MODULE 2

MOLECULAR BIOLOGY

Molecular and cellular biology is a dynamic field. There are thousands

of opportunities within the medical, pharmaceutical, agricultural, and
industrial fields (just to name a few) for a person with a concentrated
knowledge of molecular and cellular processes. This module will give you a
general introduction to these topics. While traditional biology concentrated
on studying whole living organisms and how they interact within
populations (a “top down” approach), molecular biology strives to
understand living things by examining the components that make them up
(a “bottom up” approach). Both approaches to biology are equally valid,
although improvements to technology have permitted scientists to
concentrate more on the molecules of life in recent years.

Subject Objectives

At the end of this module, the student should be able to:

 Familiarize with the processes involved in the Central Dogma of
Molecular Biology;
 Describe the applications of recombinant DNA technology.

LESSON 1 │ Central Dogma of Molecular Biology

Molecular Biology is a specialized branch of biology that employs

studying the structure and chemistry of molecules which are specifically
connected to living processes. Included in these molecules are the nucleic
acids which are the scaffolds for the production of proteins that catalyze
biological reactions. The process by which a protein is manufactured from
the instructions encoded in the genetic material of an organism is called
gene expression. The Central Dogma of Molecular Biology provides an
extensive explanation of how genetic information flow inside a biological

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system. This dogma serves as the backbone of molecular biology and is
characterized by three major stages:
1. The DNA replicates itself by utilizing the actions of several enzymes;
2. The DNA is transcribed to a messenger RNA (mRNA).
3. The mRNA carries information to the ribosomes. The ribosomes
translate the information to a chain of amino acids which forms the
proteins.

Start-up Activity

Illustrate the basic structure of DNA. What makes it a suitable “genetic

material” for most organisms?

Supplementary Ideas

DNA Replication
When a cell divides, it is important that each daughter cell receives
an identical copy of the DNA. This process is achieved by the process of DNA
replication. The replication of DNA occurs during the synthesis phase or S
phase of the cell cycle, just before it enters meiosis or mitosis.

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 The structure of the DNA, being a double helix, provided a hint of how
DNA is copied. Keeping in mind the Watson-Crick base pairing, the two
strands of DNA are complementary to each other. For instance, the strand
of DNA with a nucleotide sequence of AGCTATGA will have a complementary
sequence of TCAGTACT.

During DNA replication, each of the two

strand of the double helix serves as
templates from which new strands are
copied. The new strand will be
complementary to the parental or old
strand. This is known as semi-
conservative replication, in which the
daughter DNA is composed of a new
strand and old strand (template)

Eukaryotes are complex organisms, hence their process of DNA replication

is also a complex one that involves several enzymes and other proteins. It
occurs in three main stages : initiation, elongation and termination.

Initiation of Replication
Replication begins by unwinding the double stranded helix template/
parent DNA. This unwinding is catalyzed by the enzyme called DNA
Helicase. As the DNA opens up, a Y-shaped replication fork is formed.
Because the two strands are complementary to each other, there is a high
tendency that they will reanneal or realigned after unwinding. The prevent
rre-annealing of the strands, single strand binding proteins attached to
the DNA strands. The enzyme responsible for “copying” the DNA attaches to
the template strand. However, it cannot start polymerization without the
presence of a 3’OH end of a nucleic acid molecules. The enzyme primase
catalyzes the formation of RNA primers (short nucleic acid fragments) that
provides the 3’-OH end needed by the polymerase enzyme. As the replication
progress, a tension in regions away from the replication fork builds up. This
tension is relaxed by an enzyme called DNA Toposiomerase. Once the
required proteins and enzymes are attached, the polymerase will start the
addition of correct bases.

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Elongation
During elongation, the DNA polymerase adds nucleotide bases
complementary to the template strand. The complementarity follows the
Watson- Crick base pairing (A with T, G with C). The polymerase adds bases
in the 5’ to 3’ direction only. With this activity, one of the template strand
will have a continuous replication, while another template strand will have a
discontinuous replication. The strand with a continuous replication is called
the leading strand, while the other with a discontinuous replication is called
the lagging strand. The lagging strand produces short fragments of DNA
called Okazaki fragments. Each fragment has its own RNA primer. Once the
polymerase successfully replicated the long stretch of DNA, replication will
stop.

Termination of Replication
Among the eukaryotes, replication will stop once to replication fork
meet end to end. This signals the DNA polymerase to halt its actions once a
template DNA that is already replicated is encountered. Termination of
replication involves the removal of the RNA primers by the exonuclease
activity of the DNA polymerase. Once the primers were removed, the
proofreading activity of the enzyme. This includes removal of incorrect
bases and addition of correct bases. The nicks (unconnected regions of the
sugar phosphate backbone) in between bases are sealed by DNA ligase.
Once fully replicated, the enzymes and other proteins attached to the DNA
strand will start to disassemble.

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Transcription
Transcription is the process by which the information stored by the
DNA is copied to another molecule called the mRNA. Both prokaryotes and
eukaryotes perform fundamentally the same process of transcription, with
the important difference of the membrane-bound nucleus in eukaryotes.
With the genes bound in the nucleus, transcription occurs in the nucleus of
the cell and the mRNA transcript must be transported to the cytoplasm. The
prokaryotes, which include bacteria and archaea, lack membrane-bound
nuclei and other organelles, and transcription occurs in the cytoplasm of
the cell. In both prokaryotes and eukaryotes, transcription occurs in three
main stages: initiation, elongation, and termination.

Initiation
Transcription requires the double helix to partially unwind in the
region of RNA synthesis. This unwinding creates a transcription bubble.
Several initiation factors stabilize the bubble, while others facilitates the
recruitment of RNA polymerase. The DNA sequence onto which the proteins
and enzymes involved in transcription bind to initiate the process is called a
promoter. In most cases, promoters exist upstream of the genes they
regulate.

Elongation

Transcription always proceeds from one of the two DNA strands, which is
called the template strand. The mRNA product is complementary to the
template strand and is almost identical to the other DNA strand, called the

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nontemplate strand, with the exception that RNA contains a uracil (U) in
place of the thymine (T) found in DNA. During elongation, an enzyme called
RNA polymerase proceeds along the DNA template adding nucleotides by
base pairing with the DNA template in a manner similar to DNA replication,
with the difference that an RNA strand is being synthesized that does not
remain bound to the DNA template. As elongation proceeds, the DNA is
continuously unwound ahead of the core enzyme and rewound behind it.

Termination
Once a segment of DNA is already transcribed, the polymerase need to
be instructed to disengage in the DNA strand and liberate the newly made
DNA strand. The formation of secondary structures of the DNA template can
signal the end of transcription as the RNA polymerase will start to halt.
Also, the presence of a rho factor signals the end of transcription, Rho factor
is a protein that binds to RNA polymerase and facilitates its dissociation
with the DNA template.

mRNA Processing
Upon the completion of transcription, the mRNA transcript undergoes
several modification before it proceed to the next procedure. Modifications
include addition of guanine bases on the 5’ end of the transcript (capping),
and addition of adenine residues on the 3’ end (tailing). These modifications
confers stability and protection against degradation. Further, it is essential
that all of a pre-mRNA’s introns (non-coding region) be completely and
precisely removed before protein synthesis so that the exons (coding regions)
join together to code for the correct amino acids. This process is called RNA
splicing. Variations in splicing generate a handful of different proteins.

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Translation

Translation is a process by which the genetic code contained within a

messenger RNA (mRNA) molecule is decoded to produce a specific sequence
of amino acids in a polypeptide chain. It occurs in
the cytoplasm following transcription and, like transcription, has three
stages: initiation, elongation and termination.

Initiation

Several translation initiation factors

assemble to recruit the small subunit of the
cell’s protein machinery, the ribosome. Once
the small sub unit of the ribosome is attached
to the mRNA transcript, other initiation
factors stabilize this binding. During
translation, the mRNA is read as codon, three
adjacent nucleotide bases. Another type of
RNA, the transfer RNA (tRNA), contains a sequence that is complementary
to the codons of the mRNA transcript, this sequence of three adjacent bases
is called the anticodon. The enzyme aminoacyl synthetase activate an
amino acid by bring it to the opposite end of the anticodon of the tRNA.

The small subunit of the ribosomes binds to a region 10-11 bases

away from the start codon -AUG. The small subunit of ribosome scans the
mRNA until it encounters the start codon. Other initiation factors recruit
the large subunit to the start codon, one the ribosome initiation complex is
assembled, the elongation process will commence.

Elongation

The large subunit of the ribosome has three compartments, the A, P

and E sites. The A sites accepts the incoming tRNA bearing an amino acid,
the P site holds the growing peptide chain and E site indicates the exit of
tRNAs.

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 While the tRNA with amino acid methionine occupies the P site, the
tRNA-amino acid complex complementary to the next codon will occupy the
A site. The methionine moves from P site to the A site. This movement is
actually creates a peptide bond between the two amino acids. The enzyme
responsible for this activity is called peptidyl transferase. As the ribosome
translocate to the next codon, the tRNA holding the P site no longer has an
attached amino acids, so it will leave the ribosome via the E site. Now the
growing polypetide lies at the P site and the A site is open the binding of the
next aminoacyl-tRNA, and the cycle continues. The polypeptide chain is
build up from the N terminal to the C terminal of the chain.

Termination
Once one of the three stop codons (UGA, UAA and UAG) is
encountered at the A site, the translation process will end. No amino acids
correspond to these codons so the peptide and tRNA in the P site become
hydrolysed releasing the polypeptide into the cytoplasm. Instead, a release
factor enters the A site and signals the ribosomal subunits to dissociate
from the mrNA transcript. The poplypetide chain also undergoes several
modifications to achieve its functional form.

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Activity 3.1

1. Given the following DNA sequence :

5'-CCCTATATPROMOTERCGCGATGAAAGGGCCCTGAGGGTERMINATORAAAAA-3'
3'-GGGATATAPROMOTERGCGCTACTTTCCCGGGACTCCCTERMINATORTTTTT-5'

a. Transcribe the sequence above.

b. Translate the resulting mRNA transcript.

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 2. Translate the following mRNA
5'-CGAUGCCCGGGAAUUAAGGGAAAAA -3

LESSON 2 Recombinant DNA Technology

Recombinant DNA technology harnesses molecules of DNA from two

different species that are inserted into a host organism to produce new
genetic combinations that are of value to science medicine, agriculture and
other industries. Recombinant DNA technology has made it possible to
isolate a gene or gene array, enabling researchers to determine its
nucleotide sequences, study its mRNA transcripts, mutate it in specific
manners and insert the modified sequence into a living system.

Start-up Activity

Name a movie that involves genetically modified plants or animals. Describe

the modification that happened and its impact on the community.

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 Supplementary Ideas

DNA Cloning
In biology clone refers to a
group of organisms that
descended from a common
ancestor. This means all the
members of the clones are
genetically identical because
cell replication produces
identical daughter cells each
time. The use of the
word clone has been extended
to recombinant DNA
technology, which has provided
scientists with the ability to
produce many copies of a single
fragment of DNA, such as a
gene, creating identical copies
that constitute a DNA clone.
The procedure starts by
selecting a piece of DNA to be
inserted into a vector. The second step is to cut that piece of DNA with a
restriction enzyme and then ligate the DNA insert into the vector with DNA
Ligase. The insert contains a selectable marker which allows for
identification of recombinant molecules. An antibiotic marker is often used
so a host cell without a vector dies when exposed to a certain antibiotic, and
the host with the vector will live because it is resistant. The vector is
inserted into a host cell, in a process called transformation. One example of
a possible host cell is E. Coli. The host cells must be specially prepared to
take up the foreign DNA.

Recombinant DNA works when the host cell expresses protein from
the recombinant genes. A significant amount of recombinant protein will
not be produced by the host unless expression factors are added. Protein

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expression depends upon the gene being surrounded by a collection of
signals which provide instructions for the transcription and translation of
the gene by the cell. These signals include the promoter, the ribosome
binding site, and the terminator. Expression vectors, in which the foreign
DNA is inserted, contain these signals. Signals are species specific. In the
case of E. Coli, these signals must be E. Coli signals as E. Coli is unlikely to
understand the signals of human promoters and terminators.

Polymerase chain reaction (PCR)

Another way of making many copies of a specific section of DNA, without

the need for vectors or host cells, is through a polymerase chain reaction
(PCR). The DNA to be copied - the template DNA - is mixed with two 20 base
pair primers complementary to the end of the template DNA, nucleotides,
and a version of DNA polymerase known as Taq polymerase. (This enzyme is
stable under high temperatures, and is obtained from the thermophilic
bacterium Thermus aquaticus.) The process involves the repetition of three
steps:
 denaturation, which separates the two nucleotide strands of the DNA
molecule
 primer annealing, in which the primers bind to the single-stranded
DNA
 extension, in which nucleotides are added to the primers - in the 5' to
3' direction - to form a double-stranded copy of the target DNA

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 Each cycle takes about a few minutes, so repeated cycles can produce
large amounts of a specific DNA sequence in hours rather than days.
However, some details about the nucleotide sequence to be copied must be
known in advance, and the technique is sensitive to small amounts of
contamination.

Gene libraries

A gene library is a large collection of cloned DNA sequences from a

single genome. A genomic library, in theory, would contain at least one copy
of every sequence in an organism's genome. To investigate the structure of a
given chromosome, or to clone specific genes, libraries may be prepared
from a subset of the entire genome (for example, a single chromosome). The
first step is to break up, or 'fractionate', the genome using physical methods
or restriction enzymes. The fragments are then linked to appropriate vectors
and cloned in a suitable host cell population.
A cDNA library (complementary DNA) contains DNA prepared from the
mRNA present in a given cell population using the enzymes reverse
transcriptase, which produces single-stranded DNA from mRNA, and DNA
polymerase, which converts single-stranded DNA into double-stranded DNA.
The resulting cDNA represents the genes expressed in the cell population as
a subset of the entire genome, and can be cloned using a vector and
suitable host cell. The cDNA will not include introns or regulatory sequences
as these are removed from the RNA during processing. A cDNA library can
also be prepared using reverse transcriptase PCR (RT-PCR).

Activity 3.2

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Discuss the figure showing how human insulin is produced through
recombinant DNA technology.

Self-Reflection

Encircle
your
answer

FORM
Read each statement and check ( ) the box that reflects your work
today.
Name: Date:

Section:

Strongly Strongly
Agree Disagree
Agree Disagree

1. I found this work interesting.

2. I make a strong effort.
3. I am proud of the results.
4. I understood all the
instructions.
5. I followed all the steps.
6. I learned something new.
7. I feel ready for the next
assignment.
www.ldatschool.ca/executive-function/self-assessment/

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 Self-Assessment

1. Create a diagram showing the relationship of replication, transcription

and translation.
2. Why is recombinant DNA technology important?

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