MOLECULAR

STRUCTURES
OF DNA, RNA & PROTEIN
DNA STRUCTURE
DNA STRUCTURE
DNA STRUCTURE - ATTACHING A PHOSPHATE
GROUP
DNA STRUCTURE - ATTACHING A BASE AND
MAKING A NUCLEOTIDE
DNA STRUCTURE - ATTACHING A BASE
AND MAKING A NUCLEOTIDE
 DNA STRUCTURE - JOINING
NUCLEOTIDES INTO A DNA STRAND
DNA
DNA
RNA
PROTEIN
AMINO ACID
CENTRAL DOGMA
OF MOLECULAR
BIOLOGY
An Overview
• The central dogma of molecular biology is a
theory stating that genetic information flows only
in one direction, from DNA, to RNA, to protein, or
RNA directly to protein. (National Human
Genome Research Institute)
3 MAIN STEPS:

1. REPLICATION (DNA Synthesis)

2.TRANSCRIPTION (RNA Synthesis)
3. TRANSLATION (Protein Synthesis)
 1. REPLICATION or DNA Synthesis
• DNA strands separate and serve as templates for the production of
new DNA molecules.
A. The following are features of replication:
i. Semiconservative - the resulting DNA consists of one old and
one new strand
ii. Base pairing is maintained; Adenine pairs with Thymine,
Guanine pairs with Cytosine
iii. New DNA molecules are produced in the 5’ to 3’ direction
iv. Semidiscontinuous. The leading strand is synthesized in a
continuous manner (5’ to 3’) while the lagging strand is produced
discontinuously in short stretches called Okazaki fragments.
1. REPLICATION or DNA Synthesis
B. In lagging strand synthesis, there is a need for a primer terminus
which is provided by an RNA molecule. RNA is synthesized by
a primase or RNA polymerase. The 3’OH of the RNA is where
new DNA nucleotides are added thus new DNA is built in the
5’ to 3’ direction.
C. Enzymes in replication are as follows:
1. helicase; 4. primase or RNA polymerase;
2. gyrase; 5. DNA polymerase and
3. SSB (single strand 6. DNA ligase
binding proteins);
1. REPLICATION or DNA Synthesis
2. TRANSCRIPTION or RNA synthesis
• DNA is unwound and one strand is used as template for the
production of an RNA molecule.
• An RNA polymerase makes RNA in the 5’ to 3’ direction.
• Specific regions in the DNA called promoters allow the binding of
transcription factors which make possible the binding of RNA
polymerase.
• Three major types of RNA are:
1. messenger RNA (mRNA);
2. transfer RNA (tRNA) and
3. ribosomal RNA (rRNA).
2. TRANSCRIPTION or RNA synthesis
 3. TRANSLATION or Protein synthesis
• This occurs in the ribosome. Basic
ingredients are the various types of
RNAs produced in transcription and
some proteins or enzymes.
• The mRNA contains triplets of bases
called codons that specify an amino
acid, eg. UUU-phe.
• Various tRNAs carry amino acids
from the cytoplasm to the actual site
of translation in the ribosome.
• A tRNA has an anticodon that pair
with a codon in the mRNA.
3. TRANSLATION or Protein synthesis
• In bacteria, transcription and translation may be simultaneous.
• In eukaryotic cells, mRNA, tRNA and rRNA travel from the nucleus
to the cytoplasm through the nuclear pores.
• RNAs may undergo processing. Some unnecessary parts like
introns are removed.
• In eukaryotic mRNA, a 5’ cap and a 3’ poly A tail are added.
• Coding regions of mRNA are called exons. They specify functional
protein products.
3. TRANSLATION or Protein synthesis
• To initiate translation, the small and
the big subunits of the ribosome have
to be separated.
• Initiation factors (IF) make this
possible. They also prevent the
premature reassociation of these
subunits.
• The small subunit of the ribosome
binds the mRNA and allows the
entrance of a tRNA to the P site
bearing the first amino acid.
 3. TRANSLATION or Protein synthesis
• The big subunit then binds and
together they form an assembly
ready for the next amino acid in the
A site of the ribosome.
• In the elongation process of
translation, amino acids are linked
by peptide bond formation due to
the action of peptidyl transferase
known to be a part of the ribosome
subunit.
3. TRANSLATION or Protein synthesis
• A stop codon signals the end of translation. No amino acid
corresponds to a stop codon.
• Release factors halt the process and the polypeptide is
released.
3. TRANSLATION or Protein synthesis
• The genetic code is the
correspondence of the
mRNA codons to amino
acids.
• An amino acid is
specified by a codon
with three code letters.
The genetic code is
shown here
 GENETIC
ENGINEERING
GENETIC ENGINEERING
• involves the use of molecular techniques to modify the
traits of a target organism.
• modification of traits may involve introduction of new traits
into an organism as to enhancement of present traits by
increasing the expression of the desired gene or by
disrupting the inhibition of the desired genes’ expression.
GENETIC ENGINEERING includes
• Classical breeding - considered as the traditional way of
genetic engineering which practices the mating of organisms
with desirable qualities
• Recombinant DNA (rDNA) technology - modern technique of
genetic engineering
- the joining together of DNA molecules from two different
species
- recombined DNA molecule is inserted into a host organism
to produce new genetic combinations that are of value to
science, medicine, agriculture, and industry
Recombinant DNA (rDNA)
• the general name for a piece of DNA that has been created
by the combination of at least two strands.
• they are DNA molecules formed by laboratory methods of
genetic recombination (such as molecular cloning) to bring
together genetic material from multiple sources, creating
sequences that would not otherwise be found in the genome
• first achieved in 1973 by Herbert Boyer, of the University of
California at San Francisco, and Stanley Cohen, at Stanford
University, who used E. coli restriction enzymes to insert
foreign DNA into plasmids.
Recombinant DNA (rDNA)
Steps of Genetic Recombination Technology
1. Isolation of Genetic Material
2. Restriction Enzyme Digestion
3. Amplification Using PCR
4. Ligation of DNA Molecules
5. Insertion of Recombinant DNA Into Host
6. Isolation of Recombinant Cells
1. Isolation of Genetic Material
• The first step in rDNA technology is to isolate the desired DNA in its
pure form i.e. free from other macromolecules.
• Since DNA exists within the cell membrane along with other
macromolecules such as RNA, polysaccharides, proteins, and lipids,
it must be separated and purified which involves enzymes such as
lysozymes, cellulase, chitinase, ribonuclease, proteases etc.
• Other macromolecules are removable with other enzymes or
treatments. Ultimately, the addition of ethanol causes the DNA to
precipitate out as fine threads. This is then spooled out to give
purified DNA.
2. Restriction Enzyme Digestion
• Restriction enzymes act as molecular scissors that cut DNA at specific
locations. These reactions are called ‘restriction enzyme digestions’.
• They involve the incubation of the purified DNA with the selected restriction
enzyme, at conditions optimal for that specific enzyme.
• The technique ‘Agarose Gel Electrophoresis’ reveals the progress of the
restriction enzyme digestion.
• This technique involves running out the DNA on an agarose gel. On the
application of current, the negatively charged DNA travels to the positive
electrode and is separated out based on size. This allows separating and
cutting out the digested DNA fragments.
• The vector DNA is also processed using the same procedure.
3. Amplification Using PCR
• Polymerase Chain Reaction or PCR is a method of making multiple copies
of a DNA sequence using the enzyme – DNA polymerase in vitro.
• It helps to amplify a single copy or a few copies of DNA into thousands to
millions of copies.
• PCR reactions are run on ‘thermal cyclers’ using the following components:
1. Template – DNA to be amplified
2. Primers – small, chemically synthesized oligonucleotides that are
complementary to a region of the DNA.
3. Enzyme – DNA polymerase
4. Nucleotides – needed to extend the primers by the enzyme.
• The cut fragments of DNA can be amplified using PCR and then ligated with
the cut vector.
4. Ligation of DNA Molecules
• The purified DNA and the vector of interest are cut with the same
restriction enzyme.
• This gives us the cut fragment of DNA and the cut vector, that is
now open.
• The process of joining these two pieces together using the enzyme
‘DNA ligase’ is ‘ligation’.
• The resulting DNA molecule is a hybrid of two DNA molecules –
the interest molecule and the vector. In the terminology of
genetics this intermixing of different DNA strands is called
recombination.
• Hence, this new hybrid DNA molecule is also called a recombinant
DNA molecule and the technology is referred to as the
recombinant DNA technology.
1

3
5. Insertion of Recombinant DNA Into Host
• In this step, the recombinant DNA is introduced into a
recipient host cell mostly, a bacterial cell. This process is
‘Transformation’.
• Bacterial cells do not accept foreign DNA easily. Therefore,
they are treated to make them ‘competent’ to accept new
DNA. The processes used may be thermal shock, Ca++ ion
treatment, electroporation etc.
6. Isolation of Recombinant Cells
• The transformation process generates a mixed population of
transformed and non-trans- formed host cells.
• The selection process involves filtering the transformed host cells
only.
• For isolation of recombinant cell from non-recombinant cell,
marker gene of plasmid vector is employed.
• For examples, PBR322 plasmid vector contains different marker
gene (Ampicillin resistant gene and Tetracycline resistant gene.
When pst1 RE is used it knock out Ampicillin resistant gene from
the plasmid, so that the recombinant cell become sensitive to
Ampicillin.
Some ways in which these plasmids may be
introduced into host organisms;
1. BIOLISTICS
- In this technique, a “gene gun” is used to fire DNA-coated pellets
on plant tissues.
- Cells that survive the ‘bombardment’, and are able to take up the
expression plasmid coated pellets and acquire the ability to express the
designed protein.
Some ways in which these plasmids may be
introduced into host organisms;
2. Plasmid insertion by Heat Shock Treatment.
- Heat Shock Treatment is a process used to transfer plasmid
DNA into bacteria.
- The target cells are pre-treated before the procedure to
increase the pore sizes of their plasma membranes that make
the cells “competent” for accepting the plasmid DNA.
- After the cells are made competent, they are incubated with
the desired plasmid at about 4°C for about 30min.
Some ways in which these plasmids may be
introduced into host organisms;
2. Plasmid insertion by Heat Shock Treatment.
- The plasmids concentrate near the cells during this time.
Afterwards, a “Heat Shock” is done on the plasmid-cell solution by
incubating it at 42°C for 1 minute then back to 4°C for 2 minutes.
- The rapid rise and drop of temperature is believed to increase
and decrease the pore sizes in the membrane.
- The plasmid DNA near the membrane surface are taken into
the cells by this process.
- The cells that took up the plasmids acquire new traits and are
said to be “transformed”.
Some ways in which these plasmids may be
introduced into host organisms;
3. Electroporation.
- This technique follows a similar methodology as Heat
Shock Treatment, but, the expansion of the membrane
pores is done through an electric “shock”.
- This method is commonly used for insertion of genes
into mammalian cells.
DNA Cloning
• A clone is defined as a cluster of individual cells that come
from a progenitor.
• A clone is genetically similar to its parent cell from which it
replicates.
• DNA cloning is initiated when DNA fragments are inserted
into DNA molecules. This molecule is designed to multiply
inside a living cell, such as a bacteria.
• Clones are genetically identical because each time a cell
replicates, it produces identical daughter cells.
Genetically Modified Organisms (GMOs)
• These organisms are produced through the rDNA technology
technique.
• Genetically modified plants are produced by integrating a
gene of interest into the Ti-plasmid before inserting the
plasmid into the plant cells.
• These now possess gene that would confer traits such as
resistance to certain bacterial or fungal pests.
Genetically Modified Organisms (GMOs)
EXAMPLES:

Bt Corn - expresses a gene

from the soil-dwelling
bacterium Bacillus
thuringiensis, making them
resistant to the corn borer
disease.
Genetically Modified Organisms (GMOs)
EXAMPLES:

Golden Rice - a transgenic

variety of rice that is
engineered to produce
beta-carotene and prevent
Vitamin-A deficiency.
 Genetically Modified Organisms (GMOs)
EXAMPLES:

Insulin - medication used

in the treatment and
management of diabetes
mellitus type-1 and
sometimes diabetes
mellitus type-2
 Genetically Modified Organisms (GMOs)
EXAMPLES:

Human Growth Hormone -

taken to cure stunted
growth
Activity: DESIGNER GENE
• Hypothetically, you are given the chance to enhance/modify a
trait of certain plant/crop. These are the things that you will
consider to make a genetically modified organism (GMO)
a. Identify special trait.
b. Identify a source organism
c. Identify a target organism
d. Identify the modified/added trait
e. What benefit/s would the recombinant organism provide to
society?
 Activity: DESIGNER GENE
• Refer from the table below for this activity. See example below.
Special trait Source Target Modified trait/ Application
(gene of nterest) organism organism added trait
Large-sized fruit Jackfruit/ Lanka aratilis Lanka-sized aratiles Bigger size of fruits
can supply greater
food demand
1.

2.

3
4
5