Chem 45 Biochemistry: Stoker chapter 22 Nucleic Acids

Chapter 22
Table of Contents
Copyright © Cengage Learning. All rights reserved 2
22.1 Types of Nucleic Acids
22.2 Nucleotide Building Blocks
22.3. Nucleotide Formation
22.4 Primary Nucleic Acid Structure
22.5 The DNA Double Helix
22.6 Replication of DNA Molecules
22.7 Overview of Protein Synthesis
22.8 Ribonucleic Acids
22.9 Transcription: RNA Synthesis
22.10 The Genetic Code
22.11 Anticodons and tRNA Molecules
22.12 Translation: Protein Synthesis
22.13 Mutations
22.14 Nucleic Acids and Viruses
22.15 Recombinant DNA and Genetic Engineering
22.16 The Polymerase Chain Reaction

Types of Nucleic Acids
Section 22.1
• The Swiss physiologist Friedrich Miescher (1844-1895) discovered
nucleic acids in 1869 while studying the nuclei of white blood cells.
• The fact that they were initially found in cell nuclei and are acidic
accounts for the name nucleic acid.
• It is now known that nucleic acids are found throughout a cell, not
just in the nucleus.
• Of all biomolecules, it is only the nucleic acids that have the
remarkable property of replicating itself, thus nature chose
these molecules to serve as the repository and transmitter of
genetic information in every cell and organism.
• The genome or total DNA of a cell acts like a molecular file where
the program for an organism’s activities (maintenance, development,
growth, reproduction, and even death) are encoded.

Section 22.1
• Cells in an organism are exact replicas
• Cells have information on how to make new cells
• Molecules responsible for such information are nucleic acids
• The nucleic acids (DNA in particular) are the “informational
molecules”; into their primary structure is encoded a set of directions
that ultimately governs the metabolic activities of the living cell.
• Two types of Nucleic Acids:
• DNA: Deoxyribonucleic Acid: found within cell nucleus
– storage and transfer of genetic information
– passed from one cell to other during cell division
• RNA: Ribonucleic Acid: occurs in all parts of cell
– primary function is to synthesize the proteins

Section 22.1
• Gene is a segment of DNA which specifies the chain of amino acids that comprises
the protein molecule
– most human genes are ~1000–3500 nucleotide units long
– genome: all of the genetic material (the total DNA) contained in the
chromosomes of an organism
– human genome is about 20,000–25,000 genes
• The genetic message is transcribed by mRNA and translated by tRNA and rRNA
into thousands of different proteins.
The Central Dogma

Section 22.2
Nucleotide Building Blocks
• Nucleic Acids: polymers in which
repeating unit is nucleotide
• A nucleotide has three components:
– pentose sugar - a
monosaccharide
– phosphate group (PO4
3-)
– heterocyclic base

Section 22.2
Nitrogen-Containing Heterocyclic Bases

Section 22.2

Section 22.3
Nucleotide Formation
Nucleoside Formation
• Nucleoside: formed from condensation reaction between a five-carbon monosaccharide
and a purine or pyrimidine base derivative.
– the N9 of a purine or N1 of a pyrimidine base is attached to C1’ position of sugar (beta-
conformation) in an N-C-glycosidic linkage
• Nomenclature:
– for pyrimidine bases – suffix -idine is used (cytidine, thymidine, uridine)
– for purine bases – suffix -osine is used (adenosine, guanosine)
– prefix “-deoxy” is used to indicate deoxyribose present (e.g: deoxythymidine)

Section 22.3
• Phosphate group is
added to a nucleoside
– attached to C5’
position through a
phosphoester
bond
– condensation
reaction (H2O
released)
– named by
appending 5’-
monophosphate to
nucleoside name

Section 22.3
Nucleotide Nomenclature
1) The 5’- nucleoside monophosphate of…is called….
a) adenosine…adenylic acid or adenosine
monophosphate (AMP)
b) guanosine…guanylic acid or guanosine
monophosphate (GMP)
c) cytidine…cytidylic acid or cytidine
monophosphate (CMP)
d) uridine…uridylic acid or uridine
monophosphate (UMP)
e) deoxythymidine…deoxythymidylic acid or
deoxythymidine monophosphate
(dTMP)
2) The 5’-nucleoside diphosphates are ADP,GDP,
CDP, UDP, dTDP
3) The 5’-nucleoside triphosphates are ATP, GTP,
CTP, UTP, dTTP
* If deoxyribose is present, the prefix deoxy is used
(dAMP, dADP, dATP, dGMP, dGDP, dGTP, dCMP,
dCDP, dCTP)

Section 22.3
Nucleotide Nomenclature

Section 22.4
Primary Nucleic Acid Structure
Primary Structure
• The nucleotides of a
polynucleotide chain are linked
to one another in 3’,5’-
phosphodiester bonds
• Phosphoric acid forms a
phosphate ester to connect
the 3’-hydroxyl group of one
pentose to the 5’-carbon on
another pentose
• Sugar-phosphate groups are
referred to as nucleic acid
backbone ; found in all
nucleic acids
• Sugars are different in DNA
and RNA

Section 22.4
Polynucleotides and the
Nucleic acids
• A ribonucleic acid (RNA) is a
polynucleotide in which each
of the monomers contains
ribose, a phosphate group,
and one of the heterocyclic
bases adenine, cytosine,
guanine, or uracil
• A deoxyribonucleic acid
(DNA) is a nucleotide polymer
in which each of the
monomers contains
deoxyribose, a phosphate
group, and one of the
heterocyclic bases adenine,
cytosine, guanine, or thymine.

Section 22.4
• 5’ end has free phosphate group and 3’
end has a free OH group
• the sequence of bases is read from 5’ to 3’
• the next nucleotide binds at the 3’ end

Section 22.4
Shorthand Structure of Polynucleotides
• bases are indicated by their
initials, the ribose by a
straight line extending from
the base, and the phosphate
by P.
• the C3’ and C5’ of the ribose
or deoxyribose are indicated
by the fact that the C5’ is at
the end of the ribose line and
the C3’ is toward the middle
of the line.
• Takadiasase (mold) 
attacks “b” linkages in which
“a” is linked to a purine
nucleotide
• RNAse (bovine pancreas) 
attacks “b” linkages in which
“a” is linked to a pyrimidine
nucleotide

Section 22.5
The DNA Double Helix
• DNA, a high mol.wt., double-stranded
polynucleotide that occurs almost
exclusively in the nucleus of the cell
• primary function is storage and transfer of
genetic information which is used
(indirectly) to control many functions of a
living cell
• genetic information is encoded in the
primary structure of the DNA
• the primary structure of DNA is the
sequence of nucleotides in the chain
• the base content of DNA displays three sets
of equivalent pairs:
A + G = T + C (pu / pyr ratio = 1)
A = T
G = C
• the structure of the four bases permit
hydrogen bonding between specific base
pairs: Adenine always pairs with
Thymine and Guanine with Cytosine

Section 22.5
• the proof of this base-pairing came when
Watson and Crick proved by x-ray
diffraction that the DNA structure was a
double helix whose chains were
complementary and antiparallel
• complementary  means that A binds
to T and C to G between the chains
- the sequence of bases on one
strand automatically determines the
sequence of bases on the other
strand
• antiparallel  means that each end of
the helix contains the 5’ end of one strand
and the 3’ end of the other, so that the
chains travel in opposite directions
- only when the 2 strands are
antiparallel can the base pairs
form the H-bonds that hold them
together

Section 22.5
• The double helical secondary
structure of DNA is stabilized by a
number of factors:
• Chargaff’s rule of base pairing:
(A=T and GΞC)
%A = %T and %C = %G)
– example: human DNA
contains 30% adenine, 30%
thymine, 20% guanine and
20% cytosine
• Stacking interaction of the
hydrogen-bonded bases (the
purines and pyrimidine rings) at the
center
• Hydrophobic interior (bases) and
hydrophilic exterior (sugar-
phosphate backbone) ; contact with
bases through spiral grooves :
major and minor grooves

Section 22.5
Practice Exercise
• Predict the sequence of bases in the DNA strand
complementary to the single DNA strand shown below:
5’ A–A–T–G–C–A–G–C–T 3’

Section 22.5
Practice Exercise
• Predict the sequence of bases in the DNA strand
complementary to the single DNA strand shown below:
5’ A–A–T–G–C–A–G–C–T 3’
Answer:
3’ T–T–A–C–G–T–C–G–A 5’

Section 22.5
• the sugar-phosphate backbone of the two
strands spiral around the outside of the helix
like the handrails on a spiral staircase
• the nitrogenous bases extend into the center
at right angles to the acids of the helix as if
they are the steps of the spiral staircase
Denaturation of DNA
• The loss of helical structure due to disruption
of H–bonds is called denaturation or
melting, where the double strands separate
into single strands.
• This can be due to extremes of pH, heat, or
chemicals that disrupt H-bonds.
• DNAs which are G-C rich denature at a
higher temperature (Tm) than those which
are A-T rich.

Section 22.5
• Conformations of DNA:
• DNA can assume different conformations
because deoxyribose is flexible and the C–N-
glycosidic linkage rotates. (Recall that
furanose rings have puckered conformation)
• B-DNA – the common form as described by
Watson and Crick model
• A-DNA – when DNA becomes partially
dehydrated it assumes the A-form; observed
when DNA is extracted with solvents such as
ethanol.
• Z-DNA – named for its “zigzag” conformation;
DNA segments with alternating purine and
pyrimidine bases (esp. CGCGCG) are most
likely to adopt a Z configuration; regions of
DNA rich in GC repeats are often regulatory,
binding specific proteins that initiate or block
transcription.

Section 22.5
Types of DNA sequences:
1. Exons – the coding sequences; interrupted by
noncoding sequences
2. Introns – the noncoding sequences; from 10 to
10,000 bases long
3. Palindrome or inverted repeats
• a DNA sequence that contains the same
information whether it is read forward or
backward; e.g. “MADAM, I’M ADAM”
• tendency to form hairpin loop and a snapback
(cruciform)
• perfect palindrome forms with exact base pairs;
quasi palindrome, when not all will form hairpin
loop
4. Cruciform (or snapback)
• as their name implies, are crosslike structures
• when a DNA sequence contains a palindrome

Section 22.6
Replication of DNA Molecules
• Process by which DNA molecules
produce exact duplicates of themselves
• The two strands of the DNA double helix
unwind, the separated strands serve as
templates for the formation of new DNA
strands.
• Free nucleotides pair with the
complementary bases on the separated
strands of DNA.
• When the process is completed two
identical molecules of DNA are formed
• The newly synthesized DNA has one new
DNA strand and old DNA strand
• Two daughter DNA molecules are
produced from one parent DNA molecule,
with each daughter DNA molecule
containing one parent DNA strand and
one newly formed DNA strand.

Section 22.6
• DNA replication is semiconservative and mostly
bidirectional
• First step is the separation of the strands
– accomplished by helicase, which breaks
the H-bonds between base pairs
– positive supercoiling results when H-bonds
are broken, this is relieved by
topoisomerase
– when supercoiling is relieved, single-strand
binding protein binds to the separated
strands to keep them apart
– primase catalyzes synthesis of a 10-12
base piece of RNA to “prime” the DNA
replication
• DNA polymerase “reads” the parental strand
or template, catalyzing the polymerization of a
complementary daughter strand; the enzyme
checks the correct base pairing and catalyzes
the formation of phosphodiester linkages

Section 22.6
• There are different mechanisms for replication
of the two strands
• DNA polymerase enzyme can function only in
the 5’-to-3’ direction which can be offered only
by the 3’strand
1) the 3’ strand is called the leading strand
because it is replicated in a continuous
process in the direction of the
unwinding;
2) the 5’ strand is the lagging strand, it is
replicated in a discontinuous
mechanism and grows in segments
(Okazaki fragments) in the opposite
direction; the segments are later
connected by DNA ligase
In the Leading Strand:
• The DNA Polymerase, using dNTP’s and Mg2+,
cause the replication by base-pairing the
3’strand with free nucleotide units

Section 22.6
In the Lagging Strand
• The enzyme primase (using NTP’s and Mg2+)
puts primers on the lagging strand by forming
short RNA strands through base-pairing of the
5’strand.
• DNA Polymerase recognize, then lengthen
the primers using dNTP’s.
• The primers are then removed by
nucleotidase and further lengthening is done
by DNA Polymerase resulting to an OKAZAKI
STRAND.
• The Okazaki strands are then linked together
and sealed using the enzyme ligase leading to
the formation of a NEW STRAND
DNA replication usually occurs at multiple sites
within a molecule (origin of replication) and the
replication is bidirectional from these sites
• Multiple-site replication enables rapid DNA
synthesis

Section 22.6
Two conditions must be satisfied for replication to take place with high fidelity and accuracy:
a) normal electronic characteristics
b) normal base sequence

Section 22.7
Overview of Protein Synthesis
• Protein synthesis is directly under the
direction of DNA
• The expression of the information
contained in the DNA is fundamental to
the growth, development, and
maintenance of all organisms
• Proteins are responsible for the
formation of skin, hair, enzymes,
hormones, and so on
• Protein synthesis can be divided into
two phases.
– Transcription – a process by which
DNA directs the synthesis of mRNA
molecules
– Translation – a process in which
mRNA is deciphered to synthesize
a protein molecule

Section 22.8
Ribonucleic Acids
Differences Between RNA and DNA Molecules
• The sugar unit in the backbone of RNA is ribose; it is deoxyribose
in DNA.
• The base thymine found in DNA is replaced by uracil in RNA
• RNA is a single-stranded molecule; DNA is double-stranded
(double helix)
• A hairpin loop is produced when single-stranded RNA doubles
back on itself and complementary base pairing occurs.
• RNA molecules are much smaller than DNA molecules, ranging
from 75 nucleotides to a few thousand nucleotides

Section 22.8
Ribonucleic Acids
Types of RNA Molecules
• RNA functions primarily in the
synthesis of proteins, the molecules
that carry out essential cellular
functions
• Heterogeneous nuclear RNA (hnRNA)
- formed directly by DNA transcription.
• Messenger RNA (mRNA) - carries
instructions for protein synthesis
(genetic information) from DNA
• Small nuclear RNA (snRNA) -
facilitates the conversion of hnRNA to
mRNA.
• Ribosomal RNA (rRNA) - combines
with specific proteins to form
ribosomes - the physical site for
protein synthesis
• Transfer RNA (tRNA) - delivers amino
acids to the sites for protein synthesis

Section 22.9
Transcription: RNA Synthesis
Transcription
• biosynthesis of RNA by DNA-dependent RNA Polymerase on a DNA template; an information transfer
process where one of the two DNA strands acts as a template, which is copied into a complementary RNA
molecule
• transcription of DNA into RNA is restricted to discrete regions or genes of DNA
• when a gene is transcribed, only one strand of the DNA serves as the template for RNA synthesis: the
template strand, also called the sense strand; the nontemplate strand is called the coding strand, also
called the antisense strand.
• in the example below the blue 3’ strand is the template strand, or the sense strand. RNA polymerase
sythesizes an hnRNA (shown in green) in the 5' to 3' direction complementary to this template strand. The
opposite 5’ DNA strand (red), the nontemplate strand, is called the coding strand, or the antisense
strand. The easiest way to find the corresponding hnRNA sequence (shown in green) is to read the coding,
or antisense strand directly in the 5' to 3' direction substituting U for T.
5' T G A C C T T C G A A C G G G A T G G A A A G G 3'
3' A C T G G A A G C T T G C C C T A C C T T T C C 5'
5' U G A C C U U C G A A C G G G A U G G A A A G G 3'

Section 22.9
Steps in the Transcription Process
• RNA Polymerase recognizes promoter
sites (sequence of bases which signals
where to start) and enhancer sites (base
sequence which make recognition clearer)
on DNA.
• These sites interact to define the region
for transcription.
• Like DNA Polymerase, RNA Polymerase
requires a 5’-3’ direction which can only
be provided by the 3’strand of DNA
• The RNA Polymerase, once it has spotted
the portion to be transcribed, does the
transcription in the 5’  3’ direction and
uses NTP’s and Mg2+

Section 22.9
Steps in the Transcription Process
• Unwinding of DNA double helix by RNA
polymerase to expose some bases (a
gene):
• Alignment of free ribonucleotides along
the exposed DNA strand (template)
forming new base pairs
• RNA polymerase catalyzes the linkage of
ribonucleotides one by one to form
hnRNA molecule
• Transcription ends when the RNA
polymerase enzyme encounters a stop
signal on the DNA template:
• The newly formed hnRNA molecule and
the RNA polymerase are released
• Transcription occur with very high
accuracy and fidelity (normal base
sequence and normal electronic
character) except under conditions of
spontaneous and induced mutation

Section 22.9
Post-Transcription Processing: Formation of mRNA
• hnRNA is a primary transcript which is
processed in post-transcriptional
modification, a three step process:
– A 5' cap structure is added; this
structure is required for efficient
translation of the final mRNA
– A 3' poly(A) tail is added by poly(A)
polymerase to protect the 3' end of
the mRNA from enzymatic digestion;
prolongs the life of the mRNA
– RNA splicing removes portions of
the primary transcript that are not
protein coding
• transcripts due to introns are removed
by spliceosomes, composed of “small
nuclear ribonucleoproteins (snRNPs,
read “snurps”) ; the transcripts due to
exons are joined by ligase
• the exons in DNA are transcribed as the
codons in mRNA

Section 22.9
• Once the mRNA is formed and released from DNA, it moves into the cytoplasm
and combines with rRNA in ribosomes where protein synthesis occurs.

Section 22.10
The Genetic Code
• In translation, the base
sequence in mRNA
determines the amino
acid sequence of the
protein synthesized
• The base sequence of
an mRNA molecule
involves only 4
different bases - A, C,
G, and U
• The code is a triplet
code since it involves
3 bases per coding
unit.
• The coding unit is
called a codon.
• The genetic code is a
series of base triplets
in mRNA called
codons that code for
a particular amino
acid.

Section 22.10
The Genetic Code
• The genetic code is highly
degenerate:
– many amino acids are
designated by more than one
codon.
– Arg, Leu, and Ser are
represented by six codons.
– most other amino acids are
represented by two codons
– Met and Trp have only a
single codon.
– codons that specify the same
amino acid are called
synonyms
• There is a pattern to the
arrangement of synonyms in the
genetic code table.
– all synonyms for an amino
acid fall within a single box
unless there are more than
four synonyms

Section 22.10
The Genetic Code
• The genetic code is almost
universal:
– with minor exceptions the
code is the same in all
organisms
– the same codon specifies
the same amino acid
whether the cell is a
bacterial cell, a plant cell,
or a human cell.
• An initiation codon exists:
– the existence of “stop”
or termination codons
(UAG, UAA, and UGA)
suggests the existence
of “start” codons.
– the codon - coding for
the amino acid
methionine (AUG)
functions as initiation
codon.

Section 22.10
The Genetic Code
Practice Exercise
Sections A, C, and E of the following base sequence section of a
DNA template strand are exons, and sections B and D are
introns.
a. What is the structure of the hnRNA transcribed from this
template?
b. What is the structure of the mRNA obtained by splicing the
hnRNA?

Section 22.10
The Genetic Code
Practice Exercise
Sections A, C, and E of the following base sequence section of a
DNA template strand are exons, and sections B and D are
introns.
a. What is the structure of the hnRNA transcribed from this
template?
b. What is the structure of the mRNA obtained by splicing the
hnRNA?
Answers:
a. 3’ GCG–GCA–UCA–ACC–GGG–CCU–CCU 5’
b. 3’ GCG–ACC–CCU–CCU 5’ or 5’ UCC-UCC-CCA-GCG
3’

Section 22.10
The Genetic Code
Practice Exercise
The structure of an mRNA segment obtained from a DNA
template strand is
mRNA 3’ ACG-AGC-CCU-CUU 5’
What polypeptide amino acid sequence will be synthesized using
this mRNA?

Section 22.10
The Genetic Code
Practice Exercise
The structure of an mRNA segment obtained from a DNA
template strand is
mRNA 3’ ACG-AGC-CCU-CUU 5’
What polypeptide amino acid sequence will be synthesized using
this mRNA?
Answer: Phe-Ser-Arg-Ala

Section 22.11
Anticodons and tRNA Molecules
• During protein synthesis amino
acids do not directly interact
with the codons of an mRNA
molecule.
• tRNA molecules as
intermediates deliver the amino
acids to mRNA.
• Two important features of tRNA
– the 3’end is where an
amino acid is covalently
bonded to the tRNA.
– the loop opposite to the
open end is the site for a
sequence of three bases
called an anticodon.
• Anticodon - a three-nucleotide
sequence on a tRNA molecule
that is complementary to a
codon on an mRNA molecule.
• Codon-anticodon interaction is
antiparallel

Section 22.12
Translation: Protein Synthesis
Practice Exercise

Section 22.12
Practice Exercise
A tRNA molecule possesses the anticodon 5’ CGU 3’ . Which
amino acid will this tRNA molecule carry?

Section 22.12
Practice Exercise
A tRNA molecule possesses the anticodon 5’ CGU 3’ . Which
amino acid will this tRNA molecule carry?
Answer: Thr

Section 22.12
• Translation – a process in which mRNA
codons are deciphered to synthesize a
protein molecule
• Ribosome – an rRNA–protein complex
that serves as the site, or “workbench” for
protein synthesis:
• Ribosomal RNA (rRNA)
– constitutes about 60% of the
ribosomes (40% protein)
– structurally composed of two spherical
particles of unequal size: the smaller
has affinity for mRNA ; the larger has
an attraction for tRNA ;
– has two sites to bind tRNA
• P-site binds to the growing
peptide
• A-site binds the aminoacyl tRNA

Section 22.12
Five Steps of Translation Process
• Activation of tRNA: addition of specific amino acids to the 3’-OH
group of tRNA.
• Initiation of protein synthesis: Begins with binding of mRNA to small
ribosomal subunit such that its first codon (initiating codon AUG)
occupies a site called the P site (peptidyl site)
• Elongation: Adjacent to the P site in an mRNA–ribosome complex
is A site (aminoacyl site) and the next tRNA with the appropriate
anticodon binds to it. Peptidyl transferase links the A site and P site
amino acids via a peptide bond.
• Termination: The polypeptide continues to grow via translocation
until all necessary amino acids are in place and bonded to each
other. The process stops when a stop codon is encountered.
• Post-translational processing: Gives the protein the final form it
needs to be fully functional

Section 22.12
• (1) Activation of tRNA: addition of
specific amino acids to the 3’-OH group
of tRNA.
• The amino acid combines with a
molecule of ATP, yielding a compound
known as aminoacyl adenylate.
• The reaction is enzyme-catalyzed
• The aminoacyl adenylate remains on the
surface of the enzyme and then
undergoes reaction with the proper
tRNA molecule to form the
corresponding aminoacyl-tRNA
complex (charged tRNA or activated
tRNA).

Section 22.12
• (2) Initiation of protein
synthesis begins with the
formation of an initiation
complex
• mRNA binds to small
ribosomal subunit such
that its first codon
(initiating codon AUG)
occupies a site called the
P site (peptidyl site)
• The initiator tRNA
recognizes the initiation
codon, AUG.
• The large ribosomal
subunit binds to form the
complete, functional
ribosome.

Section 22.12
• (3) Elongation occurs in three steps that are repeated until protein
synthesis is complete:
– (3a), the binding of the aminoacyl-tRNA to the empty A-site (amino acyl-
tRNA binding site)
– (3b), peptide bond formation occurs catalyzed by an enzyme peptidyl
transferase that is part of the ribosome. Now the peptide chain is shifted
to the tRNA that occupies the A site.
– (3c), the uncharged tRNA molecule left on the P site is discharged, and
the ribosome changes position so that the next codon on the mRNA
occupies the A-site. This movement is called translocation, which shifts
the new peptidyl-tRNA from the A-site to the P-site.

Section 22.12
• (3) Elongation occurs in three
steps that are repeated until protein
synthesis is complete:
– (3a), the binding of the
aminoacyl-tRNA to the empty
A-site (amino acyl-tRNA binding
site)
– (3b), peptide bond formation
occurs catalyzed by an enzyme
peptidyl transferase that is part
of the ribosome.
– Now the peptide chain is shifted
to the tRNA that occupies the A
site.

Section 22.12
• The transfer of
an amino acid
(or growing
peptide chain)
from the P site
to the A site
during peptide
bond formation
is an example
of an acyl
transfer
reaction

Section 22.12
• (3) Elongation occurs in three
steps that are repeated until
protein synthesis is complete:
– (3c), the uncharged tRNA
molecule left on the P site is
discharged, and the ribosome
changes position so that the
next codon on the mRNA
occupies the A-site.
– This movement is called
translocation, which shifts the
new peptidyl-tRNA from the
A-site to the P-site.

Section 22.12
Five Steps of Translation
• (4) Termination: The
polypeptide continues to grow
via translocation until all
necessary amino acids are in
place and bonded to each other.
The process stops when a stop
codon is encountered.
• (5) Post-translational
processing: Gives the protein
the final form it needs to be fully
functional
– cleavage of f-met (initiation
codon); association with
other proteins; bonding to
carbohydrate or lipid groups;
S – S bonds between cys
units

Section 22.12

Section 22.12
Efficiency of mRNA Utilization
• Polysome (polyribosome):
The complex of a mRNA
and several ribosomes
• Many ribosomes can move
simultaneously along a
single mRNA molecule
• The multiple use of mRNA
molecules reduces the
amount of resources and
energy that the cell
expends to synthesize
needed protein
• In the process – several
ribosomes bind to a single
mRNA - polysomes

Section 22.12
• Since protein is not synthesized continuously but only as needed, the DNA
must normally be in a “repressed” state
• A repressor, which is a polypeptide, binds to small segment of the DNA
called the operator site
• As long as the repressor is bonded to the operator site of the DNA, no
mRNA is produced and protein synthesis is inhibited
• When a particular protein is needed, an inducer is formed
• The inducer combines with the repressor changing its shape so that it can
no longer bind to the DNA
• Once the repressor is removed from the DNA, synthesis of mRNA and
hence, protein can begin
• When sufficient protein has been synthesized, the inducer is removed and
the repressor once again binds the DNA, stopping protein synthesis
Regulation of protein synthesis

Section 22.12
Antibiotics inhibit bacterial protein synthesis
Antibiotic Effect on ribosomes to inhibit protein synthesis
Chloramphenicol Inhibits peptide bond formation and prevents the binding of tRNA’s
Erythromycin Inhibits peptide chain growth by preventing the translocation of the
ribosome along the mRNA
Puromycin Causes release of an incomplete protein by ending the growth of the
polypeptide early
Streptomycin Prevents the proper attachment of tRNA’s; mRNA misreading by binding
30S
Tetracycline Prevents the binding of tRNA’s by binding to 30S subunit
========================================================================
Several antibiotics stop bacterial infections by interfering with the synthesis
of proteins needed by the bacteria. Some antibiotics act only on bacterial
cells by binding to the ribosomes in bacteria, but do not act on human cells.

Section 22.13
Mutations
Replication, transcription, and translation occur with very high accuracy
and fidelity (normal base sequence and normal electronic character)
except under conditions of spontaneous and induced mutation
• An error in base sequence reproduced during DNA
replication
• Errors in genetic information is passed on during
transcription.
• The altered information can cause changes in amino
acid sequence during protein synthesis and thereby alter
protein function
• Such changes have a profound effect on an organism.

Section 22.13
Mutations
Spontaneous Mutations
1. Point Mutations
- substitution of a single nucleotide for
another caused by tautomeric base-
mispairs due to the ease by which rare
tautomers are formed
- C is the most mutable base due to the
very small energy difference between its
two tautomers
- in nature, there are more A-T pairs than
G-C pairs to protect us from the effect of
spontaneous mutation
A. Transition (C-A* ; G-T* ; C *- A mispair)
- a purine base is changed to another
purine; a pyrimidine base to another
pyrimidine
B. Transversion (A-A* mispair)
- a purine base is changed to pyrimidine;
a pyrimidine base changed to purine

Section 22.13
Mutations
Spontaneous Mutations
2. Frameshift mutation
-leads to a change in the
reading frame
A. Insertion
B. Deletion
In an insertion or deletion
mutation, one or more
nucleotides are added to or
deleted from the DNA
sequence.
Then a frameshift occurs
which leads to a misreading
of all the codons following
the base change.

Section 22.13
Mutations

Section 22.13
Mutations
Mutagens
• Although a number of structural features of nucleic acids promote
stabilization of base sequences, reactivity with some physical and chemical
agents can alter the electronic characteristics of the bases and other
structural units.
• Consequently, nucleic acid functions would be affected
• A mutagen is a substance or agent that causes a change in the structure of
a gene:
– Physical agents : heat, Ultraviolet, ionizing radiation (X-ray, gamma
rays)
– Chemical agents :HNO2 can convert cytosine to uracil
• Nitrites, nitrates, and nitrosamines – can form nitrous acid in cells
• Under normal conditions mutations are repaired by repair enzymes

Section 22.13
Mutations
Induced Mutations – Physical Agents
• UV radiation
• absorbed primarily by the pyrimidine bases.
– UV light causes covalent linkage of
adjacent pyrimidine bases forming
cyclobutane pyrimidine dimer
– when T is irradiated with UV, excitation of
pi electrons to antibonding MO’s will result
in the formation of T diradicals. Coupling
of T diradicals may result in the formation
of thymine cyclobutane dimers
– Failure to repair this defect can lead to
xeroderma pigmentosum; people who
suffer from this genetic skin disorder are
very sensitive to UV light and develop
multiple skin cancers
– No purine dimers since purines are more
thermodynamically stable than
pyrimidines

Section 22.13
Mutations
Induced Mutations – Physical Agents
• heat mutagenesis
- characterized by transmigration of N – C
glycosidic bonds producing neoguanosine
crosslinks
• ionizing radiation
– more often when a plant or animal is
irradiated most of the energy is deposited
in the aqueous phase. Less often will a
primary ionization occur in an organic
molecule
– a portion of damage to the living system
results from reactive particles that are
formed in the water phase and diffuse to
an organic molecule in the cell causing
secondary reactions (free radicals are
implicated in radiation damage)

Section 22.13
Mutations
Induced Mutations – Chemical Agents
• intercalating agents (PAH), alkylating agents, heavy metal ions, etc.
• one notorious source of numerous mutagens is cigarette smoke
• It contains PAH, nitrosamines, hydrazines, pyrolysates, alkoxy free
radicals, superoxide anion radicals, Cd2+, etc.
• other notorious sources are: cured foods; burnt portion of broiled
fish and meat; moldy peanuts and cereals; pesticides;
• polluted air (epoxides, SO2, ozone, Pb2+, ethylene dibromide, etc.)
• laboratory chemicals (benzene has been linked to leukemia, CHCl3,
CCl4, etc)

Section 22.13
Mutations
• Intercalating agents
- with polycyclic planar structures, like PAH,
e.g. benzo(a)pyrene, interpose between the
strands within the grove of DNA
- inhibit its replication/transcription, or cause
deletions

Section 22.13
Mutations
• Alkylating agents
- have electrophilic sites or may be
metabolized to electrophiles which
can interact with alkylating sites at
DNA; include PAH, nitrosamines,
aflatoxins, aromatic amines,
epoxides, nitrogen mustard,
nitrosoureas, etc.
- bases in DNA are nucleophiles and
as such are strongly attracted to
electrophilic compounds
• N7- alkylation leads to apurinic sites
(positivity of R is relayed to C8 & N9 thereby
enhancing the dipositivity of the N-C –
glycosidic bond & render it more less
stable)
• O6- alkylation leads to base mispairs

Section 22.13
Mutations
Chemical mutagens. (a) HNO
2(nitrous acid) converts cytosine
to uracil and adenine to
hypoxanthine. (b) Nitrosoamines,
organic compounds that react to
form nitrous acid, also lead to
the oxidative deamination of A
and C. (c) Hydroxylamine
(NH
2
OH) reacts with cytosine,
converting it to a derivative that
base-pairs with adenine instead
of guanine. The result is a C-G
to T-A transition. (d) Alkylation of
G residues to give O
6
-
methylguanine, which base-pairs
with T. (e) Alkylating agents
include nitrosoamines,
nitrosoguanidines, nitrosoureas,
alkyl sulfates, and nitrogen
mustards. Note that
nitrosoamines are mutagenic in
two ways: they can react to yield
HNO
2
or they can act as
alkylating agents.

Section 22.13
Mutations
When DNA is hit by a mutagen
Lesions repaired Cell death
Mutagen
Cells during Organogenesis
ininin
Somatic cells
Lesions escape repairs
Germ cells
CANCER
BIRTH DEFECTS
STERILITY
GENETIC DISORDER
(can be transmitted from
one generation
to the next)
DNA

Section 22.13
Mutations
Some chemical and environmental carcinogens
Carcinogen Tumor occurrence
Asbestos Lung, respiratory tract
Arsenic Skin, lung
Cadmium Prostate, kidneys
Chromium Lung
Nickel Lung, sinuses
Aflatoxin Liver
Nitrites Stomach
Aniline dyes Bladder
Vinyl chloride Liver
Benzene Leukemia

Section 22.13
Mutations
DNA Repair Mechanisms
• DNA is the only macromolecule that is repaired
rather than degraded.
• The repair processes are very efficient with fewer
than 1 out of 1,000 accidental changes resulting
in mutations.
• The rest are corrected through various repair
mechanisms before, during, or after replication
A) photoreactivation repair
uses an enzyme photolyase, which
binds the T-T cyclobutane dimer & in
the presence of visible light changes
the cyclobutane ring back into
individual pyrimidine bases

Section 22.13
Mutations
DNA Repair Mechanisms
B) excision repair
mutations are excised by a
series of enzymes that remove
incorrect bases and replace
them with the correct ones.
Base excision repair – involves a
battery of enzymes called DNA
glycosylases each of which recognizes a
single type of altered base in DNA and
catalyzes its hydrolytic removal from
the deoxyribose sugar (e.g., removing
deaminated cytosine, deaminated
adenine, alkylated bases, etc.)
Nucleotide excision repair

Section 22.14
Nucleic Acids and Viruses
Viruses
• Viruses: Tiny disease causing agents with outer protein
envelope and inner nucleic acid core
• They can not reproduce outside their host cells (living
organisms)
• Invade their host cells to reproduce and in the process
disrupt the normal cell’s operation
• Virus invade bacteria, plants animals, and humans
– Many human diseases are of viral origin, e. g. Common cold,
smallpox, rabies, influenza, hepatitis, and AIDS

Section 22.14
Nucleic Acids and Viruses
Human cancers caused by oncogenic viruses
Virus Disease
RNA viruses
Human T-cell leukemia-lymphoma virus-1 Leukemia
Human immunodeficiency virus Acquired immune deficiency (AIDS)
DNA viruses
Epstein-Barr virus Burkitt’s lymphoma (cancer of wbc)
Nasopharyngeal carcinoma
Hodgkin’s disease
Hepatitis B virus Liver cancer
Herpes simplex virus Cervical and uterine cancer
Papilloma virus Cervical and colon cancer, genital warts

Section 22.15
Recombinant DNA and Genetic Engineering
• Recombinant DNA: DNA molecules that have been
synthesized by splicing a sequence of segment DNA
(usually a gene) from one organism to the DNA of
another organism.
• Genetic Engineering (Biotechnology): A process in
which an organism is intentionally changed at the
molecular (DNA) level so that it exhibits different traits.

Section 22.15
• First genetically engineered organism are bacteria
(1973) and Mice (1974)
• Insulin producing bacteria - commercialized in 1982.
– Bacteria act as protein factories
• Many plants have now been genetically engineered and
numerous beneficial situations have been created.
– Disease resistance – increased crop yield
– Drought resistance – consumption of less water
– Predator resistance – less insecticide use
– Frost resistance – resist changes in temps below freezing.
– Deterioration resistance – long shelf-life.
Benefits

Section 22.15
Recombinant DNA Production using a Bacterial Plasmid
• Dissolution of cells:
– E. coli cells of a specific strain containing the plasmid of interest are treated with
chemicals to dissolve their membranes and release the cellular contents
• Isolation of plasmid fraction:
– The cellular contents are fractionated to obtain plasmids
• Cleavage of plasmid DNA:
– Restriction enzymes are used to cleave the double-stranded DNA
• Gene removal from another organism:
– Using the same restriction enzyme the gene of interest is removed from a
chromosome of another organism
• Gene–plasmid splicing:
– The gene (from Step 4) and the opened plasmid (from Step 3) are mixed in the
presence of the enzyme DNA ligase to splice them together.
• Uptake of recombinant DNA:
– The recombinant DNA prepared in step 5 are transferred to a live E. coli culture
where they can be replicated, transcribed and translated.

Section 22.15
• Transformed cell can reproduce a large number of
identical cells –clones:
– Clones are the cells that have descended from a
single cell and have identical DNA
• Given bacteria grow very fast, within few hours 1000s of
clones will be produced
• Each clone can synthesize the protein directed by
foreign gene it carries

Section 22.15
Recombinant DNA Production using a Bacterial Plasmid

Section 22.16
The Polymerase Chain Reaction
• The polymerase chain reaction (PCR): A method for rapidly
producing multiple copies of a DNA nucleotide sequence (gene).
• This method allows to produce billions of copies of a specific gene
in a few hours.
• PCR is very easy to carryout and the requirements are:
– Source of gene to be copied
– Thermostable DNA polymerase
– Deoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP)
– A set of two oligonucleotides with complementary sequence to
the gene (primers)
– Thermostable plastic container and
– Source of heat

Section 22.16
The Polymerase Chain Reaction

Chem 45 Biochemistry: Stoker chapter 22 Nucleic Acids

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