing
• The nascent RNA undergoes a number of chemical
modifications to become mature and functional RNA
molecule (post transcriptional modifications).
nascent RNA→ mature/functional RNA
• This will vary for different RNAs
• Some of such changes in the case of mRNA are
a) Poly A tailing
b) 5’ capping
c) Intron splicing

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Characteristics of transcription
• only one strand of the DNA molecule acts as a template-called
– Template/ antisense/noncoding strand
• The other strand is called
– non template/sense/coding strand
• RNA synthesis takes place only in the 5’ to 3’ direction.
– Thus, the antisense/template strand must be in the 3’ to 5’ direction
• During DNA replication, the entire DNA molecule makes its
own copy, but in transcription only a small portion of DNA
(gene) is copied
• The RNA molecule will have sequence complimentary to the
anti-sense strand of the DNA &similar to the sense/coding
strand of the DNA except that there will be uracil in place of
thymine
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 Translation

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 Translation
• It is the process of protein biosynthesis.
• takes place in cytoplasm with the help of tRNA, mRNA, so
many enzymes and protein factors.
• During translation the genetic information present in mRNA
molecule in the form of definite sequence of nucleotides is
translated into amino acid sequence in polypeptide (protein)
• In other words, genetic information written with 4-letter
language of nucleotides in mRNA is translated in to 20 letter
language (20 amino acids) of proteins.
• mRNA contains triplet of nucleotides (three adjacent
nucleotides) called codons that specify an amino acid.
• An mRNA containing 3000 nucleotides in the coding region
can code for a polypeptide containing 1000 amino acids.
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• The process of translation can be
described under the following headings.
1. Activation of amino acids
2. Initiation
3. Elongation
4. Termination
5. Post translational modification

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1. Activation of amino acids
• Attachment of amino acid to tRNA molecule to form
amino- acyl tRNA, also known as activated amino acid
– This rxn is catalyzed by enzymes called amino-acyl tRNA
synthetases, (eg.methionyl tRNA synthetase, glycinyl tRNA
synthetase etc)

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2.Initiation
• The following events takes place during initiation
– mRNA binds to 40S ribosomal subunit with its 5’ end
– methionyl tRNA forms base pair ( codon – anticodon
pairing) with the initiation codon (AUG)
– 60S large subunit binds to form the initiation complex
– The complex has three different sites –
1. Peptidyl site or P –site,
2. acceptor site or A- site and
3. exit site or E-site

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 The P-site contains the AUG codon of the mRNA and the first methionyl tRNA
attached to it by codon – anticodon base pairing. A-site is now empty
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3. Elongation
• The second amino acyl tRNA become attached to A site.
• A peptide bond is formed between the two amino acid to
form a dipeptide which remains attached to the A site (this
tRNA can be called dipeptidyl tRNA).
• Peptide bond formation is catalyzed by an enzyme called
peptidyl transferase ( later on it was recognized to be a
ribozyme-an rRNA molecule present in the larger
ribosomal subunit)
• Ribosome moves downstream by a distance of one codon-
--called translocation
• Now the dipeptidyl tRNA become shifted to the P site and
the A site becomes empty.

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• The new amino acyl tRNA complimentary to the next
codon (3rd) will come and occupy the A-site and the
whole process is repeated.
• The elongation process continues until certain codons
called stop codons (UAA, UAG and UGA)
will come and occupy the A-site.
– The stop codons will not have any tRNA to bind with it

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4.Termination
• Occurs when the amino acyl site becomes occupied by stop
codons
• This is because there is no amino acyl tRNA corresponding
to stop codons
• Certain protein factors called termination factors will bind
to the A Site.
• Followed by the dissociation of the ribosomal assembly and
the liberation of the polypeptide chain.

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Poly ribosome or polysome
• Several ribosomes may carryout the translation process
in the same m RNA simultaneously.
• Such mRNA containing large numbers of ribosomes
attached to it is called polyribosome or polysome

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5. Post translational modification
• Chemical & structural modifications on polypeptide
chain to convert it into a biologically active protein
– Proteolytic cleavage
– Chemical modification of amino acids such as
• Hydroxylation
• Phosphorylation,
• Glycosylation
• Metal ion incorporation etc.
– Protein folding

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