Molecular Biology (Biol 4051): A lecture note

2 Cr. Hr. (2 Theoretical Classes per Week)

Prepared by

Takele Taye (Ph.D.)

Department of Biology
College of Science and Mathematics Education
Kotebe University of Education

October 2023
Addis Ababa, Ethiopia

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General Introduction
▪ Molecular Biology deals with the molecular basis of biological activities, and
interaction among biological molecules to control cellular activities
▪ Molecular Biology deals with structure and function of proteins and nucleic acids.
▪ Molecular biology field overlaps with other subjects, particularly genetics,
biotechnology, and biochemistry.
Eukaryotic Cell
▪ Generally, Eukaryotes are more advanced than prokaryotes
▪ Eukaryotic cells are found in animals, plants, fungi and protists
▪ Unicellular organisms such as yeast and multicellular organisms
▪ Cell with a true nucleus, where the genetic material is surrounded by a membrane
▪ Eukaryotic genome is more complex than that of prokaryotes and distributed among
multiple chromosomes
▪ There are two types of eukaryotic chromosomes: autosomes and sex chromosomes
• Autosomes
✓ Paired chromosomes with the same length, shape, centromere location, and
genes
✓ Any chromosome other than sex chromosomes
• Sex chromosomes
✓ Members of a pair of chromosomes that differ between males and females
▪ Eukaryotic DNA is linear
▪ Eukaryotic DNA is complexed with histones
▪ Karyotype: Image of an individual’s complement of chromosomes arranged by size,
length, shape, and centromere location
▪ Numerous membrane-bound organelles
▪ Complex internal structure
▪ Cell division is by mitosis

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Prokaryotic Cell

▪ Unicellular organisms. These include bacteria and archaea

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 ▪ Without a nucleus; no nuclear membrane (genetic material is dispersed throughout
cytoplasm)
▪ No membrane-bound organelles
▪ Cell contains a single circular DNA contained in the cytoplasm
▪ DNA is naked (no histone)
▪ Simple internal structure
▪ Cell division is by simple binary fission.

Archaea

Archaea is prokaryotes; organisms without nucleus but some aspects of their molecular biology
are more like those of eukaryotes.

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The Genome

▪ Encoded in the DNA (for some viruses, RNA).

▪ A genome is a complete set of chromosomes within a cell
▪ Includes genes, intergenic sequences, repeats

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 ▪ Different species have different numbers of chromosomes
▪ Prokaryotes usually have a single chromosome, often a circular DNA molecule
▪ Eukaryotic chromosomes usually appear in pairs (diploid), each inherited from one
parent
• Homologous chromosomes carry the same genes
• Some genes are same in both parents
• Some genes appear in different forms called alleles
▪ All genes are present in all cells, but a given cell types only expresses a small portion
of the genes. Only a small fraction of the genes is expressed, or turned” on,” in a
particular cell.

Gene Organization

▪ A gene may be found on either strand of DNA

▪ Genes are continuous stretches (almost always) in prokaryotes
▪ Genes are (often) discontinuous stretches (exons) in eukaryotes. The intervening
regions are called introns
▪ Upstream is a binding site

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 ▪ Location of regulatory region is less predictable

1. The molecules of life in a cell, an overview

▪ All living things are made up of four classes of large biological molecules:
carbohydrates, lipids, proteins, and nucleic acids (DNA & RNA)
▪ Macromolecules are large molecules composed of thousands of covalently linked
atoms.
▪ All biological macromolecules are made up of a small number of elements: Carbon,
Hydrogen, Oxygen, Nitrogen, Phosphorus and Sulfur
▪ 96% of living organisms are made of:
• Carbon (C)
• Oxygen (O)
• Hydrogen (H)
• Nitrogen (N)
• Phosphorus (P)
▪ Molecular structure and function are inseparable
▪ Macromolecules are polymers that are built from monomers
▪ A polymer is a long molecule consisting of several similar building blocks
▪ The small building-block molecules are called monomers
▪ A dehydration reaction occurs when two monomers bond together through the loss of
a water molecule
▪ Polymers are disassembled to monomers by hydrolysis (chemical bonds are broken by
using water)

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The Diversity of Polymers

▪ Each cell has thousands of different macromolecules

▪ Macromolecules vary among cells of an organism, vary within and between species
▪ Different variety of polymers can be built from a small set of monomers

The Four Classes of Large Biomolecules

▪ All living things are made up of four classes of large biological molecules:
• Carbohydrates
• Lipids
• Protein
• Nucleic Acids

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1.1 Nucleic acids
▪ Nucleic acids store, transmit, and help to express hereditary information
▪ Consisting of long chains of monomers called nucleotides
▪ Give information to chromosomes, which is then passed from parent to offspring
▪ The amino acid sequence of a polypeptide is programmed by a gene
▪ Genes are made of DNA, a nucleic acid made of monomers called nucleotides
▪ There are two types of nucleic acids
• Deoxyribonucleic acid (DNA)
✓ DNA contains: Promoters, Genes & Junk DNA
✓ An open reading frames (ORF): a contiguous sequence of DNA starting at a
start codon and ending with a STOP codon
• Ribonucleic acid (RNA)
✓ Messenger RNA- copies a portion of unzipped DNA in the nucleus
✓ Ribosomal RNA- associates with a set of proteins to form ribosomes. Transfer
RNA attaches the amino acids to the ribosome

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 ▪ The DNA from a single human cell has length of ~ 5.9 feet.
▪ DNA provides directions for its own replication
▪ DNA directs synthesis of messenger RNA (mRNA) and, through mRNA, controls
protein synthesis
▪ Protein synthesis occurs on ribosomes
▪ The components of nucleic acids
• Each nucleic acid is made of monomers called nucleotides
• Each nucleotide consists of a nitrogenous base, a pentose sugar, and one or more
phosphate groups

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 • There are two families of nitrogenous bases
✓ Pyrimidines (cytosine, thymine, and uracil) have a single six-membered ring
✓ Purines (adenine and guanine) have a six-membered ring fused to a five-
membered ring
• In DNA, the sugar is deoxyribose; in RNA, the sugar is ribose
• Adjacent nucleotides are joined by covalent bonds that form between the —OH
group on the 3’ carbon of one nucleotide and the phosphate on the 5’ carbon on the
next
• These links create a backbone of sugar-phosphate units
• The sequence of bases along a DNA or mRNA polymer is unique for each gene

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 • RNA molecules usually exist as single polypeptide chains. Can form base pairs
internally
• DNA molecules have two polynucleotides spiraling around an imaginary axis,
forming a double helix
• In the DNA double helix, the two backbones run in opposite 5’→ 3’ directions from
each other, an arrangement referred to as antiparallel
• DNA is found in the nucleus
✓ With small amounts in mitochondria and chloroplasts
• RNA is found throughout the cell
• A DNA molecule contains many genes

• The nitrogenous bases in DNA pair up and form hydrogen bonds: adenine (A)
always with thymine (T), and guanine (G) always with cytosine (C)

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 • Complementary base pairing can also occur between two RNA molecules or
between parts of the same molecule
• In RNA, thymine is replaced by uracil (U) so A pairs with U
• Two closely related species are more similar in DNA than are more distantly related
species
▪ DNA → RNA → Protein.
▪ This unidirectional flow equation represents the Central Dogma (fundamental law) of
molecular biology.
▪ An exception to the central dogma is that certain viruses (retroviruses) make DNA from
RNA using the enzyme reverse transcriptase.

Processes in the transfer of genetic information

▪ Replication: identical copies of DNA are made

▪ Transcription: genetic messages are read and carried out of the cell nucleus to the
ribosomes, where protein synthesis occurs.
▪ Translation: genetic messages are decoded to make proteins.
▪ In eukaryotic cells nucleic acids are either DNA or RNA
▪ Diversity is dependent on the nucleotide sequence

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Nucleic Acid Function

▪ DNA: Genetic material - sequence of nucleotides encodes different amino acids

▪ Nucleic acid functions:
• Storage of genetic info (DNA)
• Transmission of genetic info (mRNA)
• Processing of genetic information (ribozymes)
• Protein synthesis (tRNA and rRNA)
▪ RNA: Involved in the transcription/translation of genetic material (DNA). Genetic
material of some viruses
• Functional:
✓ e.g. Translation machinery
✓ rRNA (ribosomal RNA)
✓ tRNA (transfer RNA)
➢ Small polynucleotide chains - 73 to 94 residues each
➢ Several bases usually methylated
➢ Each a. a. has at least one unique tRNA which carries the a. a. to the
ribosome
➢ 3' terminal sequence is always CCA-a. a.
➢ Aminoacyl tRNA molecules are the substrates of protein synthesis
• Regulatory
✓ Control of gene expression
➢ Short term control (to meet the daily needs of the organism)
➢ Long term control (gene regulation in development/differentiation)
✓ miRNA (microRNA): a small non-coding RNA molecule (containing about 22
nucleotides) found in plants, animals and some viruses, that functions in RNA
silencing and post-transcriptional regulation of gene expression.
• Gene Expression: Gene expression is a two-step process in which DNA is converted
into a protein it encodes.
✓ mRNA (messenger RNA)
✓ Copy of 1 gene for translation by ribosomes

Nucleotide Functions

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 ▪ Energy for metabolism (ATP)
▪ Enzyme cofactors (NAD+)
▪ Signal transduction (cAMP)
▪ Nucleoside 5'-triphosphates are carriers of energy
▪ Bases serve as recognition units
▪ Cyclic nucleotides are signal molecules and regulators of cellular metabolism and
reproduction
• GTP drives protein synthesis
• CTP drives lipid synthesis
• UTP drives carbohydrate metabolism

1.2. Proteins

▪ Proteins include a diversity of structures, resulting in a wide range of functions

▪ Proteins account for more than 50% of the dry mass of most cells
▪ Are found in every cell in the body
▪ The sequence of amino acids is determined by DNA
▪ Peptides: fewer than 50 amino acids
• Dipeptides: 2 amino acids
• Tripeptides: 3 amino acids
• Polypeptides: more than 10 amino acids
▪ Proteins: more than 50 amino acids
• Typically, 100 to 10,000 amino acids linked together
▪ Protein functions include structural support, storage, transport, cellular
communications, movement, and defense against foreign substances
▪ Protein’s function depends on its structure
▪ Essential protein — must be consumed in the diet
▪ Nonessential protein — can be synthesized in the body
▪ Conditionally essential protein — cannot be synthesized due to illness or lack of
necessary precursors
• Premature infants lack sufficient enzymes needed to create arginine
▪ Enzymatic proteins
• Enzymes are a type of protein that acts as a catalyst to speed up chemical reactions

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 • Enzymes can perform their functions repeatedly, functioning as workhorses that
carry out the processes of life
▪ Storage proteins
• Function: Storage of amino acids
• Examples: Casein, the protein of milk, is the major source of amino acids for baby
mammals. Plants have storage proteins in their seeds. Ovalbumin is the protein of
egg white, used as an amino acid source for the developing embryo.
▪ Hormonal proteins
• Function: Coordination of an organism’s activities
• Example: Insulin, a hormone secreted by the pancreas, causes other tissues to take
up glucose, thus regulating blood sugar concentration
▪ Defensive proteins
• Function: Protection against disease
• Example: Antibodies inactivate and help to destroy viruses and bacteria
▪ Transport proteins
• Function: Transport of substances
• Examples: Hemoglobin, the iron-containing protein of vertebrate blood, transports
oxygen from the lungs to other parts of the body. Other proteins transport molecules
across cell membranes.
▪ Receptor proteins
• Function: Response of cell to chemical stimuli
• Example: Receptors built into the membrane of a nerve cell detect signaling
molecules released by other nerve cells.
▪ Structural proteins
• Function: Support
• Examples: Keratin is the protein of hair, horns, feathers, and other skin appendages.
Insects and spiders use silk fibers to make their cocoons and webs,
respectively. Collagen and elastin proteins provide a fibrous framework in animal
connective tissues.
▪ Contractile and motor proteins
• Function: Movement
• Examples: Motor proteins are responsible for the undulations of cilia and flagella.
Actin and myosin proteins are responsible for the contraction of muscles.

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Alteration of the protein’s shape and thus functions (denaturation) through the use of

▪ Heat
▪ Acids
▪ Bases
▪ Salts
▪ Mechanical agitation

Amino acids

▪ Monomers of proteins
• Amino acids are organic molecules with carboxyl and amino groups
• Amino acids differ in their properties due to differing side chains, called R groups

Polypeptides

▪ Polypeptides are unbranched polymers built from the set of 20 amino acids
▪ A protein is a biologically functional molecule that consists of one or more polypeptides

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Hydrophobic: retreat from water

Hydrophilic: they are attracted to water

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Hydrophilic: Electrically charged

Peptide Bonds

▪ Amino acids are linked by peptide bonds

▪ A polypeptide is a polymer of amino acids
▪ Polypeptides range in length from a few to more than thousand monomers
▪ Each polypeptide has a unique linear sequence of amino acids, with a carboxyl end (C-
terminus) and an amino end (N-terminus)

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Protein Structure and Function

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 ▪ A string of a. a’s bound with peptide bonds.
▪ Once the string of a. a’s interacts with itself and its environment (often aqueous), then
a functional protein that consists of one or more polypeptides twisted, folded, and coiled
into a unique shape
▪ The sequence of amino acids determines a protein’s three-dimensional structure
▪ Protein structure: 4 Levels
• Primary structure consists of its unique sequence of amino acids
• Secondary structure, found in most proteins, consists of coils and folds in the
polypeptide chain
• Tertiary structure is determined by interactions among various side chains (R
groups)
• Quaternary structure results when a protein consists of multiple polypeptide chains

• Primary Structure
✓ Primary structure, the sequence of amino acids in a protein, is like the order of
letters in a long word
✓ Primary structure is determined by inherited genetic information

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 • Secondary Structure
✓ The coils and folds of secondary structure result from hydrogen bonds between
repeating polypeptide backbone
✓ Typical secondary structures are a coil called an α helix and a folded structure
called a β pleated sheet

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 • Tertiary Structure
✓ Tertiary structure is determined by interactions between R groups, rather than
interactions between backbone constituents
✓ These interactions between R groups include actual ionic bonds and strong
covalent bonds called disulfide bridges which may reinforce protein’s structure.
✓ IMFs (intermolecular forces) such as London dispersion forces (is a temporary
attractive force that results when the electrons in two adjacent atoms occupy
positions that make the atoms form temporary dipoles, i.e., separation of
positive and negative electric charges) and van der Waals interactions (are
driven by induced electrical interactions between two or more atoms or
molecules that are very close to each other), hydrogen bonds (IMFs), and
hydrophobic interactions (IMFs) may affect the protein’s structure

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 • Quaternary Structure
✓ Quaternary structure results when two or more polypeptide chains form one
macromolecule
✓ Collagen is a fibrous protein consisting of three polypeptides coiled like a rope

✓ Hemoglobin is a globular protein consisting of four polypeptides: two alpha and

two beta chains

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Determinants of Protein Structure

▪ In addition to primary structure, physical and chemical conditions can affect structure
▪ Alterations in pH, i.e., potential of hydrogen and measures concentration of H ions
(measure of acidic/basic solution, <7 acidic and >7 basic, =7 neutral), salt
concentration, temperature, or other environmental factors can cause a protein to untie
▪ The loss of protein’s native structure is called denaturation
▪ A denatured protein is biologically inactive
▪ Denature: Break Bonds or Disrupt IMFs

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1.3. Carbohydrates

▪ Made up of carbon, hydrogen, and oxygen in 1:2:1 ratio

▪ Produced by photosynthesis

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 ▪ Carbohydrates serve as fuel and building material. Sources of quick energy.
• Carbohydrates include sugars and the polymers of sugars
• The simplest carbohydrates are monosaccharides, or single sugars
• Carbohydrate macromolecules are polysaccharides, polymers composed of many
sugar building blocks
• Though often drawn as linear skeletons, in aqueous solutions many sugars form
rings
• Monosaccharides serve as a major fuel for cells and as raw material for building
molecules
• Sugars
✓ Monosaccharides have molecular formulas that are usually multiples of CH2O
✓ Glucose (C6H12O6) is the most common monosaccharide
✓ Monosaccharides are classified by
➢ The location of the carbonyl group (as aldose or ketose)
➢ The number of carbons in the carbon skeleton
➢ Aldose – polyhydroxyaldehyde, e.g., glucose
➢ Ketose – polyhydroxyketone, e.g., fructose
✓ Position of carbonyl group
➢ at C1, carbonyl is an aldehyde: aldose
➢ at any other carbon, carbonyl is a ketone: ketose
➢ Ketoses are less common than aldoses
➢ All monosaccharides (both aldoses and ketoses) and most disaccharides are
reducing sugars.
➢ Reducing sugar — a carbohydrate that is oxidized by Tollen’s, Fehling’s, or
Benedict’s solution.
➢ Tollen’s: Ag+ — Ag (silver mirror)
➢ Fehling’s or Benedict’s: Cu3+ (blue) — Cu2+ (red ppt)

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A disaccharide is formed when a dehydration reaction joins two monosaccharides. This
covalent bond is called a glycosidic linkage

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Polysaccharides

▪ Polysaccharides, the polymers of sugars, have storage and structural functions

▪ The structure and function of a polysaccharide are determined by its sugar monomers
and the positions of glycosidic bonds
▪ Storage Polysaccharides
• Starch, a storage polysaccharide of plants, consists entirely of glucose monomers
• Plants store surplus starch as granules within chloroplasts and other plastids
• The simplest form of starch is amylose
• Glycogen is a storage polysaccharide in animals
• Humans and other vertebrates store glycogen mainly in liver and muscle cells

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Structural Polysaccharides

▪ The polysaccharide cellulose is a major component of the tough wall of plant cells
▪ Like starch, cellulose is a polymer of glucose, but the glycosidic linkages differ
▪ The difference is based on two ring forms for glucose: alpha (α) and beta (β)
▪ Polymers with α glucose are helical
▪ Polymers with β glucose are straight
▪ In straight structures, H atoms on one strand can bond with OH groups on other strands
▪ Parallel cellulose molecules held together this way are grouped into microfibrils, which
form strong building materials for plants
▪ Enzymes that digest starch by hydrolyzing α linkages cannot hydrolyze β linkages in
cellulose
▪ Cellulose in human food passes through the digestive tract as insoluble fiber
▪ Some microbes use enzymes to digest cellulose
▪ Many herbivores, from cows to termites, have symbiotic relationships with these
microbes

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 ▪ Chitin, another structural polysaccharide, is found in the exoskeleton of arthropods
▪ Chitin also provides structural support for the cell walls of many fungi

Types of Carbohydrates

▪ Simple carbohydrates
• Monosaccharides
• Disaccharides
▪ Complex carbohydrates
• Oligosaccharides
• Polysaccharides
✓ Glycogen
✓ Starches
✓ Fibers

Monosaccharides

Glucose

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 ▪ Carbohydrate form used by the body, referred to as “blood sugar”
▪ Basic sub-unit of other larger carbohydrate molecules
▪ Found in fruits, vegetables, honey

Fructose

▪ Sweetest of the sugars

▪ Occurs naturally in fruits & honey, “fruit sugar”
▪ Combines with glucose to form sucrose

Galactose

▪ Combines with glucose to form lactose, “milk sugar”

Disaccharides

▪ Sucrose (table sugar)

• glucose + fructose
▪ Lactose (milk sugar)
• glucose + galactose
▪ Maltose (malt sugar)
• glucose + glucose

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Complex carbohydrates

▪ Oligosaccharides
• Short carbohydrate chains of 3 to 10 monosaccharides
• Found in legumes and human milk
• Examples:
✓ Raffinose
✓ Stachyose
▪ Polysaccharides
• Long carbohydrate chains of monosaccharides linked by glycosidic bonds
✓ Alpha (α) bonds (starch)
✓ Beta (β) bonds (found in fiber)
• Starch
✓ Plant storage form of carbohydrate
✓ Long branched or unbranched chains of glucose
➢ Amylose
➢ Amylopectin
• Glycogen
✓ Highly branched chains of glucose units

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 ✓ Animal storage form of carbohydrate
➢ Found in liver and muscle
➢ Humans store ~ 100g in liver; ~ 400g in muscle
• Fiber
✓ Dietary Fiber
➢ Non-digestible carbohydrates (chains of monosaccharides) and lignin that
are intact and intrinsic in plants (includes oligosaccharides)
➢ Dietary fiber found in all plants
➢ Types of non-starch polysaccharides include:
❖ Cellulose: an insoluble substance which is the main constituent of plant
cell walls and of vegetable fibers such as cotton. It is a polysaccharide
consisting of chains of glucose monomers.
❖ Hemicellulose: any of a class of substances which occur as constituents
of the cell walls of plants and are polysaccharides of simpler structure
than cellulose.
❖ Pectin: a group of water-soluble carbohydrate substances that are found
in the cell walls and intercellular tissues of certain plants.
❖ Gums & mucilage: Gums readily dissolve in water, whereas, mucilage
form slimy masses. Gums are pathological products, whereas mucilage
are physiological products.
❖ B-glucans: comprise a group of β-D-glucose polysaccharides naturally
occurring in the cell walls of cereals, bacteria, and fungi.
❖ Chitin & chitosan: Chitosan is a linear polysaccharide composed of
randomly distributed β-(1→4)-linked D-glucosamine (deacetylated
unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is a fibrous
substance consisting of polysaccharides, which is the major constituent
in the exoskeleton of arthropods and the cell walls of fungi.
❖ Lignans: are a large group of polyphenols found in plants.
✓ Functional Fiber
➢ Isolated, non-digestible carbohydrates that have beneficial physiological
effects in humans

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1.4. Lipids

▪ Lipids are a diverse group of hydrophobic molecules

▪ Soluble in nonpolar (no charges at the end because finely distributed electrons
symmetrically cancelled out each other) organic solvents
▪ Lipids are hydrophobic because they consist mostly of hydrocarbons, which form
nonpolar covalent bonds
▪ Water is a polar covalent molecule (unequal sharing of electrons between atoms leads
to asymmetrical shape, i.e., positive charge on hydrogen pole and negative charge on
the oxygen pole)
▪ Chemical structure: glycerol + fatty acids
▪ Lipids are composed of C, H, O
• Long hydrocarbon chains (H-C)
▪ Family groups
• Fats
• Phospholipids
• Steroids

Fat

▪ Fats are constructed from glycerol and fatty acids

▪ Glycerol is a three-carbon alcohol with a hydroxyl group attached to each carbon
▪ A fatty acid consists of a carboxyl group attached to a long carbon skeleton
▪ Fats separate from water because water molecules form hydrogen bonds with each other
and exclude the fats
▪ In a fat, three fatty acids are joined to glycerol by an ester linkage, creating a
triacylglycerol, or triglyceride (the fats and oils that are found in animals and plants are
triglycerides)
▪ Fatty acids vary in length (number of carbons) and in the number and locations of
double bonds
▪ Saturated fatty acids have the maximum number of hydrogen atoms possible and no
double bonds
▪ Unsaturated fatty acids have one or more double bonds

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 ▪ Fats made from saturated fatty acids are called saturated fats, and are solid at room
temperature
▪ Most animal fats are saturated
▪ Fats made from unsaturated fatty acids are called unsaturated fats or oils and are liquid
at room temperature.
▪ Plant fats and fish oils are usually unsaturated
▪ Fat Structure:
• Glycerol (3C alcohol) + fatty acid
✓ Fatty acid = long HC “tail” with carboxyl (COOH) group “head”

▪ Building Fats
• Triacylglycerol
✓ 3 fatty acids linked to glycerol
✓ Ester linkage between OH & COOH

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Fats store energy

▪ Long HC chain
• Polar (difference in electronegativity between the bonded atoms) or non-polar?
• Hydrophilic or hydrophobic?
▪ Function:
• Energy storage
✓ Twice carbohydrates
• Lipids are concentrated source of energy. One gram fat gives 9 K calories.
• It serves as a cushion for the vital organs and protects them from external shocks or
injuries.
• Lipids are the structural materials of cells and membranes
• Lipids serves as insulator for our body
• Lipids are the carrier/reservoir of fat-soluble vitamins (A, D, E & K)
• In food preparations lipids serves as a binding agent. It also enhances the
palatability of foods

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Phospholipids

▪ In a phospholipid, two fatty acids and a phosphate group are attached to glycerol
▪ The two fatty acid tails are hydrophobic, but the phosphate group and its attachments
form a hydrophilic head
▪ Structure:
• glycerol + 2 fatty acids + PO4
✓ PO4 = negatively charged
▪ Hydrophobic or hydrophilic
• Fatty acid tails = Hydrophobic
• PO4 head = Hydrophilic
• Hydrophilic heads attracted to H2O
• Hydrophobic tails hide from H2O
• Phospholipids create a barrier in water
✓ Define outside vs. inside
✓ They make cell membranes

Steroids

▪ Steroids are lipids characterized by a carbon skeleton consisting of four fused rings

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 ▪ Cholesterol, an important steroid, is a component in animal cell membranes
▪ Structure:
• Four fused C rings
✓ Different steroids created by attaching different functional groups to rings
✓ Different structure creates different function
• Examples: cholesterol, sex hormones

Cholesterol

▪ Important cell component

• Animal cell membranes
• Precursor of all other steroids
✓ Including vertebrate sex hormones
• High levels in blood may contribute to cardiovascular disease
• Important component of cell membrane
✓ Keeps cell membranes fluid & flexible

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2. Synthesis and structure of nucleic acids (DNA and RNA)

2.1 DNA

2.1.1 DNA structure and properties

▪ A polymer of deoxyribonucleotides
▪ The bacterial chromosome is a double-stranded, circular DNA molecule
▪ Eukaryotic chromosomes have linear DNA molecules associated with a large amount
of protein
▪ In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the
nucleoid
▪ Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic
cells
▪ Histones are proteins that are responsible for the first level of DNA packing in
chromatin
▪ DNA provides directions for its own replication
▪ DNA directs synthesis of messenger RNA (mRNA) and, through mRNA, controls
protein synthesis
▪ Each cell has about 2 meters of DNA.
▪ The average human has 75 trillion cells.
▪ The average human has enough DNA to go from the earth to the sun more than 400
times.
▪ Double-stranded
▪ Individual deoxynucleoside triphosphates are linked by phosphodiester bonds
• Esterification
• Link 3’ carbon of one ribose with 5’ carbon of another
▪ A double helical structure
• Common axis for both helices
• Handedness of helices
• The two DNA strands are antiparallel
• The two DNA strands are not identical, but they are complementary.
• The DNA double helix is held together mainly by hydrogen bonds

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 ✓ Hydrogen bonding: a chemical bond in which a hydrogen atom of one molecule
is attracted to an electronegative atom, especially a nitrogen, oxygen, or fluorine
atom, usually of another molecule.
✓ Base stacking: The bases in DNA are planar and have a tendency to "stack".
➢ Major stacking forces:
❖ hydrophobic interaction
❖ van der Waals forces
• A DNA molecule consists of two strands of nucleotide monomers coiled into a
double helix
• Two double-helix strands are held together by hydrogen bonds between nucleotide
bases
• DNA nucleotide
✓ A five-carbon sugar (deoxyribose)
✓ Three phosphate groups
✓ One nitrogen-containing base (adenine, thymine, guanine, or cytosine)
▪ Periphery of DNA
• Sugar-phosphate chains
▪ Core of DNA
• Bases are stacked in parallel fashion
• Chargaff’s rules
✓ A=T
✓ G=C
✓ Complementary base-pairing (pyrimidine always pairs with purine)
✓ AT ≠ GC
✓ Generally, GC~50%, but extremely variable
✓ Example
➢ Slime mold~22%
➢ Mycobacterium~73%
✓ Differs between species
➢ e.g. humans 30% A/T & 20% G/C
➢ E. coli 26% A/T & 24% G/C
✓ Distribution of GC is not uniform in genomes

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 ▪ Three major forms of DNA
• B-DNA
✓ Right-handed helix
✓ Turn every 3.4 nm.
✓ Each turn contains 10 base pairs (the distance between each two successive
bases is 0.34 nm)
✓ Contain two grooves;
✓ Major groove (wide): provide easy access to bases
✓ Minor groove (narrow): provide poor access
• A-DNA
✓ Less common form of DNA, more common in RNA
✓ Right handed helix
✓ Each turn contains 11 bp/turn
✓ Contain two different grooves:
✓ Major groove: very deep and narrow
✓ Minor groove: very shallow and wide (binding site for RNA)

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 • Z-DNA
✓ Radical change of B-form
✓ Left handed helix, very extended
✓ It is GC rich DNA regions
✓ The sugar base backbone form Zig-Zag shape
✓ The B to Z transition of DNA molecule may play a role in gene regulation

▪ Minor groove is narrow: 12 Å across

▪ Major groove is wide: 22 Å across

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2.1.2 The molecular basis of DNA replication

▪ Replication: DNA exactly copies itself (occurs within the nucleus)

▪ A cell replicates its DNA before it divides
▪ Each strand of the double helix serves as a template for synthesis of a new,
complementary strand
▪ DNA replication results in two double-stranded DNA molecules identical to the parent
▪ Any mistake in copying: mutation
▪ During DNA replication, the double-helix unwinds
▪ DNA polymerase uses each strand as a template to assemble new, complementary
strands of DNA from free nucleotides
▪ DNA ligase seals any gaps to form a continuous strand
▪ Complementary base pairing makes replication possible
▪ A DNA molecule is a template for making the other strand
▪ The copying of DNA is remarkable in its speed and accuracy
▪ Replication begins at special sites called origins of replication, where the two DNA
strands are separated, opening up a replication “bubble”

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 ▪ A eukaryotic chromosome may have hundreds or even thousands of origins of
replication
▪ Replication proceeds in both directions from each origin, until the entire molecule is
copied
▪ At the end of each replication bubble is a replication fork, a Y-shaped region where
new DNA strands are elongating

2.1.3 Enzymes involved in DNA replication

▪ Helicases are enzymes that untwist the double helix at the replication forks
▪ Single-strand binding protein binds to and stabilizes single-stranded DNA until it can
be used as a template
▪ Topoisomerase corrects “over winding” ahead of replication forks by breaking,
swiveling, and rejoining DNA strands

▪ DNA polymerase: DNA replication enzyme; assembles a new strand of DNA based on
sequence of a template DNA
▪ DNA ligase: Enzyme that seals breaks in double-stranded DNA

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 ▪ DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add
nucleotides to the 3’ end
▪ The initial nucleotide strand is a short RNA primer
▪ An enzyme called primase can start an RNA chain from scratch and adds RNA
nucleotides one at a time using the parental DNA as a template
▪ The primer is short (5–10 nucleotides long), and the 3’ end serves as the starting point
for the new DNA strand
▪ Most DNA polymerases require a primer and a DNA template strand
▪ The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second
in human cells

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 ▪ As each monomer of dATP joins the DNA strand, it loses two phosphate groups as a
molecule of pyrophosphate
▪ The antiparallel structure of the double helix (two strands oriented in opposite
directions) affects replication
▪ DNA polymerases add nucleotides only to the free 3’end of a growing strand; therefore,
a new DNA strand can elongate only in the 5’ to 3’ direction
▪ Along one template strand of DNA, the DNA polymerase synthesizes a leading strand
continuously, moving toward the replication fork

2.1.4 The process of DNA replication (initiation, elongation, termination)

▪ DNA replication requires:

• A strand of DNA to serve as a template
• Substrates — deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP).
• DNA polymerase — an enzyme that brings the substrates to the DNA strand
template
• A source of chemical energy to drive this synthesis reaction
▪ To elongate the other new strand, called the lagging strand, DNA polymerase must
work in the direction away from the replication fork
▪ The lagging strand is synthesized as a series of segments called Okazaki fragments,
which are joined together by DNA ligase
▪ Nucleotides are always added to the growing strand at the 3' end (end with free -OH
group).
▪ The hydroxyl group reacts with the phosphate group on the 5' C of the deoxyribose so
the chain grows

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 ▪ Energy is released when the bound linking 2 of the 3 phosphate groups to the
deoxyribonucleoside triphosphate breaks
▪ Remaining phosphate group becomes part of the sugar-phosphate backbone

Step 1: Unwinding and exposing strands

▪ DNA strands are unwound and opened by enzymes called HELICASES

▪ Helicases act at specific places called ORIGINS OF REPLICATION
▪ Synthesis of new DNA strands proceeds in both directions from origin of replication
resulting in a bubble with REPLICATION FORKS at each growing point.

Step 2: Priming the strand

▪ In order to begin making a new strand, a helper strand called a PRIMER is needed to
start the strand.
▪ DNA polymerase, an enzyme, can then add nucleotides to the 3' end of the primer.
▪ Primer is a short, single strand of RNA (ribonucleic acid) and is complimentary to the
DNA template strand.
▪ Primers are formed by enzymes called PRIMASES.
▪ Prokaryotes (bacteria) have a single bubble
▪ Eukaryotic chromosomes have many bubbles
▪ Enzyme Helicase unwinds and separates the 2 DNA strands by breaking the weak
hydrogen bonds
▪ Single-Strand Binding Proteins attach and keep the 2 DNA strands separated and
untwisted
▪ Enzyme Topoisomerase attaches to the 2 forks of the bubble to relieve stress on the
DNA molecule as it separates
▪ The Lagging Strand is synthesized discontinuously against overall direction of
replication
▪ This strand is made in MANY short segments. It is replicated from the replication fork
toward the origin

Step 3: Strand elongation

▪ DNA Polymerase III catalyzes elongation of new DNA strands in prokaryotes

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 ▪ Two molecules of DNA polymerase III clamp together at the replication forks, each
acting on one of the strands
▪ One strand exposed at its 3' end produces a daughter strand which elongates from its 5'
to 3' end and is called the LEADING STRAND. This strand is synthesized
continuously and grows from 5' to 3'.
▪ The second daughter strand is called the LAGGING STRAND and is antiparallel to the
leading strand. Its template is exposed from the 5' to 3' end but it must direct the 5' to
3' synthesis of the lagging strands, since nucleotides are added at the 3' end of the chain.
▪ The lagging strand is constructed in small, backward directed bits consisting of
discontinuous sections of 100 to 200 nucleotides in eukaryotes and 1000 to 2000
nucleotides in prokaryotes, called OKAZAKI FRAGMENTS.
▪ When an Okazaki fragment forms:
• DNA polymerase I removes the RNA primer and replaces it with DNA adjacent to
the fragment leaving one bond between adjacent fragments missing.
• A second enzyme called a DNA LIGASE catalyzes the formation of the final bond.
• Telomerase is a reverse transcriptase that contain an RNA template, adds
nucleotides to the 3’end of the lagging-strand template and thus prevents shortening
of lagging strands during replication of linear DNA molecules.

2.1.5 DNA mutation and repair

▪ DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides
▪ In mismatch repair of DNA, repair enzymes correct errors in base pairing
▪ DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and
certain molecules (smoking cigarette)
▪ In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of
DNA

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 ▪ DNA polymerase initially makes about 1 in 10,000 base pairing errors
▪ Enzymes proofread and correct these mistakes
▪ The new error rate for DNA that has been proofread is 1 in 1 billion base pairing errors

Most common forms of DNA damage:

▪ Bulges due to deletions or insertions

▪ Missing, altered, or incorrect base
▪ UV-induced pyrimidine dimers (a pair of pyrimidines)
▪ Strand breaks at phosphodiester bonds or within deoxyribose rings
▪ Covalent cross-linking of strands

DNA damage & repair

▪ Chemicals & ultraviolet radiation damage the DNA in our body cells
▪ Cells must continuously repair damaged DNA
▪ Excision repair occurs when any of over 50 repair enzymes remove damaged parts of
DNA
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 ▪ DNA polymerase and DNA ligase replace and bond the new nucleotides together

2.2 RNA

2.2.1 Types and structure of RNAs

▪ It is linear polynucleotide
▪ It is generally single stranded
▪ The pentose sugar is ribose
▪ Uracil (U) replace Thymine (T) in the pyrimidine bases
▪ Although RNA is generally single stranded, intra-molecular H-bond base pairing occur
between complementary bases on the same molecule (secondary structure)
▪ Is considerably smaller than DNA
▪ It can take 3 levels of structure:
• Primary: sequence of nucleotides
• Secondary: hairpin loops (base pairing)
• Tertiary: motifs and 3D folding

Types of RNA

▪ Messenger RNA (mRNA):

• Carries genetic information copied from DNA in the form of 3-base code, each of
which specifies a particular amino acid.

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 • It comprises only about 5% of the RNA in the cell.
• It is widely varied in size and base sequences.
• It contains coding regions to be translated in addition to other untranslated
regulatory regions at 5′- and 3′-ends.
• Special structural characteristics of eukaryotic mRNA include a long sequence of
adenine nucleotides (a “poly-A tail”) on the 3′-end of the RNA chain, plus a “cap”
on the 5′-end consisting of a molecule of 7-methylguanosine attached “backward”
(5′→5′) through a triphosphate linkage.

▪ Transfer RNA (tRNA):

• It reads the code on the mRNA.
• Each amino acid has its own tRNA, which binds to it and carries it to the growing
end of a polypeptide chain.
• tRNA are the smallest (4S) of the three major species of RNA (A svedberg unit
(symbol S, sometimes Sv) is a non-metric unit for sedimentation rate). Together,
tRNA make up about 15% of the total RNA in the cell. There are 2 important
features of tRNA:
✓ They contain unusual bases e.g., dihydrouracil (D) and pseudouracil (ψ).

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 ✓ They have extensive complementary intrachain base-pairing leading to
extensive looping and folding giving rise to the secondary and tertiary structures
of t-RNA.
• Each tRNA can recognize the signal or the message on the mRNA by its anticodon
sequence (anticodon loop), so that it brings up the specific amino acid covalently
attached to its 3′-end to add it to the growing polypeptide chain.
• The four base-paired regions and three loops are characteristic of all tRNAs, as is
the base sequence of the amino acid attachment site at the 3’ end. The anticodon
triplet is unique to each tRNA type. (The asterisks mark bases that have been
chemically modified, a characteristic of tRNA.)
• D-loop recognition site for the enzyme aminoacyl-tRNA-synthetase.
• T-arm or T-loop recognition site for tRNA-ribosome complex

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 ▪ Ribosomal RNA (rRNA):
• Associated with a set of proteins to form the ribosomes
• Ribosomes move along the mRNA molecule catalyze the assembly of amino acids
into protein chain
• They also bind tRNAs that have the specific amino acids
• In prokaryotic cells, there are three major species of rRNA (23S, 16S, and 5S),
while in eukaryotes, there are four rRNA species (28S, 18S, 5.8S, and 5S).

2.2.2 The process of transcription

▪ RNA is made on a DNA template

▪ DNA regions that can be transcribed into RNA are called structural genes
▪ The information contained in DNA is passed to form messenger RNA (mRNA) by
transcription (“rewriting”).
▪ Either strands can be used as a template. The transcription direction on different strands
is opposite. This feature is referred to as the asymmetric transcription
▪ In eukaryotic genes there are different polymerases for the synthesis of rRNA, tRNA,
and mRNA.

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 • RNA polymerase I: It synthesizes the precursor of the 28S, 18S, and 5.8S r-RNA in
the nucleolus.
• RNA polymerase II: It synthesizes the precursors of m-RNA in addition to snRNA
• RNA polymerase III: It produces the small RNA, including tRNA, 5S ribosomal
RNA, and some snRNA.
▪ In addition, a number of transcription factors are involved. These factors have two main
functions:
• They recognize which genes are to be transcribed.
• They help in the assembly of the transcription complex at the promoter region of
that gene
▪ For transcription factors to recognize and bind to their specific DNA sequences, the
chromatin structure in that region must be altered to allow access to the DNA
▪ The mechanism of transcription is quite similar to replication because DNA is the
template upon which RNA is formed. The major difference is that:
• The enzyme is RNA polymerase instead of DNA polymerase
• Sugar units of the nucleoside used to make RNA is ribose rather than deoxyribose,
and U (uracil) substitutes for T (Thymidine) in RNA.

The process of transcribing a typical gene in a eukaryotic cell is divided into 3 phases;
initiation, elongation and termination.

▪ Initiation: begins with binding of RNA polymerase in the DNA promoter region. In
eukaryotic cells, each class of polymerases has specific transcription factors and
specific promotor areas. When a DNA sequence in a specific gene is to be transcribed
to mRNA using RNA polymerase II, the ‘promoter’ consensus sequence is called
TATA or Hogness box that is located about 25 nucleotides to the left of the transcription
start site. Another consensus sequence is found between 70 and 80 nucleotides to the
left as well and called CAAT box.
▪ Each transcriptable region is called operon
• One operon includes several structural genes and upstream regulatory sequences (or
regulatory regions)
• The promoter is the DNA sequence that RNA-pol can bind. It is the key point for
the transcription control

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Transcription in eukaryotes

Initiation

▪ Transcription initiation needs promoter and upstream regulatory regions.

▪ The cis-acting elements are the specific sequences on the DNA template that regulate
the transcription of one or more genes.

In some eukaryotic genes, TATA box is substituted by a GC-rich region (GC box). Such
sequences in the promoter region serve as binding sites for proteins known as transcription
factors, which in turn interact with each other and with RNA polymerase II. Eukaryotic RNA
polymerase II does not itself recognize and bind the promoter but is brought to the promoter
by the general transcription factor, TFIIF. In addition to binding DNA, specific transcription
factors also bind other proteins (“coactivators”), recruiting them to the transcription complex.

▪ In order to enhance initiation, there are probably thousands of specific DNA sequences
away from the transcription start site (toward both 5′- and 3′-ends) called as Enhancers
or Response elements that bind to specific proteins called Activators which interact
with the Transcription complex to stimulate the transcription.

Elongation:

▪ Once the promoter region has been recognized and bound by the transcription complex,
local unwinding (melting) of DNA helix occurs. The process generates supercoils in
the DNA that can be relieved by (DNA topoisomerases I and II). RNA polymerase II
begins to synthesize a transcript of the DNA sequence. The elongation phase is said to
begin when the transcript (typically starting with a purine) exceeds 10 nucleotides in
length and the enzyme starts to leave the promoter region and moves along the template
DNA strand in 5′ → 3′ processive manner.

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Termination:

▪ The elongation of the single-stranded RNA chain continues until a termination signal
is reached. Termination can be intrinsic (spontaneous) or dependent upon the
participation of a protein known as the ρ (rho) factor. This leads to stop transcription
and the DNA double helix to zip up again.

2.2.3 Enzymes involved in transcription

▪ The enzyme responsible for the RNA synthesis is DNA-dependent RNA polymerase
• The prokaryotic RNA polymerase is a multiple-subunit protein of ~480kD
✓ The holoenzyme of RNA-pol in E. coli consists of 5 different subunits: α, αʹ′ β
βʹ′ and ω (two alphas, one beta, one beta-prime and one omega).
✓ α and αʹ determine the DNA to be transcribed
✓ β Catalyze polymerization
✓ βʹ′ Bind & open DNA template
✓ ω recognize the promote
• Eukaryotic systems have three kinds of RNA polymerases, each of which is a
multiple-subunit protein and responsible for transcription of different RNAs

2.2.4 Post-transcriptional modifications

▪ In prokaryote, the primary transcript is equivalent to the mRNA molecule.

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 ▪ The RNA strand that has been produced from DNA transcription is known as the
primary transcript. It is a linear RNA copy of the transcriptional unit (i.e. DNA segment
between the specific initiation and the termination sequences). The primary transcript
of rRNA and tRNA are transcriptionally modified by cleavage of the original transcript
by ribonucleases. tRNA will be further modified to give each species its unique identity.
▪ r-RNA of both prokaryotic and eukaryotic cells are synthesized from long precursor
molecules called (preribosomal RNAs). The 28S, 18S, and 5.8S rRNA of eukaryotes
are produced by the cleavage of the primary transcript by ribonuclease enzyme.

▪ Functional t-RNA is produced from a longer precursor molecule (pre-tRNA) after

modification. An intron (14 nucleotide intron) must be removed from the anticodon
loop, and sequences at both the 5′- and the 3′-ends of the molecule must be trimmed.
Other posttranscriptional modifications include addition of CCA sequence by
nucleotidyltransferase to the 3′-terminal end of tRNA, and modification of bases at
specific positions to produce “unusual bases” like (D= Dihydrouracil, ψ= pseudouracil,
and m= methylated bases).

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Eukaryotic mRNA posttranscriptional modification

The mRNA molecule synthesized in eukaryotic nuclei by RNA polymerase II is a collection

of the precursor molecules of mRNA called as heterogeneous nuclear RNA (hnRNA). The
primary transcripts are extensively modified in the nucleus after transcription. These
modifications usually include:

▪ 5′ “Capping”: This process is the first of the processing reactions for hnRNA. The cap
is a 7-methylguanosine attached “backward” to the 5′-terminal end of the mRNA,
forming an unusual 5′→5′ triphosphate linkage. The creation of the guanosine
triphosphate part of the cap requires the nuclear enzyme guanylyl transferase.
Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-
7-methyltransferase. The presence of 7-methyl guanosine triphosphate cap is very
essential in starting the mRNA translation (protein synthesis).

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 ▪ Most eukaryotic mRNA have a chain of 40–200 adenine nucleotides attached to the 3′-
end of mRNA primary transcript. This poly-A tail is not transcribed from the DNA, but
rather is added after transcription by the nuclear enzyme, polyadenylate polymerase,
using ATP as substrate. The mRNA is cleaved downstream of a consensus sequence,
called the polyadenylation signal sequence (AAUAAA), found near the 3′-end of the
RNA, and the poly-A tail is added to the new 3′-end. These tails help stabilize the
mRNA and facilitate their exit from the nucleus. After the mRNA enters the cytosol,
the poly-A tail is gradually shortened.
▪ Removal of introns: Maturation of eukaryotic mRNA usually involves the removal of
RNA sequences, which do not code for protein (introns, or intervening sequences) from
the primary transcript. The remaining coding sequences, the exons, are joined together
to form the mature mRNA. The process of removing introns and joining exons is called
splicing. The molecular machine that accomplishes these tasks is known as the
spliceosome.

RNA functions

▪ Structural
• e.g., rRNA, which is a major structural component of ribosomes
▪ Catalytic
• RNA in the ribosome has peptidyl transferase activity
✓ Enzymatic activity responsible for peptide bond formation between amino
acids in growing peptide chain

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 • Also, many small RNAs are enzymes "ribozymes “
▪ Regulatory
• Recently discovered important new roles for RNAs
• In normal cells:
✓ In "defense" - esp. in plants
✓ In development
e.g., siRNAs, miRNA

3. Protein Synthesis (Translation)

▪ In prokaryotes transcription and translation is coupled in space and time

▪ Proteins are made of 20 amino acids linked by peptide bonds
▪ Polypeptide backbone is the repeating sequence of the N-C-C-N-C-C… in the peptide
bond
▪ The side chain or R group is not part of the backbone or the peptide bond

3.1 The process of translation

▪ A mRNA molecule is bound to a ribosome

▪ The transfer RNA (tRNA) molecules bring amino acids to the ribosome one at a time
▪ Each tRNA identifies the appropriate codon on the mRNA and add this amino acid to
the growing protein chain.

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 ▪ The first tRNA is released and the ribosome moves one codon length along the message,
allowing the next tRNA to come into place, carrying its amino acid. Energy in the form
of ATP is required at each step in the movement.

Steps in protein synthesis

▪ Bacterial ribosomes have 3 sites that bind aminoacyl-tRNAs

• A site (aminoacyl)
• P site (peptidyl)
• E site (exit) (largely on 50s)
▪ Formation of the initiation complex (in bacteria)
▪ First step in elongation (in bacteria)
• Binding of the second aminoacyl-tRNA
▪ Second step in elongation (in bacteria): formation of the first peptide bond
• Peptide bond is formed
▪ Third step in elongation (in bacteria): translocation
• Ribosome moves one codon towards the 3’end of mRNA.
• The didpeptidyl-tRNA is now entirely in the P site
• A site is open (for incoming tRNA)
• Uncharged tRNA moves first to E site, then leaves.
▪ Termination of protein synthesis in bacteria
• Occurs in response to a termination codon in A site.
• A Release Factor binds to A site, leads to hydrolysis of ester linkage between
nascent polypeptide and the tRNA.
• Polypeptide thus released.
• mRNA, deacylated tRNA, and release factor leave ribosome. Ribosome dissociates
into 30S and 50S subunits.

Translation in Eukaryotes

▪ Initiation factors deliver 40S ribosomal subunit and Met-tRNA to 5’ methylated cap on
mRNA. The 40S subunit then “scans” down mRNA and docks at first AUG codon.

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 ▪ Initiation factor interaction with 5’ cap is facilitated by prior recruitment of initiation
factor to the mRNA via polyA binding protein (PABP) bound to the mRNA’s 3’ polyA
tail.
▪ Elongation: Peptide(n)-acyl-tRNA + Aminoacyl-tRNA ---> tRNA(uncharged) +
Peptide (n + 1)-acyl-tRNA
▪ Elongation Factors and GTP are needed for tRNA Recruitment and Translocation

Termination: Release Factors recognize stop codons at A Site. When release factor encounters
stop codon, water is used instead of amino group of charged tRNA as nucleophile to attack
acyl bond of peptidyl-acyl-tRNA. Hydrolysis releases peptide from tRNA and ribosome.

3.2 Post-translational modifications

▪ Covalent or generally enzymatic modifications of proteins during or after the synthesis

of proteins

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 ▪ PTMs are important at various levels, including the 3D structure, interactions,
subcellular location and function
▪ Regulate protein activity and protein interactions
▪ Post-translational modification (PTM): The chemical modifications that take place at
certain amino acid residues after the protein is synthesized by translation are known
as post-translational modifications. These are essential for normal functioning of the
protein. Some of the most commonly observed PTMs include:
• Phosphorylation
✓ The process by which a phosphate group is attached to certain amino acid side
chains in the protein, most commonly serine, threonine, and tyrosine.
✓ Control protein activity and structure
✓ Protein-protein and protein/nucleic acid interactions
✓ Kinases phosphorylate, phosphatases dephosphorylate
✓ Kinases are major drug targets
• Glycosylation
✓ Ser, Thr, Asn
✓ The attachment of sugar moieties to nitrogen or oxygen atoms present in the
side chains of amino acids like asparagine, serine or threonine.
✓ Regulated by glycosyl transferases
✓ Control protein structure, stability, and trafficking. Regulate protein activity
• Carboxylation
✓ Most common is carboxy-glutamate
✓ Vitamin K, CO2, O2 dependent.
✓ e. g. Prothrombin
• Hydroxylation
✓ Pro, Lys
✓ This PTM is most often found on proline and lysine residues which make up
the collagen tissue. It enables crosslinking and therefore strengthening of the
muscle fibres.
✓ Proline hydroxylation is important in transcriptional control and protein
structure
✓ Hydroxylation and subsequent crosslinking of lysine residues in collagen cause
conformational restriction and stabilize the coil-coil structure
• Acetylation
✓ N-terminus, Lysine side chains

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 ✓ Affects chromatin structure and gene expression
• Methylation
✓ Glutamate, arginine, lysine
✓ Methylation affects chromatin structure and gene expression
✓ Methylation is mediated by S-adenosyl-methionine (SAM)
• Acylation: The process by which an acyl group is linked to the side chain of amino
acids like asparagine, glutamine or lysine.
• Alkylation: Addition of alkyl groups, most commonly a methyl group to amino
acids such as lysine or arginine. Other longer chain alkyl groups may also be
attached in some cases.

3.3 Structure and types of proteins (in terms of function)

▪ Make up about 15% of the cell

▪ Have many functions in the cell
• Enzymes

• Structural proteins: are fibrous proteins. The most familiar of the fibrous proteins
are probably the keratins, which form the protective covering of all land vertebrates:
skin, fur, hair, wool, claws, nails, hooves, horns, scales, beaks and feathers.
• Transport: A transport protein (variously referred to as a transmembrane pump,
transporter, escort protein, acid transport protein, cation transport protein, or anion
transport protein) is a protein that serves the function of moving other materials
within an organism.

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 • Motor: Motor proteins are a class of molecular motors that are able to move along
the surface of a suitable substrate. They are powered by the hydrolysis of ATP and
convert chemical energy into mechanical work
• Storage: Storage proteins serve as biological reserves of metal ions and amino acids,
used by organisms. They are found in plant seeds, egg whites, and milk. Ferritin is
an example of a storage protein that stores iron. Iron is a component of heme, which
is contained in the transport protein hemoglobin and in cytochromes.
• Signaling: proteins involved in transmission of signals (signal transduction) across
the membrane of cells.
• Receptors: An intracellular protein or protein fraction having a high specific affinity
for binding agents known to stimulate cellular activity, such as a steroid hormone
or cyclic AMP.
• Gene regulation: regulatory protein (gene-regulatory protein) Any protein that
influences the regions of a DNA molecule that are transcribed by RNA polymerase
during the process of transcription. These proteins, which include transcription
factors, therefore help control the synthesis of proteins in cells. "regulatory protein."
• Special functions: Proteins are large, complex molecules that play many critical
roles in the body. They do most of the work in cells and are required for the
structure, function, and regulation of the body's tissues and organs.
▪ Globular proteins
• Compact shape like a ball with irregular surfaces
• Enzymes are globular
▪ Fibrous proteins – usually span a long distance in the cell
• 3-D structure is usually long and rod shaped
▪ Antibodies
• Y-shaped molecules with 2 binding sites at the upper ends of the Y
• The loops of polypeptides on the end of the binding site are what imparts the
recognition of the antigen
• Changes in the sequence of the loops make the antibody recognize different
antigens – specificity

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4. Regulation of gene expression

4.1 Regulation of gene expression in prokaryotes (e.g., the lac. and trp. operons)

Operon: a coordinated unit of gene expression consisting of one or more related genes and the
operator and promoter sequences that regulate their transcription. The mRNAs thus produced
are “polycistronic’—multiple genes on a single transcript.

Operon — A unit of bacterial gene expression and regulation, including structural genes and
control elements in DNA recognized by regulator gene product(s).

▪ Operons control transcription in bacterial cells

• Operon: promoter + additional sequences that control transcription (operator) +
structure genes
• Regulator gene: DNA sequence encoding products that affect the operon function,
but are not part of the operon. A gene that codes for a product (typically protein)
that controls the expression of other genes (usually at the level of transcription).
• Structural gene — A gene that codes for any RNA or protein product other than a
regulator.

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 ▪ Negative and positive control; inducible and repressible operons
• In negative regulation, a repressor protein binds to an operator to prevent a gene
from being expressed.
• In positive regulation, a transcription factor is required to bind at the promoter to
enable RNA polymerase to initiate transcription.
• Inducible operons: Transcription is usually off and needs to be turned on.
• Repressible operons: Transcription is normally on and needs to be turned off.
• Negative inducible operons: The control at the operator site is negative. Molecule
binds to the operator, inhibiting transcription. Such operons are usually off and need
to be turned on, so the transcription is inducible.
✓ Negative regulation: The product of the I gene, the repressor, blocks the
expression of the Z, Y, and A genes by interacting with the operator (O).
• Inducer: small molecule that turns on the transcription
• The inducer (lactose or IPTG — isopropyl β-D-1-thiogalactopyranoside) can bind
to the repressor, which induces a conformational change in the repressor, thereby
preventing its interaction with the operator (O). When this happens, RNA
polymerase is free to bind to the promoter (P) and initiates transcription of the lac
genes.
• Negative repressible operons: The control at the operator site is negative. But such
transcription is usually on and needs to be turned off, so the transcription is
repressible.
• Corepressor: a small molecule that binds to the repressor and makes it capable of
binding to the operator to turn off transcription

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4.2 Regulation of gene expression in eukaryotes

▪ Eukaryotic gene expression can be regulated at the transcriptional, processing, or

translational stages.
▪ Both intracellular signaling and intercellular communication are important for
transcriptional regulation in eukaryotes (cell surface to nucleus).

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 ▪ Positive and negative regulator proteins called transcription factors bind to specific
regions of DNA and stimulate or inhibit transcription.
▪ Alternate splicing of RNA
• Splicing: removing introns by spliceosomes
• Alternate splicing of transcripts makes it possible for a single gene to encode several
polypeptides.
• Alternative splicing: is a very common phenomenon in higher eukaryotes. It is a
way to get more than one protein product out of the same gene and a way to control
gene expression in cells.
• A prominent mechanism to generate protein diversity.
▪ Alternative polyadenylation = where the polyA tail is added
▪ Alternative polyadenylation and splicing can occur together.
• Examples:
✓ Human calcitonin (CALC) gene in thyroid and neuronal cells
✓ Sex determination in Drosophila

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 ▪ Cytoplasmic Control of mRNA Stability
• mRNA degradation
• mRNA stability is influenced by several factors
✓ The poly(A) tail
✓ The sequence of the 3’UTR
✓ Chemical factors (e.g., hormones)
✓ Small interfering RNAs (siRNAs) or microRNAs (miRNAs)
▪ Eukaryotic gene expression can be induced by environmental factors such as heat and
by signaling molecules such as hormones and growth factors.

Transcription control of gene regulation is regulated by:

Promoters

▪ Occur upstream of the transcription start site.

▪ Some determine where transcription begins (e.g., TATA), whereas others determine if
transcription begins.

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 ▪ Promoters are activated by specialized transcription factor (TF) proteins (specific TFs
bind specific promoters).
▪ One or many promoters (each with specific TF proteins) may occur for any given gene.
▪ Promoters may be positively or negatively regulated.

Enhancers

▪ Occur upstream or downstream of the transcription start site.

▪ Regulatory proteins bind specific enhancer sequences; binding is determined by the
DNA sequence.
▪ Loops may form in DNA bound to TFs and contact upstream enhancer elements.
▪ Interactions of regulatory proteins determine if transcription is activated or repressed
(positively or negatively regulated).

Short-term - transcriptional control of galactose-

utilizing genes in yeast:
• 3 genes (GAL1, GAL7, & GAL 10) code
enzymes that function in the galactose
metabolic pathway.
• GAL1 galactokinase
• GAL7 galactose transferase
• GAL10 galactose epimerase
• Pathway produces d-glucose 6-
phosphate, which enters the glycolytic
pathway and is metabolized by genes
that are continuously transcribed.
• In absence of galactose, GAL genes are
not transcribed.
• GAL genes rapidly induced by galactose
and absence of glucose.
• Analogous to E. coli lac operon repression
by glucose.

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5. Functional rearrangement of genes

▪ The process by which part or all a gene is moved from its normal location in the genome
to another site
▪ A structural alteration of a chromosome that causes a change in the order of its gene
▪ Chromosomal rearrangement occurs by deletion, duplication, inversion, and
translocation
▪ Chromosome rearrangements
• deletion: loss of segment
• duplication: gain of segment
• inversion: reversal of region
• translocation: movement of segment to another chromosome
▪ Balanced rearrangement: inversion
• Change in gene order, but gain or loss of DNA
• Inversion loop formed at meiosis I
• Paracentric: centromere outside inversion
✓ crossing-over in inversion heterozygote results in one dicentric chromatid and
one acentric fragment
✓ reduced number of viable gametes
• Pericentric: inversion spans centromere
✓ crossing over in inversion results in gene imbalance
✓ reduced number of viable gametes
▪ Balanced rearrangement: translocation
• Change in gene order, but no gain or loss of DNA
• Reciprocal translocations: exchange between two nonhomologous
chromosomes
• Cross-shaped configuration at meiosis I
• Crossing-over results in gene imbalance, semi-sterility
▪ Imbalanced rearrangement: deletion
• Loss of segment of DNA
• Intragenic deletion: small deletion within gene
• Multigene deletion
✓ many genes deleted

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 ✓ often severe consequences
o gene imbalance
o expression of deleterious recessive mutation (pseudodominance)
▪ Imbalanced rearrangement: duplication
• Gain of segment of DNA
• Source of new genes and gene families
• Tandem duplication: adjacent duplications
• Insertional duplication: duplicate gene inserted elsewhere in genome
• May be consequence of unequal crossingover
▪ Result from chromosome breakage with subsequent re-union in a different
configuration.
▪ Reconstitution of a functional gene from gene segments separated in the genome.
▪ Gene rearrangement
• One that occurs at DNA level.
• Another that occurs at RNA level.
• Speciation can also occur through chromosome rearrangement

Genes and Regulatory Elements

▪ Structural genes: encoding proteins. DNA regions that can be transcribed into RNA are
called structural genes.
▪ Regulatory genes: encoding products that interact with other sequences and affect the
transcription and translation of these sequences
▪ Regulatory elements: DNA sequences that are not transcribed but play a role in
regulating other nucleotide sequences
• Matches to transcription factor binding sites
• Affect the transcription of nearby genes
• Many regulatory elements are not conserved across species
• Constitutive expression: continuously expressed under normal cellular conditions
• Positive control: stimulate gene expression
• Negative control: inhibit gene expression
▪ Regulatory elements on DNA (cis-acting): cis-acting are present on the same DNA
molecule with the gene they are regulating as opposed to trans-acting elements that
regulate distantly located genes.

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 • Positive = enhancers
• Negative = silencers
▪ Regulatory elements not on DNA (protein factors; trans-acting)
• Positive = activators
• Negative = repressors
▪ Points where genes can be regulated
• through the alteration of DNA or chromatin structure
• at the level of transcription
• mRNA processing
• regulation of RNA stability
• translation control
• posttranslational modification

Gene Regulation in Eukaryotic Cells Takes Place at Multiple Levels

▪ DNA Methylation
▪ DNA methylation of cytosine bases adjacent to guanine nucleotides (CpG)–CpG
islands

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Dicer part of RNase III family. Cleves double stranded RNA (dsRNA) and pre-microRNA
(pre-miRNA) into short double-stranded RNA

5.1 A family of proteins that can be created to bind to almost any molecule

▪ Antibodies (immunoglobulins) are made in response to a foreign molecule i.e., bacteria,

virus, pollen… called the antigen
▪ Bind together tightly and therefore inactivates the antigen or marks it for destruction
▪ Y-shaped molecules with 2 binding sites at the upper ends of the Y. Variable Domains
Antigen-Binding Activity and Constant Domains Functional Activities

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 ▪ The loops of polypeptides on the end of the binding site are what imparts the recognition
of the antigen
▪ Changes in the sequence of the loops make the antibody recognize different antigens –
specificity

5.2 Rearrangement of antibody genes

How Does Rearrangement Occur?

▪ Rearrangement occurs between specific sites on the DNA called Recombination Signal
Sequences (RSSs).
▪ Rearrangement is catalyzed by two Recombination Activating Genes: RAG-1 and
RAG-2.

Glycoprotein.

▪ Produced by plasma cells.

▪ Recognize & bind antigens.
▪ Leads to:
• Phagocytosis
• Complement activation
• Antibody dependent cell cytotoxicity (ADCC).

Functions of antibody
▪ Opsonization.
▪ Immune Adherence.
▪ Transcytosis.
▪ Complement Activation.
▪ Virus and Toxin Neutralization.
▪ Antibody-Mediated Cellular Cytotoxicity.
▪ Direct lysis of Microbes.

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6. Applications of Molecular Biology

6.1 Applications of molecular biology in various fields

▪ Medicines and cures: The use of rDNA allows scientists to produce many products that
were previously available only in limited quantities, for example, insulin. Until the
1980s the only source of insulin for people with diabetes came from animals
slaughtered for meat and other purposes. The supply was never adequate to meet
demand, and this drove up prices. Then, in 1982, the sale of insulin produced by
genetically altered organisms was approved. Since 1982 several additional products,
such as human growth hormone, have been made with rDNA techniques.
▪ One of the most exciting potential applications of genetic engineering is the treatment
of genetic disorders through gene therapy. Genetic engineering offers the potential to
provide individuals with correct copies of a gene, which could make possible a cure for
that condition. In the 1980s scientists began clinical trials of a procedure known as
human gene therapy to replace defective genes.
▪ Cloning: A clone is a cell, group of cells, or organism that contains genetic information
identical to that of the parent cell or organism. It is a form of asexual reproduction. The
first clones produced by humans as long as 2,000 years ago were plants developed from
grafts and stem cuttings.
▪ In the feeding: Cultivation, cross-breeding and genetic engineering techniques have
helped increase production, eliminate weak and unwanted characteristics and introduce
disease-resistant varieties into crops, fruits and vegetables. Selective breeding has also
dramatically improved livestock, performance of food products such as poultry, milk,
honey and many other foods.
▪ In agriculture: The destruction of harmful insects and the use of modern agricultural
methods. By studying the nature, occurrence and reproduction of these pests, farmers
can increase the yield of crops using control measures.
▪ In health: Biology has made it possible to understand the causes of many diseases.
Methods of controlling, curing disease, and formulating drugs have been made possible.
The cause of malaria was unknown. It was determined that malaria is not caused by bad
air but a protozoan is the causal agent and is spread by the bite of the female Anopheles
mosquito. There are endless applications of biology in solving health problems. For
example, analgesics have the effect of calming pain while antiseptics eliminate or stop

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 the growth of microorganisms. Vaccines have been developed to fight against many
diseases, which has allowed the reduction of mortality rates. Also, through genetic
studies health professionals can identify certain abnormalities in babies before they are
born and treat these conditions.
▪ In industry: Sericulture (natural silk production) and pisciculture (fish farming) are
rapidly growing industries and rely exclusively on knowledge of the biological
sciences.
▪ In humans: It aims to improve the lifestyles of human beings through controlled
inheritance, genetic engineering, study of vitamins and hormones, cancer research and
the environment etc. It is now possible to manipulate the conditions of nature to get the
most out of it.
▪ In solving problems of modern civilization: Population growth and industrialization
have led to a number of problems. For example, numerous studies have been conducted
to determine the effects of air pollution on man, plants and animals. In the area of family
planning, multiple chemicals are initially used in animals to alter their reproductive
cycles. Subsequently, these findings are applied in contraceptive methods and
fertilization techniques.
▪ In cultural beliefs: Genetic studies have contributed to the rejection of cultural myths.
In the past only women were considered sterile and responsible for not being able to
have children. At present it is quite clear the co-responsibility of the man. Nowadays,
it is more than evident that problems in the health and in the concentration of sperms
of the man can condition the reproductive possibilities in a pair. Likewise, the belief
that the woman was responsible for the assignment of sex in offspring has been denied
through biology. Numerous studies have shown that the sex of the children is
determined by the sperm of the man and not by the ovules of the woman.
▪ In the understanding of the human body: What elements affect the structure and weight
of the human body? What is the reason for the existence of multiple races? What
motivates the snoring? These are some of the questions frequently raised by society.
▪ In the Justice: Criminals often leave evidence of their identity at the scene of the crime:
for example, hair follicles, blood or skin cells. The police can use genetic information
to demonstrate whether or not an individual was present at the scene of a crime. For
example, police can use fingerprints to catch criminals.

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 ▪ In the ecosystem: The study of ecosystems and how they condition the behavior of
societies. This science also warns about the dangerous consequences that are generated
when creating imbalances in the environment.

Some of the molecular biology techniques

▪ Nucleic acid hybridization (Southern Blotting)

• Named after Edward M. Southern who developed this procedure in 1975
• Allow investigators to determine the molecular weight of a restriction fragment
• To measure relative amounts in different samples
• To locate a particular sequence of DNA within a complex mixture
• Southern blotting methodology
✓ Digest DNA with appropriate restriction enzyme
✓ Run digest on an agarose gel
✓ Denature the DNA
✓ Transfer the denatured DNA to the membrane
✓ Add labeled probe to the membrane
✓ Detection
• Some of the application of southern blotting
✓ To look for or to confirm the presence of a gene often in conjugation with PCR
✓ To test for the presence of a specific allele of a gene
✓ To aid in restriction fragment analyses RFLP
▪ Polymerase chain reaction (PCR)
• PCR- Amplify minute amounts of target DNA within a few hours
• Typically amplify nucleic fragments of up to 10 kilo base pairs(kb), although some
techniques allow for amplification of fragments up to 40 kb in size.
• Developed by Nobel laureate biochemist Kary Mullis in 1984
• Discovered of thermostable polymerase work at 100oc: Taq polymerase
• PCR Materials
✓ DNA template that contains the DNA region (target) to be amplified.
✓ Two primers, which are complementary to the DNA regions at the 5' (five
prime) or 3' (three prime) ends of the DNA region.
✓ A DNA polymerase such as Taq polymerase or another DNA polymerase with
a temperature optimum at around 70°C.

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 ✓ Deoxynucleoside triphosphates (dNTPs; also, very commonly and erroneously
called deoxynucleotide triphosphates), the building blocks from which the DNA
polymerases synthesizes a new DNA strand.
✓ Buffer solution, providing a suitable chemical environment for optimum
activity and stability of the DNA polymerase.
✓ Divalent cations, magnesium, or manganese ions; generally, Mg2+ is used, but
Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+
concentration increases the error rate during DNA synthesis
✓ Monovalent cation potassium ions
✓ Thermal cycler
• PCR methods
✓ Capable of amplifying tiny quantities of nucleic acid.
✓ Cells separated and lysed.
✓ Double stranded DNA separated into single strands.
✓ Primers, small segments of DNA no more than 20-30 nucleotides long added.
✓ Primers are complementary to segments of opposite strands of that flank the
target sequence.
✓ Only the segments of target DNA between the primers will be replicated.
✓ Each cycle of PCR consists of three cycles:
➢ Denaturation of target DNA to separate 2 strands. This step consists of
heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA
template and primers by disrupting the hydrogen bonds between
complementary bases of the DNA strands, yielding single strands of DNA.
➢ Annealing step in which the reaction mix is cooled to allow primers to
anneal to target sequence. The reaction temperature is lowered to 50-65°C
for 20-40 seconds allowing annealing of the primers to the single-stranded
DNA template. Typically, the annealing temperature is about 3-5 degrees
Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen
bonds are only formed when the primer sequence very closely matches the
template sequence. The polymerase binds to the primer-template hybrid and
begins DNA synthesis.
➢ Extension reaction in which primers initiate DNA synthesis using a DNA
polymerase. The temperature at this step depends on the DNA polymerase
used; Taq polymerase has its optimum activity temperature at 75-80°C, and
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 commonly a temperature of 72°C is used with this enzyme. At this step the
DNA polymerase synthesizes a new DNA strand complementary to the
DNA template strand by adding dNTPs that are complementary to the
template in 5' to 3' direction, condensing the 5'-phosphate group of the
dNTPs with the 3'-hydroxyl group at the end of the nascent (extending)
DNA strand. The extension time depends both on the DNA polymerase used
and on the length of the DNA fragment to be amplified. As a rule-of-thumb,
at its optimum temperature, the DNA polymerase will polymerize a
thousand bases per minute. Under optimum conditions, i.e., if there are no
limitations due to limiting substrates or reagents, at each extension step, the
amount of DNA target is doubled, leading to exponential (geometric)
amplification of the specific DNA fragment.
➢ These three steps constitute a thermal cycle
✓ Each PCR cycle results in a doubling of target sequences and typically allowed
to run through 30 cycles, one cycle takes approximately 60-90 seconds.

6.2 Recombinant DNA technology

▪ Recombinant DNA: DNA that contains two Recombinant DNA segments not found
together in nature.
▪ Recombinant DNA technology made it possible to cut a gene out of one organism and
recombine it into the genetic machinery of another organism.

Made possible by the discovery of:

▪ Restriction enzymes — DNA cutting enzymes (molecular scissors)

▪ Plasmid DNA vectors — circular form of self-replicating DNA
• Can be manipulated to carry and clone other pieces of DNA
▪ Restriction Enzymes
• Primarily found in bacteria (they use these for defense)
• Cut DNA by cleaving the phosphodiester bond that joins adjacent nucleotides in a
DNA strand
• Bind to, recognize, and cut DNA within specific sequences of bases called a
restriction site

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 ✓ Each restriction site is a palindrome – reads same forward and backward on
opposite strands of DNA
• There are 4 or 6bp cutters because they recognize restriction sites with a sequence
of 4 or 6 nucleotides

Restriction enzymes

▪ Some cut DNA to create DNA fragments with overhanging single stranded ends called
"sticky" or "cohesive" ends
▪ Some cut DNA to generate fragments with double-stranded ends called "blunt" ends
▪ Sticky ends preferred for cloning because DNA fragments with sticky ends can be
easily joined together because they base pair with each other by forming weak hydrogen
bonds

Plasmid vectors

▪ Plasmid DNA — small circular pieces of DNA found primarily in bacteria

▪ Are considered extrachromosomal DNA because they are in the cytoplasm in addition
to the bacteria chromosome

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 ▪ Are small approximately 1 to 4 kb
▪ Can replicate independently of chromosome
▪ Can be used as vectors — pieces of DNA that can accept, carry, and replicate other
pieces of DNA

Transformation of Bacterial Cells

▪ A process for inserting foreign DNA into bacteria

• Treat bacterial cells with calcium chloride
• Add plasmid DNA to cells chilled on ice
• Heat the cell and DNA mixture
• Plasmid DNA enters bacterial cells and is replicated and express their genes
▪ Electroporation
• Apply brief pulse of high voltage electricity to create tiny holes in the
bacteria cell wall that allow the DNA to enter

Selection of recombinant bacteria after transformation

▪ Selection is a process designed to facilitate the identification of recombinant

bacteria while preventing the growth of non-transformed bacteria and bacteria that
contain plasmid without foreign DNA
• Antibiotic selection — plate transformed cells on plates containing different
antibiotics to identify recombinant bacteria and non-transformed bacteria
✓ Does not select for plasmid containing foreign DNA vs. recircularized
plasmid
• Blue-white selection
✓ DNA is cloned into the restriction site in the lacZ gene
✓ When it is interrupted by an inserted gene, the lacZ gene cannot produce
functional Beta gal
✓ When Xgal (artificial lactose) is added to the plate, if functional lacZ is
present = blue colony
✓ Non-functional lacZ = white colony = clone = genetically identical
bacterial cells each containing copies of recombinant plasmid

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