CHAPTER 5

NUCLEIC ACID
Contents
• DNA and RNA
• Biosynthesis of DNA: replication
• Biosynthesis of RNA: transcription
• Protein Synthesis: translation
LOs

At the end of the lectures, students should be able to:

1. Compare the structure and functions of DNA and

RNA
2. Explain the biosynthesis of DNA (replication)
3. Discuss the biosynthesis of RNA (transcription)
4. Explain the biosynthesis of protein (translation)
Nucleic Acids
• Nucleic acid: a biopolymer containing 3 types of monomer
units:

• a base (purine or pyrimidine)

• a monosaccharide (ribose or deoxyribose)
• phosphoric acid

• 2 types of nucleic acids:

• RNA (Ribonucleic Acid)
• DNA (Deoxyribonucleic Acid)
Pyrimidine/Purine Bases
• The structures of pyrimidine and purine
are shown here for comparison
Nucleosides
• Nucleoside: a compound that consists of pentose
sugar (ribose or deoxyribose) covalently bonded to a
base by a glycosidic bond
• Lacks phosphate group

Glycosidic
bond
Nucleotides
• Nucleotide: a nucleoside
with a molecule of
phosphoric acid

• Polymerization leads to
nucleic acids.

• Linkage is repeated (3’,5’-

phosphodiester bond)
DNA
Nucleic Acids
• Levels of structure
• 1°structure: the polynucleotide sequence
• 2°structure: the three-dimensional conformation
of the polynucleotide backbone
• 3°structure: supercoiling
• 4°structure: interaction between DNA and
proteins
DNA - 1° Structure
• Deoxyribonucleic acids (DNA): a biopolymer that consists
of a backbone of alternating units of deoxyribose and
phosphate
• the 3’-OH of one deoxyribose is joined to the 5’-OH of
the next deoxyribose by a phosphodiester bond

• Primary Structure: the sequence of bases along the

pentose-phosphodiester backbone of a DNA molecule
• base sequence is read from the 5’ end to the 3’ end
• System of notation single letter (A,G,C and T)
DNA - 2° Structure
• Secondary structure: the
ordered arrangement of
nucleic acid strands
• the double helix model of
DNA 2°structure.

• Double helix: Two

antiparallel polynucleotide
strands are coiled in a right-
handed manner about the
same axis
T-A Base Pairing
• Base pairing is complimentary

• A major factor stabilizing the double helix is base pairing by

hydrogen bonding between T-A and between C-G

• T-A base pair comprised of 2 hydrogen bonds

G-C Base Pair
• G-C base pair comprised of 3 hydrogen bonds
 Question

Identify the opposite strand for the following

DNA strand:

a) 5’ -ACGTAT- 3’
b) 5’ -AGATCT- 3’
c) 5’ -ATGGTA- 3’

Answer:
a)3’ -TGCATA- 5’
b)3’ -TGTAGA- 5’
c)3’ -TACCAT- 5’
 Question

If a DNA contains 20% adenine nucleotides,

what percentage of guanine nucleotides in the
DNA?

A. 10%
B. 20%
C. 30%
D. 40%
Answer:
(c) 30%
DNA - 3° Structure
• Tertiary structure: the three-dimensional arrangement of all
atoms of a nucleic acid (supercoiling)
• Supercoiling- Further coiling and twisting of DNA helix.
• Circular DNA: a type of double-stranded DNA in which the 5’
and 3’ ends of each stand are joined by phosphodiester
bonds
DNA - 4° Structure
• The structure of chromatin
• Each “Bead” is a nucleosome.
• Nucleosome consists of: DNA
wrapped around histone core

• Histone: a protein, found

associated with eukaryotic DNA

• Chromatin: DNA molecules

wound around particles of
histones in a beadlike structure
Denaturation of DNA

• Denaturation: disruption of 2°structure (double helix

unwinds )
• most commonly by heat denaturation (melting)
• the higher the % G-C, the higher the T m . WHY?
• Because G-C base pair comprised of 3 hydrogen
bonds  need more energy to break the bonds
• Renaturation (re-formed) is possible with slow cooling and
annealing
RNA
General Structure of RNA
• RNA
• consist of long, unbranched chains of nucleotides
joined by phosphodiester bonds between the 3’-OH of
one pentose and the 5’-OH of the next
• the pentose unit is ribose
• the pyrimidine bases are uracil (U) and cytosine (C)
• the purine bases are Adenine (A) and Guanine (G)
• in general, RNA is single stranded
Classification of RNA
• RNA molecules are classified according to their
structure and function
tRNA
• Transfer RNA, tRNA:
• the smallest kind of RNAs
• a single-stranded
polynucleotide chain (73-94
nucleotide)
• carries an amino acid at its
3’ end
• intramolecular hydrogen
bonding occurs in tRNA
rRNA
• Ribosomal RNA, rRNA:
• a ribonucleic acid found in ribosomes, the site of
protein synthesis
• only a few types of rRNA exist in cells
• in both prokaryotes and eukaryotes, ribosomes
consist of two subunits- one larger than the other (eg.
50S and 30S)
mRNA
• Messenger RNA, mRNA:mRNA
• a ribonucleic acid that carries coded genetic
information from DNA to ribosomes for the synthesis
of proteins
• single stranded
• biosynthesis is directed by information encoded on
DNA
• a complementary strand of mRNA is synthesized
along one strand of an unwound DNA, starting from
the 3’ end
DNA REPLICATION
Flow of Genetic Information in the Cell
• Mechanisms by which information is transferred in
the cell is based on “Central Dogma”
Semiconservative Replication

• Replication involves
separation of the 2 original
strands and synthesis of 2
new daughter strands using
the original strands as
templates

• Semiconservative
replication: each daughter
strand contains one template
strand and one newly
synthesized strand
• Video semi conservative replication
Direction of Replication
• DNA double helix unwinds at a specific point called an
origin of replication

• Polynucleotide chains:
• are synthesized in both directions from the origin of
replication; DNA replication is bidirectional in most
organisms
• At each origin of replication, there are 2 replication forks
(points at which new polynucleotide chains are formed)

• Circular DNA of prokaryotes:

• There is one origin of replication and 2 replication forks
Bidirectional Replication

Circular DNA Polynucleotide chains

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DNA Polymerases
• There are at least 5 types of DNA polymerase:
polymerase

1. DNA-Pol I: repair and patching of DNA

2. DNA-Pol III: responsible for the polymerization of the newly formed DNA strand
3. DNA-Pol II, IV, and V: proofreading and repair enzymes
DNA Polymerase
• DNA is synthesized from its 5’  3’ end (from the 3’  5’
direction of the template) by DNA polymerase III
• the leading strand is synthesized continuously in the 5’ 
3’ direction toward the replication fork
• the lagging strand is synthesized semidiscontinuously
(Okazaki fragments) also in the 5’  3’ direction, but away
from the replication fork
• lagging strand fragments are joined by DNA ligase

1 2 3 4 5
DNA Polymerase
• DNA polymerase function has the following
requirements:

• all four deoxyribonucleoside triphosphates: dTTP,

dATP, dGTP, and dCTP
• Mg2+
• an RNA primer - a short strand of RNA to initiate the
replication
DNA Polymerase Reaction
• The 3’-OH group at the end of the
growing DNA chain acts as a
nucleophile.

• The phosphorus adjacent to the

sugar is attacked, and then added
to the growing chain.
DNA gyrase, Helicase and SSB protein

• DNA gyrase: catalyzes reaction involving

relaxed circular DNA.

• Helicase: a helix-destabilizing protein, promotes

unwinding by binding at the replication fork.

• Single-stranded binding (SSB) protein:

stabilizes single-stranded regions by binding
tightly to them.
Primase

• The primase reaction

• RNA serves as a primer in DNA replication
• Primase - catalyzes the copying of a short stretch of
the DNA template strand to produce RNA primer
sequence
DNA polymerase III and DNA polymerase I

• Synthesis and linking of new DNA strands

• begun by DNA polymerase III
• the newly formed DNA is linked to the 3’-OH of the
RNA primer
• as the replication fork moves away, the RNA primer is
removed by DNA polymerase I
Replication Fork General Features:
Proofreading and Repair
• DNA replication takes place only once each generation in
each cell

• Errors in replication (mutations) occur spontaneously only

once in every 109 to 1010 base pairs

• Can be lethal to organisms

• Proofreading - the removal of incorrect nucleotides

immediately after they are added to the growing DNA
during replication by DNA polymerase II, IV, and V.
Animation:
• DNA Replication Fork
• Replication process (McGrahill)
TRANSCRIPTION
Flow of Genetic Information in the Cell
• Mechanisms by which information is transferred in
the cell is based on “Central Dogma”
Transcription
The Basics of Transcription

Coding strand: The DNA strand of a DNA molecule which has the same
base sequence as the RNA transcript (T is replaced with U in RNA strand)

Template strand: The DNA strand of a DNA molecule that is used as a

template for RNA synthesis.
 Question

The coding strand of a DNA contains bases

5’ -ACGTAT- 3’. Identify:
a) The template strand
b) The mRNA strand

Answer:
a)3’ –TGCATA- 5’
b)5’ –ACGUAU- 3’
Question
The template strand of a DNA molecule contains 30 As, 28
Gs, 25 Cs, and 20 Ts.

a) How many of each base is found in the complete double-

stranded DNA molecule?
b) If the template strand was copied in transcription, how
many of each base is found in the mRNA strand?

Answer:
a)50As, 50 Ts, 53Gs, 53 Cs
b)30 Us, 28 Cs, 25 Gs, 20As
Step 1: Chain Initiation
• First phase of transcription is initiation

• Initiation begins when RNA polymerase binds to

Promoter and forms closed complex
• Promoters-DNA sequence that provide direction for
RNA polymerase

• After this, DNA unwinds at promoter to form open

complex, which is required for chain initiation
Initiation and Elongation in Transcription
Step 2: Chain Elongation

• After strands separated, transcription

bubble of ~17 bp moves down the
DNA sequence to be transcribed movie

• RNA polymerase catalyzes formation

of phosphodiester bonds between
the incorp. ribonucleotides

• Topoisomerases relax supercoils in

front and behind the transcription
bubble
Chain Elongation
Transcription bubble
Step 3: Chain Termination
• 2 types of termination mechanisms:
1. Intrinsic termination- controlled by specific
sequences (termination sites)

2. Termination involves rho () protein

Rho-dependent termination sequences cause hairpin
loop to form
Post Transcriptional RNA Modification
• tRNA, rRNA, and mRNA are all modified after transcription
to give the functional form
• the initial size of the RNA transcript is greater than the final
size

• Examples of modifications:
• trimming of leader (5’-end) and trailer (3’-end) sequences
• Exon vs Intron
• addition of terminal sequences
• modification of the structure of specific bases
TRANSLATION
The Genetic Code
• Main features of the genetic code
• Triplet: a sequence of three bases (a codon) is needed to
specify one amino acid
• Nonoverlapping code: no bases are shared between
consecutive codons
• Continuous code
• Degenerate: more than one codon can code for the
same amino acid
• Universal: generally the same in all organisms
• The ribosome moves along the mRNA three bases at a
time
The Genetic Code
• All 64 codons have assigned meanings
• 61 code for amino acids
• 1 codon (AUG) for initiation signal
• 3 codons (UAA, UAG, and UGA) for termination
signals
The Genetic Code

START
Translation Requirement
• mRNA codon
• tRNA anticodon
• rRNA (ribosome)
• Various protein factors
mRNA codon and tRNA anticodon
rRNA (ribosomes)
Large subunit
• The mRNA joins to the
small ribosomal subunit.

• The large ribosomal

subunit has 3 binding Small subunit
sites, E, P, and A.
Steps of Translating the Genetic Message

• Several steps are involved

MOVIE
Step 1: Amino acid activation

• Amino acid must be activated by tRNA and aminoacyl-

tRNA synthetases to form aminoacyl-tRNA before being
incorporated into growing protein chain.

• Free energy of hydrolysis of ATP provides energy for bond

formation.

Amino acid
Step 2: Chain Initiation
• In all organisms, synthesis of polypeptide chain starts at the
N-terminal end and grows to C-terminus

• Initiation requires:
• tRNAfmet
• initiation codon (AUG) of mRNA
• 30S ribosomal subunit
• 50S ribosomal subunit
• initiation factors (IFs)
• GTP, Mg2+

• Forms the initiation complex

The Initiation Complex

N-end C-end

5’ 3’
Chain Initiation

• Requires:
• tRNAfmet contain the triplet 3’-UAC-5’
• Triplet base pairs with 5’-AUG-3’ in mRNA

• 3’-UAC-5’ triplet on tRNAfmet recognizes the AUG

triplet (the start signal) when it occurs at the beginning
of the mRNA sequence that directs polypeptide
synthesis- bind at P site
Step 3: Chain Elongation
• Uses 3 binding sites for tRNA present on the 50S
subunit of the 70S ribosome: P (peptidyl) site, A
(aminoacyl) site, E (exit) site.
• Requires
• 70S ribosome
• codons of mRNA
• aminoacyl-tRNAs
• elongation factors (EF)
• GTP, and Mg2+
Elongation Steps
• Step 1
• an aminoacyl-tRNA is bound to the A site
• the P site is already occupied by tRNAfmet
• 2nd amino acid bound to 70S initiation complex. Defined by the
mRNA
• Step 2
• the peptide bond is formed, the P site is uncharged
• Step 4
• the uncharged tRNA is released (E site)
• the peptidyl-tRNA is translocated to the P site
• the next aminoacyl-tRNA occupies the empty A site
Chain Elongation
N-end C-end

5’ 3’

NOTE:
• Translation: read the mRNA from 5’ to 3’ direction.
• So, ribosom moves toward 3-end
•Polypeptide sequence grows from N-end to C-end
Elongation step
Step 4: Chain Termination
• Chain termination requires
• stop codons (UAA, UAG, or UGA) of mRNA (STOP
CODONS HAVE NO tRNA)
• Release factor (RFs) which either binds to UAA and
UAG or UGA
• GTP which is bound to RF

• The entire complex dissociates setting free the

completed polypeptide, the release factors, tRNA,
mRNA, and the 30S and 50S ribosomal subunits
Chain Termination

N-end C-end

5’ 3’
Components of Protein Synthesis
Posttranslational Modification
• Newly synthesized polypeptides are frequently modified
before they reach their final form.
• Eg:
• leader sequences are removed
• factors such as heme groups may be attached
• disulfide bonds may be formed
• amino acids may be modified, as for example, conversion of
proline to hydroxyproline
• other covalent modifications; e.g., addition of carbohydrates
Question
An mRNA strand of 5’-AUGUUUUUAUAG-3’ was
transcripted from a DNA.

a) How many anticodons are needed to translate the

mRNA?
b) What are the triplet bases on the tRNA’s anticodon ?
c) What is the translation product of the mRNA? (refer
to the genetic code table)

Answer:
a)3 anticodons (because stop codon has no anticodon)
b)3’-UAC-5’ , 3’-AAA-5’, 3’-AAU-5’
c)Met-Phe-Leu
The Genetic Code

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