Molecular Biology (Biot.

2063)
Introduction
 Molecular Biology defined as the study of biology at molecular level. It
attempts to explain the phenomena of life starting from the
macromolecular processes that generate life.
 It study BIOMOLECULES: DNA-RNA-Proteins
 Molecular biology represents the intersection of genetics, biochemistry
and cell biology. But it also interconnected with other diverse areas of
science including microbiology, virology, immunology, bioinformatics
and physical sciences.
 Molecular biology deals with understanding
the interactions between the various systems
of a cell, including the interrelationship of
DNA, RNA and protein synthesis and
learning how these interactions are regulated.
Historical Highlights..
“History teaches everything including the future”.
Overview of the Early History of Molecular Biology:
The search for DNA is considered as the birth of molecular
biology.
 In1848, Wilhelm Hofmeister, a German botanist, observed that cell nuclei
resolve themselves into small, rod like bodies during cell division. He
called them chromosomes (colored bodies) because they can absorb dyes.
 1865 Gregor Mendel -father of genetics, established the Laws
of Inheritance through plant hybridization experiments.
 Until then it was believed that genetic material pass from
generation to generation like particles , instead of inherited from
parents.

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 1869 Friedrich Miescher, a Swiss physician, found in the nuclei of
pus cells (White blood cells) substance containing phosphorus, and
he named it nuclein. He showed that nuclein consists of an acidic
portion, which now called DNA, and a basic protein portion now
recognized as histones.
 1911 Thomas Hunt Morgan, who studied fruit fly, used light
microscope to visualize the behavior of chromosomes during mitosis
and meiosis, which led to the discovery that chromosomes are the
carriers of genes, basic units of heredity
 1911 Pheobus Aaron Theodore Lerene discovered RNA
 1941 – George Beadle and Edward Tatum identify that genes make
proteins
1950 – Edwin Chargaff discovered that the percentages of
guanine [G] and cytosine [C] bases are almost equal in any
3sample of DNA
 Later found also percentages of Adenine(A) is equal to Thymine(T)-
 “Chargaff’s rule”

A=T & C = G

 Question
 If there is 20% of Cytosine in the genome of an organism, how
much Adenine is present?

Answer: There would be 30% Adenine

 Cytosine (20%) = Guanine (20%)
 Adenine (30%) = Thymine (30%)
 Therefore, 60% A-T and 40% C-G

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1952-1953 James D. Watson and Francis H. Crick
discovered the double helical structure of DNA.
Then in 1986 sequencing the genome of deferent
organisms started : Genomic era
In general historical development of Molecular Biology
can be divided in to five stages:

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 Internal organizations of
prokaryotic and eukaryotic cell
What is Life made of?
 All living things whether Prokaryotes or Eukaryote are made of
Cells
Cells are fundamental working units of every living
system.
Every organism is composed of one of two
fundamentally different types of cells:
 Prokaryotic cells or
 Eukaryotic cells.

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Cells: Chemical composition-by weight
 70% water
 7% small molecules
 salts
 23% macromolecules
 Lipids
Proteins
 amino acids Polysaccharides
 Nucleotides lipids

 Cell is a smallest structural unit of an organism that is

capable of independent functioning
All cells share some common features: Governed by their
genetic material.

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 All Cells have common Cycles: born- eat- grow- replicate then die!

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Prokaryotes and Eukaryotes
There are two types of cells: Prokaryotic and Eukaryotic

Ribosome translates mRNA into a

polypeptide chain (protein).
The ER modifies proteins, makes
macromolecules, and transfers
substances throughout the cell

Fig. Prokaryotic cell

Nucleus only in eukaryotic

cells and it contains most of
the cell's genetic material.

Mitochondrion manufactures
adenosine triphosphate (ATP), which
is used as a source of energy.

 Approximately100 trillion (1014) cells in a

human organism
9 Fig. Eukaryotic cell  200 different forms of cells
 According to the most recent evidence, there are three main branches of life .
 Prokaryotes include Archaea (“ancient ones”) and bacteria.
Archaea are one of the three branches of the tree of life.
•They are prokaryotic, unicellular organisms.
• They live in most of earth’s extreme ecological niches, constituting 20% of the
biosphere.
 Eukaryotes are kingdom Eukarya and includes plants, animals, fungi
and certain algae.

Fig. Three Domains of Life

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 Prokaryotes and Eukaryotes

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Prokaryotes and Eukaryotes

Prokaryotes
 Eubacterial (blue green algae)
and archaebacteria
 only one type of membrane--
plasma membrane forms
 the boundary of the cell
 The smallest cells known are
bacteria
 E. coli cell
 3x106 protein molecules
 1000-2000 polypeptide species.
Eukaryotes
 plants, animals, Protista, and fungi
 complex systems of internal
membranes forms
 organelle and compartments
 The volume of the cell is several
hundred times larger
 Hela cell
 5x109 protein molecules
 5000-10,000 polypeptide species

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Prokaryotic and Eukaryotic Cells Chromosomal differences
Prokaryotes
 The genome of E.coli contains
amount of 4X106 base pairs
 > 90% of DNA encode protein
 Lacks a membrane-bound nucleus.
 Circular DNA and supercoiled
domain
 Histones are unknown
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Eukaryotes
 The genome of yeast cells contains
1.35x107 base pairs
 A small fraction of the total DNA
encodes protein.
 Many repeats of non-coding
sequences
 All chromosomes are contained in a
membrane bound nucleus
 DNA is divided between two or
more chromosomes
 A set of five histones
 DNA packaging and gene expression
regulation
 The Genetic Material
 The question that what exactly is the genetic material has challenged
scientists for long time DNA or proteins, or both together, are the
carriers of the genetic information?
 Until the early 1950s proteins were thought to be carriers of heredity,
as DNA contain only four different building blocks (ATCG), but
proteins are made up of 20 different amino acids. Proteins therefore
seem more likely to offer a greater diversity for genetic material.
 .
 .
 .

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 The search for Genetic Material
Logic- There must be genetic material for the inheritance of
traits or characteristics from generation to generation to
generation to generation…..which keep a particular trait in a
given family.
To do this the genetic material must be:
i. Stable enough to store information for long period
ii. Able to replicate accurately
iii. Flexible/able to change in order to allow evolution

In the early 1900s, chromosomes were shown to be the carriers of

hereditary information. In eukaryotes they are composed of
both DNA and protein, and most scientists initially believed
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that protein must be the genetic material.
Since chromosome consists of protein and nucleic acids these
two were candidates to be labeled as genetic material
 Protein: 20 different amino acid
 Nucleic acid: 4 different nucleotides

Complexity of life  very complicated  protein or nucleic

acid to account for the level of complexity?....wrongly proteins
were preferred candidates.
Griffith’s Transformation Experiment
Frederick Griffith’s 1928 experiment with Streptococcus
pneumoniae in mice showed that something passed from heat
killed pathogenic S. pneumoniae (smooth colony formers) into
nearby live nonpathogenic S. pneumoniae ( rough colony
formers), which changed them into pathogenic strains
He called this agent the transforming principle, but at that time the
nature of the agent and the molecular mechanism of transformation was
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unknown.
 The colonies made by pathogenic /virulent strains appear smooth on agar
plates, because they can produce and excrete polysaccharide capsule.
 The capsule protect the bacteria and allow them to survive in vertebrate hosts, including mice, that they
can infect and kill.
 Non-pathogenic /nonvirulent strains form rough colonies which are mutants
that cannot make capsule and sometimes can arise from pathogenic strains .
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 Avery’s Transformation Experiment
 The experiment which indicated that DNA is genetic material and
responsible for transformation
In 1944, Avery et al identified the transforming principle from
S. pneumoniae. Their approach was to break open dead cells,
chemically separate the components (e.g., protein, nucleic
acids) and determine which was capable of transforming live
S. pneumoniae cells.
 Only the nucleic acid fraction was capable of transforming the
bacteria.
Critics noted that the nucleic acid fraction was contaminated
with proteins. The researchers treated this fraction with
either RNase or protease and still found transforming
activity, but when it was treated with DNase, no transformation
occurred, indicating that the transforming principle was DNA.
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The experiment which showed that DNA, not RNA or protein, was the transforming principle

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 The Hershey-Chase Bacteriophage Experiment
 More evidence for DNA as the genetic material came in 1953 with
Alfred Hershey and Martha Chase’s work on E. coli infected with
bacteriophage T2. Bacteriophage /phage are virus that infect
bacteria.

20Bacteriophage T2 Lytic life cycle of a virulent phage, such as T2

 In one part of the experiment, T2 proteins were labeled with 35S,
and in the other part, T2 DNA was labeled with 32P. Then each
group of labeled viruses was mixed separately with the E. coli host.
After a short time, phage attachment was disrupted and the location
of the label determined

 The 35S-labeled protein was found outside the infected cells, while
the 32P-labeled DNA was inside the E. coli, indicating that DNA
carried the information needed for viral infection.
 This provided experimental evidence for the idea that DNA is genetic
material and responsible for inheritance of traits from generation to
generation.

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Hershey-Chase experiment demonstrating DNA is genetic material

In conclusion this
and Avery’s experiment
demonstrated that
DNA is genetic material.

Animation
22 Video
 Structure and function of nucleic acids
The nucleic acids deoxyribonucleic acid/DNA and ribonucleic
acid/RNA are polymers composed of monomers called
nucleotides
Each nucleotide has three parts:
I. A nitrogenous base
II. A pentose (5-carbon) sugar.
III. A phosphate group
The pentose sugar in RNA is ribose, and in DNA it’s
deoxyribose, from which DNA gets its full name,
deoxyribonucleic acid
 Bases
 Types:- adenine and guanine (fused five- and six-membered
heterocyclic compounds (double rings)) – Purines
 cytosine & thymine (six-membered single rings )-Pyrimidines.
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 A fifth pyrimidine base, called uracil (U), takes the place of
Purines ( A and G) and Pyrimidines (C and T/U)

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Nucleotides without a phosphate group are known as
nucleosides

25 (nucleotide)
The function of DNA is to store the genetic material and
transfer to the subsequent generation countless time.
4 “letters” base pairs. AGTC (adenine, guanine, thymine,
cytosine ) A=T and C≡G

Sugar

Phosphat
e

Base (A,T, C or
G)

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DNA has a double helix structure- which was discovered by
J. Watson and F. Crick through model building experiment.
However, it is not symmetric. It has a “forward” and
“backward” direction-
 The two strands are anti-parallel. The ends are labeled 5’ and 3’
after the carbon atoms in the sugar component.
3’ TTAGCGTTA 5’
5’AATCGCAAT 3’

DNA always reads 5’ to 3’ for replication and transcription

The DNA double helix is stabilized by hydrogen bonds
between the bases attached to the two strands.3 H-bonds
between G and C, 2 H-bonds between A and T.
Base pairing

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One major difference between DNA and RNA is the
sugar, 2-deoxyribose in DNA being replaced by the
alternative pentose sugar ribose in RNA

ribose

RNA is a biologically important type of molecule that

consists of a long chain of nucleotide units.
 Similar to DNA, Each nucleotides of RNA consists of a
nitrogenous base, a ribose sugar, and a phosphate.
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Difference between RNA & DNA

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 Polymerization and Base pairing
Nucleotides are polymerized in linear fashion through
enzymatic condensation to form long polymers.
The Sugar moieties of two adjacent nucleotides are joined
together through a bond mediated by the phosphate group-
Phosphodiester bond.
Only the alpha phosphate is involved in the formation of the
bond
 nucleoside 5’-triphosphate is used as substrate in the
condensation reaction.
The 3’-OH group of first nucleotide binds with 5’-PO4 of
the second nucleotide.

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During polymerization reaction inorganic pyrophosphate
( consisted of beta and gamma phosphate of second NTP)is
released.
The alpha phosphate of the nucleotide form bridge between the
3’ of the first nucleotide and 5’of the second nucleotide . This
same reaction is repeated many times to polynucleotide/nucleic
acid.
 Therefore, the first nucleotide contains free 5’-phosphate groups and this end
of polynucleotide is called 5’-end or head of the nucleic acid
The last nucleotide
contains free OH group
at the 3’-end and is
known as 3’-end or
tail of the nucleic acid.

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Different Forms of DNA
 Three major forms
 B-DNA
 A-DNA
 Z-DNA
B-DNA: is biologically the most common DNA form
 right-handed (20 angstrom (A°) diameter)
 complementary base-pairing (Watson-Crick)
 A-T
 G-C

Ideal B-DNA has 10 bases pairs per helical turn and it is

characterized by: narrow minor groove and wide major
groove.
A-DNA : observed under dehydrating conditions
right-handed helix
wider and flatter than B-DNA
11.6 bp per helical turn.
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 when relative humidity
is ~ 75%
B-DNA  A-DNA (reversible)
A-DNA has been observed in 2
contexts:
At active site of DNA polymerase
(~ 3 bp )
Gram (+) bacteria undergoing
sporulation
Z-DNA
left-handed helix
12 base pairs per helical turn
deep minor groove and no discernible
major groove
reversible change from B-DNA to Z-DNA
in localized regions may act as a “switch”
to regulate gene expression
seen in conditions of high salt
concentrations
36  High salt concentration reduces
repulsions between closest phosphate
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 Structures of DNA
 Primary Structure - Linear array of nucleotides
 Secondary Structure – double helix
 Tertiary Structure - Super-coiling, stem-loop formation
 Quaternary Structure – Packaging into chromatin

Primary structure of DNA Can be determined by sequencing

the nucleotides in a given DNA sample for example by Chain-
termination method developed by Sanger which involves in
vitro replication of target DNA.
eg. AAATCGCCG

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Secondary Structure – double helix
Secondary structure of DNA is anti-parallel double helix
which is right handed except Z form of DNA.

Tertiary Structure can be formed through supper coiling of

the secondary structure or by forming cruciform structures.
 Supercoils
 In duplex DNA, ten bp per turn of helix (relaxed form)
 DNA helix can be over-wound.
 Over winding of DNA helix can be compensated by supercoiling.
 Super coiling prevalent in circular DNA molecules and within local regions of
long linear DNA strands
 Enzymes called topoisomerases or gyrases can introduce or remove supercoils
 In vivo most DNA is negatively supercoiled.
 Therefore, it is easy to unwind short regions of the molecule to allow access for
enzymes
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 Cruciforms occur in palindromic regions of DNA
 Can form intrachain base pairing
 Negative supercoiling may promote cruciforms

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Cruciform structure
Quaternary Structure:
Quaternary structure of DNA associated with packaging in to
chromosome.
The quaternary structure refers to a higher-level of
organization of nucleic acids. Moreover, it refers to
interactions of the nucleic acids with other molecules.
The most commonly form of higher-level organization of
nucleic acids is seen in the form of chromatin which leads
to its interactions with the small proteins histones.
Also, the quaternary structure refers to the interactions between
separate RNA units in the  ribosome or spliceosome.

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In chromosomes, DNA is tightly associated with proteins known as
histones.
 Human DNA’s total length is ~2 meters!
 This must be packaged into a nucleus that is about 5 micrometers
in diameter
 This represents a compression of more than 100,000×!
 It is made possible by wrapping the DNA around protein spools
called nucleosomes and then packing these in helical filaments
 Chromatin, the nucleoprotein complex, consists of histones and
nonhistone chromosomal proteins
 High % histone proteins: H1, H2A, H2B, H3 and H4
 Histone octamers are major part of the “protein spools”
 Nonhistone proteins are regulators of gene expression

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 DNA Denaturation and Renaturation
 Base pairing is due to noncovalent weak bonds: H-bonds, hydrophobic interactions due to
base stacking
These interactions are weakened by:
 High temperature
 High pH
 Some chemicals (urea, formamide)
 Low Na+ concentration
 The splitting of double stranded DNA to single stranded

 Double Strand DNA can be denatured by heat (get strand separation)

 Can determine degree of denturation by measuring absorbance at 260 nm.
 Conjugated double bonds in bases absorb light at 260 nm.
 Base stacking causes less absorbance.
 Increased single strandedness causes increase in absorbance
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 DNA denaturation or melting : separation of the two complementary
strands of DNA
Melting temperature or Tm: the temperature required to
separate the two strands. H bonds are broken by thermal energy.
Melting temperature is affected by:
- GC content of DNA (GC rich DNA requires higher
temperatures to be denaturated)
-ion concentration (the double helix is destabilized by low Na+
ion
concentration, melting temperature is lower)
- alkalinity of solution, formamide concentration favors
denaturation

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 RENATURATION OF DNA
To favor re-annealing of long molecules, reassociation must
be performed at temperatures just below Tm. In these
conditions imperfect matches can again dissociate to allow the
strands to align correctly.
At low temperatures the separation of mismatched bases is
prevented due to limited diffusion.
Only simple DNA molecules can re-anneal quickly at low
temperature (4 °C).
Renaturation kinetics depends on: DNA concentration,
time, ionic strength, complementarity of single stranded
molecules

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Renaturation kinetics depends on the random collisions of
complementary single strand molecules.
 Each DNA is characterized by a particular renaturation
kinetics
In standard conditions, DNA concentration (Co) and reaction
time (t) are the two parameters for DNA renaturation.
The parameter defining reassociation kinetics is Cot,unit of
measurement: moles x sec/l.
Cot1/2 are the conditions required for renaturation of 50% of the
initial molecules.
Cot1/2 increases increasing the length of the starting DNA and
the complexity of the DNA or genome.
Reassociation reactions can be accelerated by increasing DNA
concentration and/or reaction time.
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 NUCLEIC ACID HYBRIDIZATION

Renaturation reaction between nucleic acids of different

origin. It is due to base pairing between complementary bases.
The ability to form double stranded molecules provides a test
to
measure complementarity between different molecules.
Molecules can be perfectly or partially complementary

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Nucleic acid hybridization is the basis for several
fundamental techniques in molecular biology :
- Library screening (one partner on filter and one in solution,
DNA/DNA)
- Southern blotting (one partner on filter and one in solution,
DNA/DNA)
- northern blotting (one partner on filter and one in solution,
RNA,DNA)
- in situ hybridization (one partner on slide and one in solution,
chromosomes, chromatin/ DNA probe)
- microarray (one partner on slide and one in solution,
DNA/DNA or cDNA)

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