Chapter 9-NUCLEIC ACIDS

Acknowledgement
• Addisa Ababa University
• Haramaya University
• Hawassa University
• Jimma University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-
Ethiopia
 Chapter learning objective
• At the end of this chapter the student will be able to :-
• Explain classification and chemistry of nucleic acids-
RNA and DNA
• Discuss metabolism of nucleotides
• Explain clinical significance (Hyperuricemia and gout
and Orotic aciduria
• Explain DNA replication
• Explain transcription
• Explain translation
 Outline
8.1. Classification and chemistry of nucleic acids
8.2. Metabolism of nucleotides
8.3. Clinical significance (Hyperuricemia and gout
and Orotic aciduria
8.4. DNA replication
8.5. Transcription
8.6. Translation
• 8.1. classification and biochemistry of nucleic
acid
• Nucleic acids are required for the storage and
expression of genetic information
• Two types of nucleic acids are present in all
mammalian cells including humans.
• They are DNA-deoxy ribonucleic acid and RNA-
ribonucleic acid.
• DNA is present not only in chromosomes in the
nucleus of eukaryotic organisms but also in
mitochondria and the chloroplasts of plants
• Nucleotides are the monomeric units of the nucleic
acids, DNA (deoxyribonucleic acid) and RNA
(ribonucleic acid).
• Each nucleotide consists of a heterocyclic
nitrogenous base, a sugar, and phosphate.
• DNA contains the purine bases adenine (A) and
guanine (G) and the pyrimidine bases cytosine (C)
and thymine (T). RNA contains A, G, and C, but it
has uracil (U) instead of thymine.
• In DNA, the sugar is deoxyribose, whereas in RNA it
is ribose.
• Prokaryotes have a single chromosome, but may
also contain nonchromosomal DNA in the form of
plasmids
• RNA is present in nucleus and cytoplasm
• Nucleic acids are acidic substances containing
nitrogenous bases, sugar and phosphorus.
• Both DNA and RNA are polymers of nucleotides
(polynucleotides) joined together by
phosphodiester linkage.
• Diester linkage of phosphate joins 3' OH and 5' OH
belonging to two separate sugars
• Nucleotide sequence of a polynucleotide is
known as primary structure of nucleic acid and it
confers individuality to polynucleotide chain.
• Polynucleotide chains are represented in 5' → 3'
direction only.
• However, the phosphodiester linkage runs in 3'
→ 5' direction.
• Each poly nucleotide chain has two ends. The 5'
end carrying phosphate is on the left hand side
and 3' end carrying unreacted hydroxyl is on the
right hand side.
• Primary structures of DNA and RNA exist in
single stranded DNA and RNA organisms.
• Therefore, in DNA and RNA, letters A, G, C, T
stands for nitrogen bases and sugar is deoxy
ribose if the polynucleotide is a segment of DNA
and sugar is ribose if it is RNA segment.
• Hydrolysis of nucleotides produce nitrogen
bases, sugars and phosphate.
• Nucleotides contain two types of nitrogenous
bases:
-purine
-pyrimidine
• In the case of purine nucleosides, the sugar is
attached to N-9 of purine ring where as in
pyrimidine nucleosides the sugar is attached to
N-1 of pyrimidine ring.
• Nucleotides are phosphorylated nucleosides.
Purine deoxyribonucleotide
 Structure of DNA
• Deoxyribonucleic acid (DNA) is polymer of
deoxyribonucleotides attached to each other by
phosphodiester linkages.
• Each deoxyribonucleoside is composed of nitrogen
bases and a sugar deoxyribose.
• The nitrogenous bases are purines and pyrimidines.
• The two purine bases are adenine and guanine.
• The three pyrimidine bases are cytosine, thymine
and uracil.
Primary Structure of DNA
• The deoxyribonucleotides are linked together by
phosphodiester bonds between the 3'–hydroxyl of
the sugar of one nucleotide through a phosphate
molecule to the 5'–hydroxyl on the sugar of
another nucleotide.
• The sugar–phosphate linkages form the backbone
of the polymer to which the variable bases are
attached.
• The nucleotide polymer has a free phosphate
group attached to 5'–position of sugar and a free
3’–hydroxyl group.
• The sequence of the polymer is written in the 5’
to 3’ direction with abbreviations to different
bases
Secondary Structure of DNA
• The secondary structure of DNA is formed when
the two strands of DNA are paired together
• In the secondary structure of DNA, the two
strands are anti-parallel. That means, the 5’ ----
3’ of one strand is in opposite direction to the
other strand.
• The bases are stacked in the inside of the two
strands.
• The bases of one strand pairs with the bases of
the other strand of the same plane such that
adenine always pairs with thymine with two
bonds. Guanine always pairs with cytosine with
three bonds.
• The negatively charged phosphate group and
the sugar units expose themselves to the
outside of the chain.
• The two strands of DNA coil around a single axis
forming right handed double helix.
• The planes of the sugars are at right angles to
that of the bases.
• The helical structure repeats after 10 residues
on each chain.
• Watson - Crick Model of DNA is also referred as
B-DNA, which is the most stable under
physiological conditions.
• The mitochondrial DNA is circular and there can
be formation of Z-DNA and C-DNA which can be
performed during either replication or
transcription.
 The Structure of RNA
• The building unit of RNA is ribonucleotide.
• Ribonucleotide contains “O” in the carbon 2’
sugar ribose.
• Uracil is found in RNA while Thymine is found in
DNA.
• The nuclear DNA is in secondary structure, but
RNA is in primary structure.
• Only tRNA after post transcriptional process can
be changed to tertiary structure.
structure of Ribose and Deoxy-ribose in nucleotides
• The three RNAs that have important role in
protein synthesis are:
1. Messenger RNA (mRNA)
2. Transfer RNA (tRNA)
3. Ribosomal RNA (rRNA)
• They differ from each other by size, function and
stability.
Messenger RNA (mRNA)
• It accounts for 5% of cellular RNA.
• mRNA contains cap at the 5’ end of the chain and
poly-A tail at 3’- end.
• Cap characterizes 7-methylated guanosine tri
phosphate (m7 GTP).
-Cap protects from exonuclease attack.
-also used for recognition during protein synthesis
• Poly- A (a polymer of adenylate) characterizes
about 200 successive adenylate residues.
-serves to protect the mRNA form exonuclease
attack.
-serves for the transport of the mRNA form
nucleus to cytosol.
• They consist of 1000-10,000 nucleotides
• mRNA molecules have different life spans that
ranges from few minutes to days.
• In prokaryotes 5' end of mRNA contains a
sequence rich in A and G. Such sequence is known
as Shine-Dalgarno sequence. It helps attachment
of mRNA with ribosome during protein synthesis.
• Some prokaryotic mRNA has secondary structure.
Intrastrand base pairing among complementary
bases allows folding of linear molecule. As a result
hairpin, or loop like secondary structure is formed.
Transfer RNA (tRNA)
• tRNA accounts for 15% of total cell RNA.
• They are the smallest of all the RNAs. Usually
consist of 50-100 nucleotides.
• All tRNA have 4 arms.
1. Amino acid arm: the one that carries amino acid
2. DHU (dihydrouridine) arm: the one that binds
with active center of the enzyme aminoacyl
tRNA synthetase.
3. TϕC (ribothymidine-pseudouridine-cytidine) arm:
the one that binds to ribosome during protein
synthesis. Greek alphabet ϕ (Psi) stands for
pseudo uridine.
4. Anticodon arm: which pairs with the codon of
mRNA during protein synthesis.
• Secondary structure of all the tRNAs is in the
form of clover leaf
• An amino acid arm is where amino acid is
attached to 3'-OH of adenosine moiety of tRNA.
• ACC is the common base sequence at the 3'-end.
• An anti-codon arm recognizes codon on mRNA.
• The 5' end of tRNA is phosphorylated and residue
is guanosine.
Ribosomal RNA (rRNA)
• rRNA is highly methylated as compared to the
other RNAs.
• rRNA, ribosomal proteins and Mg++ constitute
ribosome.
• Each ribosome consists of two big and small
subunits.
• rRNA accounts for 80% of total cellular RNA.
• In ribosomes, rRNA is found in combination with
protein. It is known as ribonucleoprotein.
• The length of r-RNA ranges form 100-600
nucleotides.
• rRNAs differ in sedimentation coefficients (S).
1. In prokaryotic cells as 23s, 16s, and 5s
2. In eukaryotic cells as 28s, 18s, 5.8s and 5s.
• S is for Svedberg unit which is related to
molecular weight and shape
components of 70s prokaryotic ribosome
Secondary structure of 16S rRNA
 8.2. Catabolism of Nucleic acids

• Pancreatic enzymes called nucleases hydrolyze

both DNA and RNA to nucleotides and then to
nucleoside and phosphoric acid in the intestinal
tract.
• Nucleic acid is also hydrolyzed by lysosomal
enzymes inside tissues.
• The nucleoside is absorbed in to blood and
transported to peripheral tissues. Excess nitrogen
bases are further degraded.
• Finally adenine and guanine are converted to uric
acid in our body which is excreted through urine.
9.3. clinical correlates
• Since uric acid precipitates, excess uric acid
causes kidney stone in kidney and gout in joints
• On degradation of pyrimidine bases:
1. Cytosine and uracil are converted to
ammonia, carbondioxide and beta-alanine
2. Thymine to NH3, CO2, H2O and α-methyl β
alanine.
 BIOSYNTHESIS OF NUCLEIC ACIDS
• Genetic information stored in DNA in the form of
nucleotide sequence flows from DNA to DNA,
DNA to RNA then from RNA to protein.
• This flow of genetic information is the central
dogma of molecular genetics.
• Usually involves three different processes.
1. Replication
• Is synthesis of new DNA or information copying
• In this process information is transmitted from
parent to daughter. The new DNA is identical to
parent DNA
2. Transcription
• Is synthesis of RNA from DNA or information
transfer
• In this process, information is transferred from
DNA to RNA
3. Translation
• Synthesis of proteins using information present
in RNAs or information decoding.
• In this process, information present in RNA in
the form of nucleotide sequence, is converted
into sequence of amino acids
9.4. DNA REPLICATION
• Is synthesis of DNA
• It is the way in which the genetic information can
pass from parental cell to daughter cell.
• As stated before, the double helical structure of
DNA depends on the base complementarity.
• Also this complementarity represents the
fundamental basis for the formation of new DNA
strands from the parent DNA strand in a semi
conservative manner.
• In this process, two daughter DNA’s are
produced, each has one parent strand
(conserved) and newly synthesized strand.
• The enzymes involved are template-directed
polymerases that can synthesize the complementary
sequence of each strand with extraordinary fidelity.
• Steps in prokaryotic DNA synthesis
-separation of the two complementary DNA strands
and formation of the replication fork
-RNA primer synthesis
-chain elongation
-excision of RNA primers and their replacement by
DNA
Separation of the two complementary DNA strands
and formation of the replication fork
• The parental double helical DNA must separate
(melt) in order to replicate
• DNA replication begins at unique nucleotide
sequence, origin of replication
• In prokaryotic cells origin is at one site. In
eukaryotic cells origin is at many sites.
• These sites include a short sequence composed
almost exclusively of AT base pairs
• As the two strands unwind and separate they
form a “V” where active synthesis occurs,
replication fork
Proteins required for DNA strand separation
( prepriming complex)
• DnaA protein: bind to specific nucleotide
sequences at the origin of replication.
-It separates the DNA strand forming localized
regions of single-stranded DNA
• DNA helicases: bind to single-stranded DNA near
the replication fork and then move into the
neighboring double stranded region, forcing the
strand apart in effect.
• Single-stranded DNA-binding (SSB) proteins
(helix-destablizing proteins): bind only to the
single-stranded DNA and keep the two strands
of DNA separated in the area of replication origin
to provide the single stranded template required
by polymerase.
-Also protect the DNA from nucleases that
cleave single-stranded DNA
Solving the problem of supercoils
• As the two strands are separated there will be
appearance of positive supercoils (supertwists)
ahead of the replication fork
• Topoisomerases are responsible for removing
supercoils in the helix
• Type I DNA topoisomerases: reversibly cut a
single strand of the double helix.
-
have both nuclease (strand-cutting) and
ligase (strand-resealing) activities
• Type II DNA topoisomerases: bind tightly
to the DNA double helix and make
transient breaks in both strands
Direction of DNA replication
• The DNA polymerase responsible for
copying the DNA templates are only able to
“read” the parental nucleotide sequences in
the of 3' → 5' direction and synthesize the
new DNA in the 5' → 3‘
• Leading strand: the strand that is being
copied in the direction of the advancing
replication fork and is synthesized almost
continuously
• Lagging strand: the strand that is being copied in
the direction away from the replication fork,
synthesized discontinuously
-these short stretches of discontinuous DNA
(Okazaki fragment) are eventually joined to
become a single continuous strand
RNA primer
• DNA polymerase cannot initiate synthesis of a
complementary strand of DNA on a totally
single-stranded template, rather require an RNA
primer
• RNA primer: a short, double-stranded region
consisting of RNA base-paired to the DNA
template with a free hydroxyl group on the 3' -
end of the RNA strand.
Chain elongation
• Prokaryotic and eukaryotic DNA
polymerase elongate a new DNA strand
by adding deoxyribonucleotides, one at a
time, to the 3' –end of the growing chain
• DNA polymerase III: catalyze DNA chain
elongation. It remains bound to the template
strands as it moves along.
-The new strand grows in the 5' → 3‘, antiparallel
to the parental strand
-the nucleotide building blocks are 5‘-
deoxyribonucleoside triphosphate
Proofreading: the nucleotide sequence of DNA is
replicated with as few errors as possible
-misreading of the template sequence could result in
deleterious, perhaps lethal, mutations
-the proof reading activity requires an exonuclease
that move 3‘→ 5' direction , not 5' → 3‘ like the
polymerase activity, because excision must be done
in the reverse direction from that of synthesis.
Excision of RNA primers and their replacement
by DNA
• DNA polymerase III continues to synthesize
DNA on the lagging strand until it is blocked by
proximity to an RNA primer.
• DNA polymerase I has a 5' → 3‘ exonuclease
activity that is able to hydrolytically remove
the RNA primer and fill the gap (5' → 3‘
polymerase activity)
• RNase H catalyzes this reaction in eukaryotes
DNA ligase
• Catalyzes the final phosphodiester linkage
between the 5'-phosphate group on the DNA
chain synthesized by DNA polymerase III and
the 3‘-hydroxyl group on the chain made by
DNA polymerase I
DNA repair mechanism
 9.5. Transcription
• Transcription is the process of RNA Synthesis
directed by a DNA template.
• Following their synthesis, mRNAs are translated
into sequence of amino acids. rRNA, tRNA and
additional small RNA molecules perform
specialized structures and regulatory functions
and are not translated.
• It occurs in three phases:-
1. Initiation
2. Elongation
3. Termination
1.Initiation
• Involves the binding of the RNA polymerase
holoenzyme to a region on the DNA that
determines the specificity of transcription of
that particular gene (Promoter region).
• The left side of that particular point is called
upstream. The promoter is found at this
upstream.
• The RNA polymerase is made up of σ- (sigma)
subunit 2 α-and 2β-subunits.
• Those that are recognized by RNA polymerase
σ (sigma) factors include
Pribnow box: a stretch of 6 nucleotides (5'-
TATAAT- 3') centered about 8-10 nucleotides
to the left of the transcription start site
- a base in the promoter region is assigned a
negative number if it occurs prior to
(“upstream” of) the transcription start site
- -35 sequence: a second consensus
nucleotide sequence (5'-TTGACA- 3‘), is
centered about 35 bases to the left of the
transcription start site
• In between the consensus and TATA box the
base sequences are highly variable and it is this
sequence that can be recognized by σ (sigma)
subunit or RNA polymerase in prokaryotic cell
and by transcription factor in eukaryotic cell.
• To initiate transcription the sigma subunit of
RNA polymerase recognizes the promoter site
on DNA and separates the two strands.
• After the two strands of DNA are separated the
RNA Polymerase starts the pairing of purine
nucleotide .
• RNA polymerase does not require a primer and
has no known endonuclease or exonuclease
activity.
2. Elongation
• Once RNA synthesis is started, there occurs the
step by step addition of ribonucleoside
triphosphates (ATP, GTP, CTP and UTP) at 3’ –
OH end of RNA and releases pyrophosphate
each time a nucleotide is added.
• The forward movement of RNA polymerase
continuous until a termination signal is reached.
• The area of DNA transcribed by polymerase
rewinds back to the double helix.
3. Termination
• The process is carried out by two ways
a. The RNA transcript must be able to form a
stable hair pin turn that slows down the progress
of RNA polymerase and causes it to pause
temporarily (rho independent termination).
b. An additional protein ρ (rho) factor may be
required for the release of the RNA product (ρ-
dependent termination) .
• This factor binds to the C-rich region near the 3’-
end of the newly synthesized RNA, and migrates
along behind the RNA polymerase in the 5' → 3‘
direction until the termination site is reached,
then the factor displaces the DNA template
strand, facilitating the dissociation of the RNA
molecule.
• These two termination mechanisms are carried
out in prokaryotic cells. But in eukaryotic cells
termination may occur by transcription factors
themselves.
9.6. Translation

• The genetic information which flows from DNA to

mRNA will be translated to the universal
language called protein.
• During transcription the base sequence of DNA
determines the base sequence of mRNA.
• Under translation the base sequence of mRNA
determines the amino acid sequences in protein.
• The pathway translates the three-letter alphabet
of nucleotide sequence on mRNA into the twenty
letter alphabet of amino acids that constitute
proteins
• Takes place in 3 stages:
1. Initiation
2. Elongation
3. Termination
1. Initiation of translation
• There are two mechanisms by which the
ribosome recognizes the nucleotide sequence
that initiate translation
a. Shine-Dalgarno sequence: (5’-UAGGAGG-3’) is
located 6 to 10 bases upstream of the AUG
codon on the mRNA molecule, near its 5’-end.
• The 16S rRNA component of the 30S
ribosomal subunit has a nucleotide sequence
near its 3’-end that is complementary to all or
part of the Shine-Dalgarno sequence
• Eukaryotes do not have Shine-Dalgarno
sequence. The 40S ribosomal sub units binds to
the cap structure of mRNA and moves until it
encounters the initiator AUG codon.
b. Initiation codon: the codon AUG is recognized
by a special initiator tRNA. The recognition is
facilitated by IF-2
• In eukaryotes there are at least 10 IF’s are
known (eIF)
2. Elongation of protein chain
• Involves the addition of amino acids to the
carboxyl end of the growing chain
• During elongation the ribosome moves from the 5’-
end to the-3’ end of the mRNA that is being
translated
• Delivery of the aminoacyl-tRNA is facilitated by
elongation factors ( EF-Tu, EF-Ts).
• Elongation cycle takes place in 3 steps:
a. Aminoacyl-tRNA is delivered to A site
b. Formation of peptide bond
c. Translocation
3. Termination stage
• The movement of the ribosome consequently
reaches to the termination site of mRNA.
• Termination occurs when one of the three stop
codons moves into the A site
• Release factor 1(RF-1) recognizes UAG and
UAA; RF-2, UGA and UAA; RF-3, binds GTP
and stimulates RF-1 and RF-2
• The factors cause the newly synthesized protein
to be released and also dissociation of the
ribosome from the mRNA
• No tRNA has anticodons that pairs with the stop
codons.