Chapter 6

NUCLEIC ACID

Subtopic

DNA Structure
DNA Replication
DNA Transcription
DNA Translation

Learning Outcome
• Understand the fundamental of nucleic acids
• Learn how does nucleotides from nucleic acids
• Able to distinguish between DNA and RNA
• Able to identify the structural features and function of nucleic acids, DNA and RNA

Introduction
1. Levels of structure in nucleic acids are viewed same as protein structure levels.
a. Primary structure: order of bases in polynucleotide
b. Secondary structure: 3D conformation of the backbone
c. Tertiary structure: supercoiling of the molecule

2. Nucleic acids are made from monomers called nucleotides, linked through phosphodiester bonds.
3. In biological systems, they serves as information-carrying molecules or, in the case of some RNA
molecules are catalysts
4. Nucletiodes: Nucleotides are monomer of nucleic acid that consist of one phosphate group, one 5-
carbon sugar (ribose or deoxyribose) and one nitrogenous base (ATGC/ AUGC), all covalently
bond together
5. Polymers of nucleotides is called poly-nucleotides
6. Nitrogenous base are made of pyrimidine and purine aromatic ring

7. Function of nucleotides
 Serve as storage for cell genetic information
 Bases serve as recognition units
 Serves as signal molecules and regulators of cellular metabolism and reproduction:
i. ATP is central to energy metabolism
ii. GTP drives protein synthesis
iii. CTP drives lipid synthesis
iv. UTP drives carbohydrate metabolism
8. How nucleotides combine to become nucleic acid?
a. Nucleotides join together through phosphodiester linkages between the 5' and 3' carbon
atoms to form nucleic acids.
b. The 3' -OH of the sugar group forms a bond with one of the negatively charged oxygen of
the phosphate group attached to the 5' carbon of another sugar.
c. When many of these nucleotide subunits combine, the result is the large single-stranded
polynucleotide or nucleic acid
 9. DNA : Serves as a cellular database. Contain information about all the polypeptides a cell can
potentially make
10. Why DNA is deoxyribose and RNA is not?
 Ribose sugar contain OH group on carbon number 2 and 3 which makes it more susceptible
to hydrolysis
 Deoxyribose lack OH- group on carbon 2 making it more stable and thus reliable to carry
genetic material
 RNA can be broken down when not in used.

DNA Double helix theory

1. Erwin Chargaff figures out:
 DNA varied from one species to another, in particular relative amount of A,T,G,C, bases
 In any DNA, the amount of A’s are similar to T’s and amount of G’ is similar to C’s
2. Rosalind Franklin, a chemist and experimental researcher, provide x-ray diffraction data of DNA
3. With Franklin data and Chargaff findings, James Watson and Francis Crick proposed that
DNA :
 double helix and ;
 base pairs are bonded by H-bonds

2 hydrogen bonds:
 A-T
 A-U
3 Hydrogen bonds
 G-C

4. DNA double helix consist of 2 polynucleotide chain wrapped around each other to form a helix
5. Sugar phosphate backbone is the outerpart of the helix
6. Phosphate group at the backbone are high-negatively charge and polar
7. Chain run antiparallel direction from 5’ to 3’ and the other 3’ to 5’
8. Base pair are complementary A-T, and G-C
9. Pair of bases, one on each strand are held in alignment by H-bond
10. The helix structure is stabilized by the H-bond. H- bond are important in holding the double helix
together
 DNA with high G-C content is more stable compare to low G-C content
11. Stacking of the base in DNA contributes the largest part of DNA stabilization energy
Base stacking refers to attractive non-covalent interactions between aromatic rings of the bases
12. By convention, the top strand is written from the 5’ end to 3’end and bottom from 3’ to 5’

DNA sequence:
5’ ATCGATTGAGCTCTAGCG 3’
3’ TAGCTAACTCGAGATCGC 5’

RNA sequence:
5’ AUCGAUUGAGCUCUAGCG 3’
13. DNA Supercoil
 In eukaryotes, the supercoiled DNA is complexed with proteins known as histones
 The DNA is tightly bound to all type of histones except H1
 H1 is the ‘linker histones’ that function to binds the nucleosome at the entry and exit sites
of the DNA, thus locking the DNA into place
 DNA wrapped around a histone is called a nucleosome

14. DNA denaturation

 Denaturation of DNA:
A process where two stands of the double helix is separated by heating the sample in a
solution.
 Heat denaturation is a way to obtain single strand of DNA
 The process will break the H-bond that hold the base pairs together and disrupt the base
stacking interactions
 Can be observed by UV light at 260 nm wavelength region
 Temperature for denaturation by heat to occur depends on its base composition
 Higher temperature is needed to denature DNA rich in G-C pairs
 Renaturation of denatured DNA is possible by slow cooling
 The separate strands can recombine and forms to same base pairs

15. RNA
RNA is needed to convert “DNA information” into polypeptide sequences. In some viruses, RNA
serves as the primary database with no DNA involvement
1. rRNA is located in the cytoplasm of a cell, where ribosomes are found.
 Site of protein synthesis
2. tRNA contain 73-94 nucleotides
 Carrier of amino acids for protein synthesis
3. mRNA carries genetic information from DNA in the nucleus to ribosome in cytoplasm
where protein synthesis takes place
 mRNA transcribes the genetic code from DNA into readable form and use it to
make protein
16. Differences between DNA and RNA

17. Central dogma shows the flow of how genetic information is transferred in the cell:

DNA Replication
Introduction
1. Occurs prior to cell division (mitosis and meiosis)
2. Sequence of bases in DNA encodes for genetic information
The duplication of DNA is essential to produce new daughter cell with the same DNA.
3. The actual gene product is produce when RNA is replicate from DNA template through
transcription process
4. The base sequence of the synthesized RNA is used to produce amino acids sequence through
translation process
5. DNA replication is the process of producing two identical replicas that has the same base
sequence as the original DNA. Takes place in nucleus
6. Type of DNA Replication:

a. Semi conservative replication: Produce two pair of DNA that each contains one original
strands and one new strand.

b. Conservative Replication: Produce two pair of DNA, one pair contains only original
DNA, the other pair only new DNA

c. Dispersive Replication: Produce two pair of DNA that made of the original and new
DNA. Original strand integrity disrupted

7. DNA Replication Process

a. DNA double helix unwinds at specific point called the origin of replication (by enzyme
helicase.
 Prokaryotes usually have 1 origin of replication
 Eukaryotes can have multiple origin of replication
 One Origin of replication will have 2 replication forks
 Why DNA double helix did not completely unwind during the replication +-process?
 When the DNA double helix unwind:
 Nucleases enzyme will attack single stranded DNA
 Single Stranded Binding protein (SSB)
 prevent DNA from winding back
 protect the single-stranded DNA from being digested by nucleases
b. DNA Topoisomerase enzyme will relieve the DNA strand while being unwind by
Helicase enzyme
c. Single Stranded Binding protein (SSB)
 prevent DNA from winding back
 protect the single-stranded DNA from being digested by nucleases
 remove secondary structure from the DNA to allow other enzymes to function
effectively upon it.

d. In DNA replication, synthesis occurs in both direction which are also called
bidirectional replication
 Each strand is replicated, from each origin, from two replication forks move in the
opposite direction on antiparallel templates.
 Both new polynucleotide chains are synthesized in the 5’ to 3’ direction
  Two antiparallel strand must be synthesized in the same direction

e. Once the two strands are separated, primase enzyme will add complementary short strand
called RNA primers to synthesis new DNA

f. The leading strand receives one RNA primer while the lagging strand receives several RNA
primer.
g. The leading strand is continuously extended from the primer by DNA polymerase. While the
lagging strand is extended discontinuously from each primer, forming Okazaki fragments.
h. DNA polymerase will matches complementary nucleotides from RNA primer for the leading
and lagging strands and form phosphodiester bond between nucleotides.
i. When this is complete, a single nick (gap) on the leading strand and several nicks on the
lagging strand can be found. DNA ligase will help to patch the gap on the new strand.
 A nick is a discontinuity in a double stranded DNA where there is missing
PHOSPHODIESTER BOND between adjacent nucleotides
j. Proofreading correction is done by DNA polymerase I
  Proofreading refers to the removal of incorrect nucleotides immediately after they
are added to the growing DNA during replication process

8. How does proofreading helps?

 Spontaneous mutation of bases, insertion or deletion of nucleotides leads to incorrect
nucleotide growing on DNA chain in every 10 to 10 base pairs
 DNA uses its exonuclease activity to remove the incorrect nucleotide and improves the
fidelity of the replication
 Replication resumes when the correct nucleotide is added by DNA polymerase I
9. How replication can proceed along the DNA if the 2 strands are going in the opposite directions?
 daughter strand called the “Leading Strand” is formed continuously from 5’ to 3’ end on
a DNA strand template
 The other strand called “Lagging strand” is formed discontinuously from 5’ to 3’ end on
the other DNA strand template in small fragments called the Okazaki fragments.
 The Okazaki fragments is later linked by DNA Ligase
10.

DNA Transcription

1. DNA transcription is the process where by the RNA is formed from the DNA that carries genetic
codes for
a. Protein production
b. Formation of rRNA and tRNA
2. The RNA produced by transcription is what we call the messenger RNA (mRNA)
3. Once messenger RNA is produced, it will then exit the nucleus (in eukaryotes) and enter the
cytoplasm for translation process
4. Takes place in nucleus
5. Transcription of DNA occurs in 3 main stages:
a. INITIATION of transcription on the template DNA strand,
b. subsequent ELONGATION of the RNA chain, and
c. TERMINATION of transcription, accompanied by the release of RNA polymerase and
the completed RNA product from the DNA template
6. DNA Transcription in Prokaryotes
a. Initiation
 RNA Polymerase in Prokaryotes has σ, α, β, ω section

 σ - unit role is recognition of specific promoter. Other units act as active site for
RNA polymerization .
 σ - unit of RNA polymerase directs to the promoter region.
 RNA polymerase binds to the promoter on DNA double helix and forms a closed
complex. This initiates DNA to unwind at promoter to form open complex for
elongation process.
b. Elongation
 DNA Template strand unwind, forming transcription bubble
 Topoisomerase relieve the DNA strand at both end of the transcription bubble while
being unwind
 σ –subunit will unbound
 RNA polymerase catalyzes formation of phosphodiester bonds between the new
incorporated ribonucleotides on RNA strand from 5’ to 3 ‘
 c. Terminus
 2 type of termination mechanism:
a) Rho-independent termination
- controlled by specific sequences called termination sites.
- a short complementary GC-rich sequence (followed by several U residues) will
form a "brake" that will help release the RNA polymerase from the template (at
the weakly poly-U stretch).
- Inverted repeats in the DNA sequence being transcribed can lead to mRNA
molecule forms a hairpin loop.
- Common method to terminate transcription.
b) Rho-dependent termination
- Requires rho (termination protein) to recognizes and binds to termination site
- This binding destabilizes the interaction leading to dissociation of the
polymerase from the mRNA strand

7. Summary of DNA transcription in prokaryotes

8. Transcription Regulation in Prokaryotes

9.

**Operon – genes that codes enzyme of a certain metabolic pathway

These genes are not transcribed all the time but triggered by an inducer and
inhibits by repressor
a. Ex: the production of β-galactosidase only takes place in the presence of lactose in E.coli
b. Thus, β-galactosidase is an inducible enzyme and lactose is an inducer

10. DNA Transcription in Eukaryotes

Eukaryotic transcription more complicated than Prokaryotes . And it uses more type of protein in
the process (Transcriptional factors protein).
It is carried out by one of three RNA polymerases (RNAP II), and proceeds in three sequential
stages:

RNA Polymerase in Eukaryotes

a. Initiation
 RNA polymerase do not bind on their own to DNA template
 RNA Polymerase II requires transcriptional factors (TF) to form a initiator
transcription complex to initiate transcription
– These transcriptional factors (TF) are proteins that involve in DNA
Transcription process
 TF’s + RNA Polymerase II will form a initiator transcription complex
 The complex are bind at the DNA template to find the promoter
 Once the promoter is found, DNA will unwind, and transcription begin
– Diagram of initiation complex

 Most common promoter sequence is TATAAAT

– A-T is most common in sequence because they only bonded by 2H bond, thus
easier to unwind

b. Elongation
 RNA polymerase II catalyzes formation of phosphodiester bonds between the new
incorporated ribonucleotides on RNA strand from 5’ to 3‘ end

c. Termination
 RNA polymerase II will come across consensus sequence AAUAAA before finally
stops at the actual end
 The consensus sequence AAUAAA may be 100-1000 bases away from the actual
end
 RNA polymerase II terminate the process at the actual end with assistance from RNA
Polymerase I and III
  Initiator complex are release from DNA template strand and is recycled for another
round of transcription
11. If the coding strand were transcribed, would the protein produce be the same?
 If the coding strand were transcribed and translated, the resulting amino acid sequence would
NOT be the same.
 RNA polymerase will not recognize the template and the process may not happen.
 If the base sequence is transcribed it would result in a different peptide

12. Where the site of transcription initiation does takes place?

 DNA sequence that signals the start of RNA transcription is called a promoter
- Bacteria can have hundreds of it on a DNA strand
- Eukaryotic cells can have thousands
- RNA Polymerase and Transcription complex (for eukaryotes) will bind at promoter
and transcribe the coding region at the Transcription Start Site
13. Post-transcriptional modification
Messenger RNA in eukaryotes requires excessive processing
a. Capping of 5’ end
 5 prime "cap" (a guanosine nucleotide methylated at the 7th position) – to give
stability to mRNA

b. Polyadenylating - Adding poly-A

 Protect from nuclease degradation
c. Splicing – removal of introns in pre-mRNA
DNA Translation
1. How does DNA carry genetic codes?
a. As we now know, the “language” of DNA only has four alphabets – A, T, C and G
b. Similar to our language, these four alphabets can make up “genetic words” but the words
in genetics only has three alphabets – codons
c. This combination of three nucleotides encodes for a specific amino acids.
d. The amino acids produced will form proteins that will then be used for biological
processes.

2. The Genetic Code

a. A codon is a sequence of three DNA or RNA nucleotides that corresponds with a specific
amino acid or stop signal during protein synthesis
b. Each codon is a triplet of nucleotide
c. There are total 64 codons in the genetic code
– 61 codes for amino acids and the other 3 is for nonsense codon (stop codon)
d. Initiator Codon
– AUG is the initiator codon in majority of protein
e. There are 3 out of 64 codon who does not encode for any amino acid
– There are called termination codon or stop codon or nonsense codon
– The stop codons :UAA, UAG, UGA
– They encode for no amino acid
– The ribosome would pause the protein synthesis when encounter the stop codons
 The Genetic Code

3. In transcription, DNA strand was used as a template to produce mRNA. After transcription, the
mRNA migrates from nucleus to cytoplasm for translation process.
The coding mRNA sequence can be described as a unit of three nucleotides called a codon
4. Three stages;
a. Initiation – the use of start codon AUG
b. Chain elongation – the building of amino acid sequence from the codons
c. Termination – the completion of the mRNA sequence with one of the stops codons UAG,
UAA or UGA
5. tRNA has to be activated before DNA Translation starts
 tRNA bind with activated amino acid and form aminoacyl-tRNA
 Also known as “Aminoacyl-tRNA synthethase reaction” .
Have 2 processes:
i. Amino Acid + ATP → Aminoacyl-AMP + Ppi
ii. Aminoacyl-AMP + tRNA →Aminoacyl-tRNA +AMP
 The steps is catalyzed by Aminoacyl-tRNA synthethase enzyme
 Aminoacyl-tRNA is used translation for carrying the amino acid and anti-codon

6. Initiation
 Chain initiation by binding of ribosome subunits; large ribosomal subunit and small
ribosomal subunit
 mRNA binds at the “mRNA binding site” at ribosome
 The ribosomal binding sequence on mRNA is usually located 8-10 bases upstream from the
start codon, AUG
 Ribosomal binding sequence is the sequence helps to signal the ribosome to the start codon
for translation
a. in prokaryotes, Shine-Dalgarno sequence
b. in eukaryotes, Kozak sequence
 Ribosome has 3 binding site for DNA Translation process
7. Elongation
 The next aminoacyl-tRNA that carries anticodon attached to A site
 Triplet of tRNA bases (anticodon) forms H-bond with codon on mRNA
 Additional proteins called Elongation Factors (EF) and GTP are required
a. To guide aminoacyl-tRNA to the A site properly
b. To align the anticodon to the mRNA codon
 Peptide bond formation:
a. The α-amino group of the amino acid at the A-site performs nucleophilic attack
on the carbonyl group of the amino acid linked to the tRNA in the P site
b. Both amino acid are now linked with dipeptidyl-tRNA at the A site
c. And the tRNA at the P site has no amino acid (we called it “uncharged tRNA”)
 Translocation
a. The uncharged tRNA moves from P to E site which be later released
b. Peptidyl-tRNA moves from A site to the vacated P site
c. mRNA moves along the mRNA binding site as well
8. Termination
a. The end of translation occurs when the ribosome reaches STOP codons (UAA, UAG,
UGA).
b. The codons are not recognized by tRNA. Release factors protein recognize these codons
when they arrive at the A site and block the binding of new aminoacyl-tRNA at the A site
c. RF ‘s protein and GTP — releases the polypeptide from the ribosome.
d. The ribosome splits into its subunits, which can later be reassembled for another round of
translation process

9. Summary of translation
a. Activation of amino acid by tRNA.
b. Chain initiation by binding of ribosome subunits to mRNA and binding of tRNA-amino acid
complex to mRNA
c. Chain elongation by movement of ribosome subunits along mRNA; release of tRNA and
formation of peptide bond between amino acids.
d. Chain termination by dissociation of ribosome subunits from mRNA and release of
polypeptide chain.