Nucleic Acids

NUCLEIC ACIDS NUCLEOTIDES

➜ Nucleic Acids are biopolymers made up of ➜ Primary function is to synthesize the proteins

BIOPOLYMERS and their MONOMERIC ➜ Nucleic Acids: Polymers in which repeating unit
UNITS is nucleotide
(building blocks)
➜ Nucleotides are part of many coenzymes
Starch Glucose
Proteins Amino Acids (metabolic pathways)
Nucleic Acids Nucleotides ➜ Serves as donors of phosphoryl groups
DNA deoxyribonucleotides
RNA ribonucleotides ➜ They could act as second messengers and
monomeric units of NUCLEOTIDES allosteric regulators (in enzymes)

➜ Nucleic Acids are found in the nucleus and are

acidic in nature
➜ Two important functions:

1. To hold genetic information

2. To perform variety of other functions

TWO TYPES OF NUCLEIC ACIDS

1. DNA (Deoxyribonucleic Acid)

➜ Found within the cell nucleus (chromosomal
DNA)
A NUCLEOTIDE HAS THREE COMPONENTS:
➜ Can also be found in other parts of the cell:
• Pentose Sugar: Monosaccharide
 mitochondria (mitochondrial DNA)
 chloroplast (chloroplast DNA) • Phosphate Group (PO43-)
 In prokaryotes, cytoplasm (Cytoplasmic • Heterocyclic Base
DNA)
FALSE: DNA is exclusively found in the nucleus
➜ Storage and transfer of genetic information
➜ Passed from one cell to other during cell division
➜ DNAs are the chemical basis of heredity, and it
is grouped into genes.

 Genes – the fundamental units of genetic

information
2. RNA (Ribonucleic Acid)
➜ Occurs in all parts of the cell
 PYRIMIDINE – single-ring bases
PENTOSE SUGAR
Three pyrimidine derivatives:
➜ RNA and DNA differ in the identity of the sugar
unit in their nucleotides.  thymine (T)
 Ribose is present in RNA  cytosine (C)
 2-deoxyribose is present in DNA  uracil (U)

STRUCTURAL DIFFERENCE: PURINE – composed of two fused rings

Two purine derivatives:
 adenine (A)
 guanine (G)
Found in both DNA and RNA:
 Adenine (A)
 guanine (G)
 cytosine (C)
Found only in RNA:
 —OH group present on carbon 2’ in ribose  Uracil (U)
 —H atom in 2-deoxyribose Found only in DNA:
➜ The numbers are primed (‘) – to distinguish the
atoms of the sugar from the nitrogenous bases  Thymine (T)

PHOSPHATES
because they are also numbered (not primed).
➜ Phosphate - is derived from phosphoric acid
(H3PO4)
➜ Under cellular pH conditions, the phosphoric

acid is fully dissociated to give a hydrogen

phosphate ion (HPO42-)
NITROGEN-CONTAINING
HETEROCYCLIC BASES

➜ Heterocyclic compounds containing carbons and

other elements
➜ They are planar in structure to facilitate stacking
➜ The formation of a nucleotide from sugar, base,
➜ There are a total of five bases (four of them in and phosphate is visualized below.
most of DNA and RNAs)
 NUCLEOTIDE FORMATION
➜ Phosphate attached to C-5’ and base is attached
to C-1’ position of pentose
➜ Condensation reaction – water is removed from
the bond
➜ Bond linking the base to the sugar: beta-
glycosidic bond
➜ Bond linking the sugar to the phosphate: ester
bond
➜ Nucleoside - consists of only base and sugar
NUCLEOTIDE NOMENCLATURE
➜ In PCR (polymerase chain reaction), they are
called dNTPs or Deoxynucleoside triphosphates.
These are the substrate in replication.
➜ If there are two/three phosphate groups the
nucleotide name would be (ex: dADP –
diphosphate; dATP - triphosphate)
➜ In nomenclature, pyrimidine bases end with
“-dine”, while purine bases end with “-sine”
➜ Sugar-phosphate groups are referred to as
nucleic acid backbone (sides of the helix
structure; hydrophilic – exposed in the water
environment) (Found in all nucleic acids)
➜ bases are at the center - hydrophobic interior of
the helix
➜ Sugars are different in DNA and RNA
PRIMARY NUCLEIC ACID STRUCTURE
➜ A ribonucleic acid (RNA) is a nucleotide
polymer 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.
➜ Structure: Sequence of nucleotides in DNA or
RNA
➜ Structure of polynucleotides – directional
molecules
➜ Primary structure is due to changes in the
bases
➜ Phosphodiester bond at 3’ and 5’ position
➜ 5’ end has free phosphate and the 3’ end has a
free OH group
➜ Sequence of bases read from 5’ to 3’
➜ 4 nucleotides (TETRANUCLEOTIDE)
➜ 3’5’-Phosphodiester linkage bonds
nucleotides together
➜ Phosphodiesterase enzyme could catalyze the
hydrolysis of phosphodiester bonds (also
exonucleases and endonucleases)
➜ DNA’s structure is more stable than RNA’s
➜ Each end of the nucleotides/polymer is distinct
(5’ end and the 3’ end)
➜ Backbone: Phosphate-Sugar (Nucleic acids)
➜ Backbone: Peptide bonds (Proteins)
NUCLEIC ACID: 3’5’-phosphodiester bonds
PROTEIN: amide linkages/ peptide bonds
➜ The secondary structure involves two
polynucleotide chains coiled around each
other in a helical fashion
➜ the poly nucleotides run anti-parallel
(opposite directions) to each other, (5’  3’
and 3’  5’)
➜ 5’ is attached to 3’ and 3’ is attached to
5’ (forming the 3’5’-phosphodiester bond)
➜ The bases are located at the center and
hydrogen bonded
➜ Complementary base pairs:
 Adenine = Thymine
 Guanine Ξ Cytosine
THE DNA DOUBLE HELIX
➜ H bonds stabilize the helical structure in
DNA; also the interior hydrophobic interactions
between the bases
➜ Base composition: %A = %T and %C = %G
(Chargaff's rule) [%purines = %pyridines]
Example: Human DNA contains 30% adenine,
30% thymine, 20% guanine and 20% cytocine
➜ Major Groove: Blue helix-backbone
➜ Minor Groove: Orange helix-backbone
the
➜ 3 forms of the double-stranded helix:
1. Alpha Form
A-DNA is a right-handed helix, in which for every
360 deg turn of the helix, there are 11 nucleotides.
2. Beta Form
Beta DNA is a right-handed helix, in which for every
360 deg turn of the helix, there are 10 nucleotides.
3. Z Form
Z DNA is a left-handed helix, in which for every 360
deg turn of the helix, there are 12 nucleotides/base
pairs.
DNA SEQUENCE
➜ the sequence of bases on one polynucleotide is
complementary to the other polynucleotide
➜ Complementary bases are pairs of bases in a
nucleic acid structure that can hydrogen bond to
each other.
➜ Complementary DNA strands are strands of
DNA in a double helix with base pairing such that
each base is located opposite its complementary
base.
Example :
➜ List of bases in sequential order in the direction
from the 5’ end to 3’ end of the segment:
5’-A-A-G-C-T-A-G-C-T-T-A-C-T-3’
➜ Complementary strand of this sequence will be:
3’-T-T-C-G-A-T-C-G-A-A-T-G-A-5’
BASE PAIRING
➜ One small and one large base can fit inside the
DNA strands:
 Hydrogen bonding is stronger with A-T and
G-C
 A-T and G-C are called complementary
REPLICATION OF DNA MOLECULES
bases
➜ Replication: Process by which DNA molecules
produce exact duplicates of themselves
➜ Occurs during the S phase of the cell cycle
➜ Replication is known to be a semi-conservative
process
 Old strands act as templates for the
synthesis of new strands (complementary
daughter strand)
 DNA polymerase checks the correct base
pairing and catalyzes the formation of
phosphodiester linkages
 The newly synthesized DNA has one new
DNA strand and old DNA strand
➜ DNA polymerase enzyme can only function in
the 5’-to-3’ direction
 DNA polymerase III catalyzes DNA
elongation using 5’ deoxyribonucleoside
triphosphate (dNTP) as substrates.
 DNA polymerase has proofreading ability.
It proofreads the newly synthesized DNA
using its 3’5’ exonuclease activity.
 DNA polymerase is only able to read the
template in a 3’5’ direction but the synthesis
is in a 5’3’ direction.
 DNA polymerase I enzyme removes the
primers using 5’3’ exonuclease activity and
is responsible for filling gaps in the Okazaki
fragments
➜ Therefore one strand (top; leading strand )
grows continuously in the direction of unwinding
 The DNA is synthesized in opposite
direction in both strands. (Leading and
lagging strand)
➜ The lagging strand grows in segments (forms
Okazaki fragments) in the opposite direction
➜ The segments are latter connected by DNA
ligase
 DNA ligase enzyme seals the mix between
the okazaki fragments and catalyzes the
final phosphodiester bonds.
➜ DNA replication usually occurs at multiple sites
within a molecule (origin of replication)
➜ Multiple-site replication enables rapid DNA
synthesis
 In eukaryotic cells, there are multiple
origins of replication. Meanwhile in
prokaryotic cells, there is only one.
 The origin of replication is indicated by a
short sequence composed almost
exclusively 80 base pairs
 The origin of replication is recognized by
ori-binding proteins (OBP), where most 80
base pairs are located.
 Once recognized, the helicase enzyme
unwinds the double-helix ahead of the
advancing replication fork.
➜ DNA replication is bidirectional from these sites
(replication forks)
 Replication forks are single-strand binding
proteins that maintain the separation of the
parental strands
 DNA topoisomerase enzyme remove
supercoils that would interfere with the
further unwinding of the helix.
ex: DNA topoisomerase 1 “subelase?”
& DNA topoisomerase 2 “gyrase”
 Primase enzyme synthesizes short chains
of RNA called primers, these are needed by
DNA polymerase to begin DNA elongation.
CHROMOSOMES
➜ Upon DNA replication the large DNA molecules
interacts with histone proteins to fold long DNA
molecules.
 Histone – positively charged proteins that
forms helix bonds with a negatively charged
DNA (ionic interaction)
 Histone serves as a structural core, in
which 8 DNAs are wrapped, creating a
nucleosome. This will condense to form the
chromosomes
➜ The histone–DNA complexes are called
chromosomes
➜ A chromosome is about 15% by mass DNA and
85% by mass protein.
➜ Cells of different kinds of organisms have
different numbers of chromosomes.
Example: Number of chromosomes in a human
cell 46, a mosquito 6, a frog 26, a dog 78, and a
turkey 82
➜ Chromosomes occur in matched (homologous)
pairs.
OVERVIEW OF PROTEIN SYNTHESIS
Example: The 46 chromosomes of a human cell
constitute 23 homologous pairs
➜ Protein synthesis is directly under the direction
of DNA
➜ 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
isdeciphered to synthesize a protein
DIFFERENCES BETWEEN RNA AND
DNA MOLECULES
molecule
➜ 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)
➜ RNA molecules are much smaller than DNA
TYPES OF RNA MOLECULES
molecules, ranging from 75 nucleotides to a few
thousand nucleotides
1. Heterogeneous nuclear RNA (hnRNA)
➜ Also known as the primary transcript
➜ Formed directly by DNA transcription.
➜ Post-transcription processing converts the
hnRNA to mRNA
The primary transcript still contains exons and
introns. Introns are the non-sensical regions of
hnRNA, while the exons are the sensical regions.
Non-sensical regions are regions that cannot be
translated into proteins. And once the introns are
removed (facilitated by snRNA), the mRNA will be
formed. The exons will bond together to also form
mRNA.
2. Messenger RNA
➜ Carries instructions for protein synthesis (genetic
information) from DNA
mRNA is usually used as a template for protein
synthesis
➜ The molecular mass of mRNA varies with the
length of the protein
 mRNA is known to be massive. This is
because they carries genetic information
from the nuclear DNA to the cytosol.
 mRNA is modified after transcription (Post
Transcriptional Modification) through
capping (5’ end) using the molecule 7-
methylguanosine by a triphosphate linkage
and poly A-tailing (3’ end), its function is to
preserve the information carried by the
mRNA
3. Small nuclear RNA
➜ Facilitates the conversion of hnRNA (primary
transcript) to mRNA. [removal of introns and
leaving the exons to be spliced to form the mRNA]
➜ Contains from 100 to 200 nucleotides [They are
involved in processing and gene regulation]
4. Ribosomal RNA (rRNA)
➜ Combines with specific proteins to form
ribosomes - the physical site for protein synthesis
➜ Ribosomes have molecular masses on the order
of 3 million
 “rampant RNA” – the most common type
of RNA
 Associated with several proteins as a
component for ribosomes [which has 3
distinct sizes of prokaryotic cells & 4 distinct
sizes in eukaryotic cells]
4. Transfer RNA (tRNA)
➜ Delivers amino acids to the sites for protein
synthesis
➜ tRNAs are the smallest (75–90 nucleotide units)
 “tiny RNA”
 Serves as an adaptor molecule that carries
specific amino acid to the site of the protein
synthesis
 Each amino acids has its own tRNA (has
atleast 1)
➜ Replication & Transcription – occurs in the
nucleus
➜ The product of transcription is mRNA
➜ hnRNA (the primary transcript), with the help of
snRNA, it is converted to mRNA.
➜ Once mRNA is formed, passes through the
nuclear pore and from the nucleus it will come out
to the cytoplasm, where protein synthesis takes
place.
➜ Translation – occurs in the cytoplasm
➜ The information contained by the mRNA is
translated to proteins with the help of rRNA and
tRNA
Catalyzing polymerases and transreplication in
transcription comparisons:
 The nucleic acids synthesized by mRNA
polymerase are from 5’ to 3’ direction
 The required template in DNA during
replication is copied from 3’ to 5’
 Reading is 3’ to 5’ but synthesis is from
5’ to 3’
  DNA polymerase would catalyze DNA
 RNA polymerase would catalyze RNA
 For DNA, there are 2 strands which are
called coding strand and template strand,
which is copied during mRNA synthesis.
 Required substrates by the DNA
polymerase: dNTP, dATP, dGTP, CTP,
dTTP
 Required substrates by the RNA
polymerase: ATPs, GTPs, CTPs, UTPs
 Required primer by the DNA polymerase:
short structures of RNA primers
 Required primer by the RNA polymerase:
None
 Proofreading activity is done 3’ to 5’, using
TRANSCRIPTION: RNA SYNTHESIS
exonucleus acitivity enzyme (only in DNA
polymerase; RNA polymerase has none)
➜ Transcription - A process by which DNA directs
the synthesis of mRNA molecules
➜ Two-step process:
1. synthesis of hnRNA
2. editing to yield mRNA molecule
➜ Gene - A segment of a DNA base sequence
responsible for the production of a specific
hnRNA/mRNA molecule
➜ Genome - All of the genetic material (the total
DNA) contained in the chromosomes of an
organism
Initiation (1st step):
 RNA polymerase (multimeric enzyme –
several polypeptide chain) possesses 5’ to
3’ polymerase activity
Requires the ff:
 The sigma factor recognizes the nucleotide
sequence (promoter region), which
indicates the beginning of the length of the
DNA to be copied or the start of the
transcription.
 In replication, the origin binding proteins
STEPS IN TRANSCRIPTION PROCESS
(oriBPs) recognizes the origin of
replication.
 Sometimes, the rho factor is also required
for termination but only for some genes.
➜ Unwinding of DNA double helix to expose some
bases (a gene):
 Once the sigma factor recognizes the
promoter region, the RNA polymerase binds
to it. Then the unwinding of the double-helix
starts.
➜ The unwinding process is governed by RNA
polymerase
 RNA polymerase copies one strand of the
DNA double-helix. Pairing Cs with Gs, As
with Us.
 The substrates here are ribonucleoside
triphosphates. (deoxyribonucleotide
triphosphate in DNA)
 Does not requires a primer & no known
proofreading activity
➜ 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 mRNA molecule
➜ Transcription ends when the RNA polymerase
enzyme encounters a stop signal on the DNA
template:
➜ The newly formed RNA molecule and the RNA
polymerase enzyme are released
STEPS:
1. Initiation – recognition of the promoter site
POST-TRANSCRIPTION PROCESSING:
FORMATION OF MRNA
2. Elongation –
3. Termination –
*This could be accomplished by the RNA
polymerase alone or a Rho factor would be
required.
➜ Involves conversion of hnRNA to mRNA
 The product of transcription is hnRNA
(primary transcript) which will undergo post-
 transcriptional modification that will form
mRNA
➜ Splicing: Excision of introns and joining of
exons (driven by snRNA)
 Exon - a gene segment that codes for
genetic information
 Intron – DNA segments that interrupt a
TRANSCRIPTOME
genetic message
➜ Alternative splicing - A process by which
several different protein variants are produced from
a single gene
➜ The process involves excision of one or more
exons
➜ Transcriptome: All of the mRNA molecules that
can be generated from the genetic material in a
genome.
➜ Transcriptome is different from a genome
➜ Responsible for the biochemical complexity
created by splice variants obtained by hnRNA.
 different combinations of exons how they
are spliced together will be forming different
THE GENETIC CODE
mRNAs (and also different proteins)
➜ The base sequence in an mRNA determines the
amino acid sequence for the protein synthesized.
➜ The base sequence of an mRNA molecule
involves only 4 different bases - A, C, G, and U
➜ Codon: A three-nucleotide sequence in an
mRNA molecule that codes for a specifi c amino
acid
➜ Based on all possible combination of bases A,
G, C, U” there are 64 possible codes
CHARACTERISTICS OF GENETIC
CODE
➜ Genetic code: The assignment of the 64 mRNA
codons to specific amino acids (or stop signals)
➜ 3 of the 64 codons are termination codons
(“stop” signals)
1. They are specific and ambiguous (a specific
codon always code for the same amino acid)
2. Known to be universal (you can use the
genetic code for plants, microorganisms,
humans.)
✤ 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 corn plant cell,
or a human cell.
3. They are redundant and degenerate (a given
amino acid has more than one triplet or codon)
✤ The genetic code is highly degenerate:
➜ Many amino acids are designated by more than
one codon.
➜ Arg, Leu, and Ser - represented by six codons.
[coding for these amino acids]
➜ Most other amino acids - 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 in unless there are more than four
synonyms
➜ The significance of the “single box” pattern -
the first two bases are the same

ANTICODONS AND TRNA MOLECULES
➜ For example, the four synonyms for Proline -
CCU, CCC, CCA, and CCG.
✤ An initiation codon exists:
➜ The existence of “stop” codons (UAG, UAA, and
UGA) suggests the existence of “start” codons.
➜ The codon - coding for the amino acid
methionine (AUG) functions as initiation codon.
➜ During protein synthesis amino acids do not
directly interact with the codons of an mRNA
molecule.
➜ tRNA (adaptor molecules) as intermediaries
deliver amino acids to mRNA. [the site of protein
synthesis]
TRANSLATION: PROTEIN SYNTHESIS
➜ Two important features of the tRNA structure
➜ The 3’ end of tRNA is where an amino acid is
covalently bonded to the tRNA.
➜ The loop opposite to the open end of tRNA 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.
➜ Translation – a process in which mRNA codons
are deciphered to synthesize a protein molecule
➜ Ribosome – an rRNA–protein complex - serves
as the site of protein synthesis:
➜ Contains 4 rRNA molecules and ~80 proteins -
packed into two rRNA-protein subunits (one small
and one large)
➜ ~65% rRNA and 35% protein by mass
➜ A ribosome’s active site – Large subunit
➜ Ribosome is a RNA catalyst
➜ The mRNA binds to the small subunit of the
ribosome.
Requirements:
1. The amino acids will eventually appear and the
protein should be present (each aa has their own
tRNA)
2. There should be a prescence of the poly
competent ribosomes, protein factors, and the
energy sources (ATP and GTP)
RIBOSOMES
Eukaryotic ribozymes – will form complexes (60s
and 40s will form 80s complexes)
*60s – big ribosomal subunit
*40s – small ribosomal subunit
Prokaryotic ribozymes – 50s and 30s forming 70s
complexes
*50s – big ribosomal subunit
* 30s – small ribosomal subunit
FIVE STEPS OF TRANSLATION
PROCESS
Each ribosome has 3 binding sites:
1. A site – binds to the incoming aminoacyl tRNA
2. P site – occupied by the peptidyl tRNA
3. E site – occupied by the mt tRNA (mitochondrial
tRNA) that is about to exit in the ribosome
tRNA should have the correct charge. A
mischarged tRNA reads the usual codon but inserts
the wrong amino acid. The protein that will be
produced will be dysfunctional (either longer or
shorter)
1. Activation of tRNA: addition of specific amino
acids to the 3’-OH group of tRNA.
2. 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)
 Initiation is activated by GTP hydrolysis –
energy source, with the help of initiation
factors. They will assemble the 40S
chromosomal sub unit with the initiator
 tRNA released. The mRNA and the
ribosomal unit assemble within the complex.
 The mRNA, tRNA, and the ribosomal RNA
should come together to form the so-called
translation complex.
3. 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.
 Aminoacyl tRNA binds to the A site.
Elongation factors direct the binding of the
appropriate tRNA to the codon in the A site.
 It will try to fit but only the tRNA whose anti
codon is complementary to the codon will
be able to bind.
 After binding, the trans enzyme peptidyl
transferase catalyzes peptide bond
formation, then transfers growing
polypeptide to the amino acid A site.
 Then, the ribosome advances three
nucleotides towards the three prime end of
the RNA (translocation – moving peptidyl
tRNA to the P site)
4. Termination: The polypeptide continues to grow
via translocation until all necessary amino acids are
in place and bonded to each other.
 Releasing factors – proteins.
 Their function is to hydrolyze the peptidyl
tRNA bond when a stop codon (UAA, UAG,
UGA) occupies the A site.
 The stop codons would indicate that the
protein synthesis is completed. Then this is
released from the ribosomes, through
hydrolysis and the assembly would
dissociate.
5. Post-translational processing – gives the
protein the final form it needs to be fully functional
 There is a trimming of excess amino acids
 Addition, phosphorylation, glycosylation,
hydroxylation
 The protein synthesized by the ribosomes
EFFICIENCY OF mRNA UTILIZAITION
attached to the ER (concentrated in the
lumen) would undergo glycosylation.
 Glycosylation serves as a tag (for
identification) to address the protein to its
proper destination
 The proteins that are defective are destined
for rapid turn over. They are marked for
destruction by a ubiquitin. They are
degraded by the enzyme proteasome
 Post-translational processing happens in
the golgi apparatus. (where the proteins are
modified and altered to address the protein
to its proper destination)
➜ Polysome (polyribosome): complex of 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
MUTATION
single mRNA - polysomes.
✤ once the stop codons are encountered,
translation will stop. Releasing factors would
hydrolyze the peptidyl tRNA. Translation complex
dissociates and will undergo post-translational
modification.
➜ An error in base sequence reproduced during
DNA replication
➜ Errors in genetic information is passed on during
transcription. [could pass on to the next generation]
➜ 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.
➜ ANY PERMANENT HEREDITABLE DAMAGE
or change in the DNA base sequence
➜ Has the potential to change the base sequence
of the mRNA and the amino acid sequence in
proteins
➜ The protein could be defective or dysfunctional
because of mutation.
➜ Point Mutation – could be transition and
transversion
 Transition – purine to purine
ex: adenine is replaced by guanine or
pyrimidine (thymine) is replaced by cytosine
 Transversion – purine to pyrimidine
TYPES OF MUTATION:
1. Silent Mutation
➜ The new codon specifies the same amino
acid. A change in the third position of the codon
(base of the 3rd position) Leads to it being a
synonym of another codon, coding for the same
amino acid. Basically, there is no effect in the
protein.
2. Missense Mutation
➜ The new codon specifies a different amino
acid. There’s a possibility that this will decrease
protein function. It has variable effects such as
protein being shorter or longer that becomes
dysfunctional.
3. Nonsense Mutation
➜ The new codon is a stop codon. If this
happens, the protein that will be produced would be
shorter than normal. Therefore it will become
nonfunctional.
4. Frameshift Mutation
➜ There is a deletion or an addition of a base. If
this happens, the protein that will be produced
could become nonfunctional or could be shorter
than normal
5. Large Segment Deletion
➜ For example, the unequal crossover in myosis
leads to loss of function. The protein that will be
formed could be shorter than normal or there’s a
MUTAGENS
portion that is missing.
6. Splice Donor or Acceptor
➜ The spliced site is moved? through mutation.
The defects could be variable: ranging from the
addition or the lesion of a few amino acids or
probably the entire exon missing. If this happens,
the protein that will be formed will be nonfunctional.
➜ same with conditions like tay sachs disease,
goiter disease, beta thalassemia
7. Triplet Repeat Expansion
➜ The expansions in coding regions, the protein
(product) is produced to be longer than normal in
size. If this happens, the protein would be unstable.
Same with the coditions like huntington's disease,
fragile x syndrome, and myotonic dystrophy.
➜ Mutations are caused by mutagens
A mutagen is a substance or agent that causes
a change in the structure of a gene:
➜ Radiation and chemical agents are two
important types of mutagens
➜ Ultraviolet, X-ray, radioactivity and cosmic
NUCLEIC ACIDS AND VIRUSES
radiation are mutagenic –cause cancers
➜ Chemical agents can also have mutagenic
effects. Ex: HNO2 can convert cytosine to uracil
➜ Nitrites, nitrates (preservatives), and
nitrosamines – can form nitrous acid in cells
➜ Under normal conditions mutations are
repaired by repair enzymes
 If an individual is still young, the repair
mechanisms would be efficient. But as it
grows older (geriatrics), the repair
mechanisms would be affected. So, if there
are cases of mutations, they are no longer
repaired. This is why generally, there are
higher indicents of cancers in adults
compared to younger individuals.
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
➜ Retroviruses (ex. HIV) – they carry their
genome in the form of a single stranded RNA
molecules
➜ Using the enzyme called reverse transcriptase,
the viruses makes a DNA copy of their RNA and
they could integrate the copy into host cells. This is
why viruses have the ability to evolve and have
different variants.
➜ RNA polymerase lack proofreading activity
unlike the DNA polymerase which explains the high
mutation rate of the viruses.
➜ DNA repair occurs when there is a mismatch
and is done by DNA polymerase 1 and ligated by
DNA ligase. (identifies and corrects damage to the
DNA molecules)
Types: mismatch repair (MMR), base excision
repair (BER), nucleotide excision repair (NER),
double strand break repair.
VACCINES
✤ DNA can be repaired but not RNA because of
its lack of proofreading activity of the RNA
polymerase.
➜ Viruses attach to the host cell on the outside cell
surface and proteins of virus envelope catalyze the
breakdown of the cell membrane and forms a hole
(to enter)
➜ Viruses then inject their DNA or RNA into the
host cell
➜ The viral genome is replicated, proteins coding
RECOMBINANT DNA AND GENETIC
ENGINEERING
for the viral envelope are produced in hundreds of
copies. [using reverse transcriptase]
➜ Hundreds of new viruses are produced using the
host cell replicated genome and proteins in short
time [results in infection]
➜ Inactive virus or bacterial envelope
➜ Antibodies produced against inactive viral or
bacterial envelopes will kill the active bacteria and
viruses
 some uses mRNAs, nuclear envelope and
different parts of the virus, for the scientists
to develop the vaccine.
 PRINCIPLE OF VACCINES: Antibodies
that are produced against inactive viral or
bacterial envelopes will kill the active
bacteria and viruses in the body
RECOMBINANT DNA PRODUCTION
USING A BACTERIAL PLASMID
➜ 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
➜ The manipulation of the DNA
Genetic Engineering (Biotechnology):
➜ The study of biochemical techniques that allow
the transfer of a “foreign” gene to a host organism
and produce the protein associated with the added
gene [so that the traits that are needed would be
manifested in the protein that is translated later on]
➜ Bacterial strains such as E. coli inserted with
circular plasmids, and/or yeast cells carrying
vectors containing foreign genes are used for this
purpose
➜ Plasmids (double stranded DNA) replicate
independently in bacteria or yeast
1. 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
2. Isolation of plasmid fraction
➜ The cellular contents are fractionated to obtain
plasmids
3. Cleavage of plasmid DNA
➜ Restriction enzymes are used to cleave the
double-stranded DNA
4. Gene removal from another organism
➜ Using the same restriction enzyme the gene of
interest is removed from a chromosome of another
organism
5. 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.
6. 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 (into proteins that will be manifested
in the organism)
Genetically Modified Organisms (GMO) – such
as different rice varieties (IR8, IR5) wherein the
agricultural build would be 15 times greater
compared to the traditional rice varieties. Rice
resources in the philippines, using uptake of
recombinant DNA, they were able to produce
different varieties such as drug, pests, disease
resistants and many more.
 More examples would be products such as
seedless grapes and watermelons.
How they solve dengue and malarian problems:
 In the lab, they would produce a mosquito
wherein a certain gene is inserted in such a
way that when it is released to the wild, it
will mate with other female mosquitoes but
the larvae that will be produced will not
reach maturity.
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
POLYMERASE CHAIN REACTION
➜ Each clone can synthesize the protein directed
by foreign gene it carries
✤ Cloning – the production of recombinant DNA
molecule that is self perpetuating
 The DNA fragments are inserted into a
bacterial plasmid that contain antibiotic
resistance genes
 This plasmids can be selected for using a
media contain antibiotic and then amplified
 Restriction enzymes cleave DNA for about 4
to 6 base pairs (palindromic sequence)
allowing for insertion of a fragment into the
plasmid
 Tissue mRNA is isolated and exposed.
Using reverse trancriptase this will form
DNA
➜ The polymerase chain reaction (PCR) is 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.
 This is done in a molecular laboratory using
a PCR machine.
 This is used to synthesize many copies of
the desired fragment of DNA
 Involves several steps, which would be
repeated many many times until the needed
amount is acquired
[The DNA is denatured to generate two separate
strands. Joined cloning? excess remade DNA
primers with anneal to the specific sequence on
each found to be amplified. Then, heat stables DNA
polymerase, replicates the DNA sequence following
the primer.] [dude this doesn’t make any sense
TvT]
✤ These are three main stages:
 Denaturing – when the double-stranded
template DNA is heated to separate it into
two single strands.
 DNA SEQUENCING
 Annealing – when the temperature is
lowered to enable the DNA primers to
attach to the template DNA.
 Extending – when the temperature is
raised and the new strand of DNA is made
by the Taq polymerase enzyme.
PCR is very easy to carryout and
requirements are:
➜ Source of gene to be copied
 dideoxyribonucleotides DNA
polymerization at its base generating
sequences of various lenghts that
encompass the entire sequence
➜ This interruption of synthesis leads to the
formation of every possible nucleotide site mixture.
➜ These nucleotides are labeled using radioactive
the dNTP during their synthesis.
➜ The radiolablled nucleotides are then separated
on a gel by electrophoresis

➜ Thermostabel DNA polymerase Step 1: Cleavage of DNA using restriction

➜ 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
✤ Used to diagnose COVID-19 (RT PCR test) – the
fastest way
➜ DNA sequencing is a method by which the base
sequence in a DNA molecule (or a portion of it) is
determined.
➜ Discovered in 1977 by Fredrick Sanger
enzymes
➜ Restriction enzymes are used to cleave the large
DNA molecule into smaller fragments (100–200
base pairs).
Step 2: Separation into individual components
➜ The mixture of small DNA fragments generated
by the restriction enzymes is separated into
individual components via gel electrophoresis
techniques.
Step 3: Separation into single strands
➜ A given DNA fragment is separated into its two
strands by chemical methods to use it as a
template in step 4.

 He used this to the original sequence of the

DNA (through sanger’s reagent)
Concept in DNA sequencing:

➜ Selective interruption of polynucleotide

BASIC STEPS INVOLVED IN DNA

SEQUENCING

synthesis using 2’,3’- dideoxyribonucleotide

triphosphates (ddNTPs).

 Sequencing started from the end terminal of

the polypeptide chain going to the C
terminal: Interlin?