UNIT 2.

BIOCHEMISTRY AND MOLECULAR ASPECTS OF LIFE

Introduction

Carbohydrates are polyhydroxy aldehydes (or) ketones. The word saccharide in derived from
the greek “saccharine” meaning sugar. Monosaccharide’s are simple sugar consist of single
polyhydroxy aldehyde or ketone unit.

Oligosaccharides consist of short chains of monosaccharide units (or) resides joint by

characteristics linkage called glycosidic bonds.

The Polysaccharides are sugar polymers containing more than 20 monosaccharide units and
some have 100-1000 units some polysaccharides such as cellulose are lineal chains others such
as glycogen are branched.

Monosaccharides

The simplest of the carbohydrates the monosaccharides are either aldehyde (or) ketone with
two or more hydroxyl groups.

Glucose:-

Disaccharides

 Disaccharides consist of two Monosaccharide units

 They are crystalline, water soluble and sweet to table.

Classification of Disaccharide
 Reducing Non Reducing
(with free aldehyde) (absence of free aldehyde (or) ketone group)

Reducing Sugar Non-Reducing Sugar

1. Carbohydrates with free aldehyde Aldehyde (or) ketone group is not free but
(C-1) or free ketone (C-2) group instead untilised in bond formation
2. They are in hemiacetal (or) They are acetal or ketal form
hemiketal form
3. Do exhibit mutarotation Do not exhibit mutarotation
4. Do form osazones with Do not form oxime
phenylhydrazine
5. Do from oxime with hydroxylamine Do not from oxime
Eg. Fructose, Lactose, Glucose, Eg. Sucrose, Glycogen, Inulin
Maltose

Maltose

Occurrence:-

 It does not occur in the body

 The source is germinating cereals and Malt.
 It is the intermediate product in the breakdown of starch by the enzyme amylase in the
alimentary canal.

Structure

Maltose contains two glucose residues joined by glycosidic linkage between C-1 (anomeric
carbon) of one glucose residue which can act as a reducing agent.

Thus maltose is a reducing disaccharide.

Maltose occurs in a malt product and is produced as an intermediate product in the

digestion of starch and glycogen by the action of the enzyme - amylase.
 Lactose

Occurrence:

 It is present in milk and formed in the lactating mammary gland. Hence it is

commonly called as milk sugar.
 Lactose contains one unit of -D-galactose and one unit of -D-glucose that are
linked by a O- glycosidic linkage.

Lactose is hydrolysed to glucose and galactose by lactose enzyme in human and

in bacteria by -D-galactosidase.

Functions:

 The α-isomer of lactose crystallizes in ice-cream, making the product seem sandy in
texture.
 The more soluble -isomer is used indicts for infants.

Sucrose
Occurrence:

 It is otherwise called as “table sugar”.

 It is the common sugar of commerce and kitchen and is widely distributed in all
photosynthetic plants.
 It occurs in very large amounts in Sugarcane, beetroot, pineapple, maple fruits, carrot,
Sweet potato, honey.
 It is also present in the hector of flowers.
 It is also present in varying amounts in different plant organs. Such as fruits, seeds,
flowers, seeds.
  Sucrose is a disaccharide of glucose and fructose. It is formed by plant but not by human
beings.
 Sucrose is the commonly used “table sugar” and contributes some calories in the diet.

Structure:

 It is a disaccharide composed of one molecule of α-D-glucose and one molecule of β-D-

fructose. Which are condensed together by α(1→2) glycosidic linkage.
 The linkage exists between aldehyde group of glucose and ketogroup fructose. As a result,
no reducing group remains free in sucrose molecule.
 Since, the functional groups of both glucose and fructose are blocked in the linkage; this
disaccharide is called as non-reducing sugar.

Functions:
 It serves as nutrient.
 Invert sugar is used in candy both because of its sweetness and do not crystallize.
 It is used to produce artificial sweetener of no nutritive value. eg: saccharin. It is
developed especially for obese (or) a diabetic person. Saccharin is 400 times sweeter
than glucose

Polysaccharides. (Glycans)

Polysaccharides generally called glycans are compounds formed by the condensation of a

number of monosaccharide unit linked through glycosidic bonds. They serve as structural
components of the cell as well as function as nutrients.
 1. Homopolysaccharides (Homoglycans) which yield only one type of monomer units
hydrolysis

Ex:- cellulose, glycogen, starch

2. Heteropolysaccharides (Heteroglycans) that yields two (or) more types of monomer

unites viz hyaluronicacid chondriotin sulfate, heparin, and murrains.

Homopolysaccharides (or) Homoglycans (Storage)

Occurrence:-

 It is the most important reserve food material of the higher plants and is found in
cereals tubers (ie) Potatoes, seeds in the rhizomes and pith of plants.
 Sago starch is obtained from sago palm
 They usually occur as compact insoluble grains inside the plant cells.
 The bulk of our diet which consists of rice wheat and vegetables is a good source
of starch.

Structure:- (Amylose)

Amylose is a linear polymer of D glucose units joined by α (1→4) glycosidic linkage. It

is the reserve carbohydrate from in plants. Eg:- Potato is grains and seeds and in many fruits.
It is the storage form of glucose in plants. Starch is composed of Amylose and Amylopectin
 In amylose the glucose units are joined by α (1→4) linkage to form unbranched chains
which are in the form of a helix with 6 glucose units perturb.

Amylose forms the inner portion of starch grains and is soluble in water and less viscous.
The molecular weight are about 60,000 which are equivalent to about 300-400 glucose units and
are responsible for the development of blue colour with iodine.

Amylopectin

Amylopectin is structurally identical to those of amylose α (1→4) (glycosidic linkages) but

with side chains joining them by α (1→6) linkages.

Thus amylopectin is a branched polymer having both α (1→4) α (1→6) linkage. The branch
points in amylopectin are created by α (1→6) bonds and occur at an interval of 24-30 units of
glucose.

In amylopectin glucose molecules are arranged in highly branched form. They have much
larger molecular weight of upto million and the chains have atleast 80 branches each branch is at
interval of 24-30 glucose units.

Branch point occurs at the 6th carbon atom of glucose molecule. Hence amylopectin have
both (α1→ 4) (α1→6) glycosidic linkages of starch grains and is insoluble in H2O. It develops
violet colour with dilute iodine solution.
 Glycogen (Animal starch)

Occurrence:-

 It is called otherwise called as” animal starch”

 It is a storage polysaccharide found in liver and muscles of animals and human beings
 It is also found in fungi & yeast which have no chlorophyll system but not in green plants

Structure:-

 Glycogen is a branched chain structure with straight chain units of 8-12 α–D
glucopyranoses in [α 1→4] glycosidic linkages with branching by means of α 1→b
glycosidic bonds.

Function:-

 The function of muscle glycogen is to act as a readily available source of glucose for
energy with in muscle itself.
 Liver glycogen is concerned with storage and maintenance of the blood glucose.

Lipids
Lipids are esters of alcohols and fatty acids. The two major constitutions of lipids are
alcohol and fatty acids.

Ex:- fat, oil, wax,

Lipids may be defined as, organic substances insoluble in water but soluble in organic
solvents like, chloroform, ether, benzene
 They are ester of fatty acids with alcohol (or) are substances capable of forming such
esters and are utilizable by the living organisms.

Classification of lipids:
Lipids are classified broadly into simple lipids, Compound lipids, derived lipids. These,
lipids are sub classified into different types.

 Simple lipids
 Compound lipids
 Derived lipids

I. Simple lipid:-
These are esters of fatty acids with various alcohols Depending on the type of alcohols
these are Sub classified as:-

1. Neutral fats (or) triglycerides

2. Waxes

Glycerol Triacylglycerol

Waxes:-
Waxes are hard esters of long chain saturated fatty acid of 14-36 carbon atoms with long
chain monohydroxy alcohol of nearly 36 – Carbon atoms.

True waxes:-

These are esters of fatty acids with high molecular weight monohydric long chain
alcohols.
  Bees- wax and Spermacetic oil (from wholes)

Widely used in pharmaceutical, Cosmetics, and Other industries in manufacture of

lotions, ointments, polish

Other waxes:-
Estes of fatty acids with alcohol

1. Cholesterol form cholesterol ester.

2. Retinol ( vit – A) vit ‘A’ ester (Retinol esters)
3. Cholecalciferol (Vit –D ) forms vit – D- ester

Honey comb vertebrates, birds, Rhododendron plant leaf and protein that transport insoluble
lipids through the blood between different organs and tissues

Lipoprotein consist of a lipids core containing non-polar tri acylglycerol & cholesterol
ester surrounded by a single surface layer of amphipathic Phospholipids and free cholesterol
molecule with Some protein. (apo)

Derived lipids:
After hydrolysis of simple (or) compound lipids they obtained that derived lipids: eg:-
 Fatty acids
 Glycerol
 Steroids
 Cholesterol
 Vit ‘A’ & ‘D’

Fattyacids:-

Fattyacids are carboxylic acids with hydrocarbon chains (-CH2-CH2-CH2-) and represented
by a chemical formula R-COOH ( R – hydrocarbon chain ) The batty acids are amphipathic in
nature each has hydrophilic (COOH) hydrophobic (hydrocarbon) groups in the structure.
 Fatty acids occur mainly as ester of glycerol in neutral fats and oils. Fatty acids serve as a
Major fuel for most cells and they are precursors of all other classes of lipids.

Functions of fatty acids:

 Fatty acids have 3-major physiological function.

1. Serve as building blocks of phosphor and glycolipids.
2. These ampipathic molecules are important components of biological membranes.
3. Prostaglandins → intracellular messenger.

Vitamins – classification and functions

It is a known fact that we require energy in order to perform different activities. We get
these energies from the food we eat. Apart from the normal food that we take, our body requires
a certain number of compounds in small amounts for the proper functioning and deficiency of
these compounds may cause diseases. These compounds are known as vitamins. They are
chemical compounds that are required in small amounts with our regular diet in order to carry
out certain biological functions and for the maintenance of our growth.

What are vitamins?

The name “vitamin” is sourced from "vital amines" since it was originally thought that these
substances were all amines.

• Vitamins are a class of organic compounds that are important for normal growth and nutrition;
required in small quantities in the diet because they cannot be synthesized by the body. These
are therefore essential nutrients for living system.

• Vitamins participate in regulation of the energy-producing processes. With the exception

of vitamin D and K, vitamins cannot be synthesized by the human body and must be obtained
from the diet.
Types of vitamins
Vitamins are generally classified as water soluble vitamins and fat soluble vitamins.

Fat Soluble vitamins

Vitamin A, D, E and K are fat soluble. These are stored in adipose tissues and hence are called
fat soluble vitamins.

Water Soluble vitamins

Vitamins in B-group and vitamin C are water soluble and cannot be stored in our body as they
pass with the water in urine. These vitamins must be supplied to our body with regular diets.

Functions

Based on their role in biological processes and their affect different vitamins have different
functions, their function can be best understood by knowing about their deficiency diseases.
Given below is the list of vitamins and their deficiency diseases:

1. Vitamin A – Hardening of cornea in eye, night blindness.

2. Vitamin B1 – Deficiency may cause beri beri, dwarfism.
3. Vitamin B2-Deficiency can cause disorders in digestive system, skin burning sensations,
cheilosis.
4. Vitamin B6– Deficiency of B6 causes convulsions, conjunctivitis, and sometimes
neurological disorders.
5. Vitamin B12– Its deficiency can cause pernicious anaemia and decrease in red blood
cells in haemoglobin.
6. Vitamin C- It is a water soluble vitamin, its deficiency causes bleeding in gums and
scurvy.
7. Vitamin D- It is obtained by our body when exposed to sunlight. Its deficiency causes
improper growth of bones, soft bones in kids , rickets.
8. Vitamin E- Deficiency of vitamin E leads to weakness in muscles and increases the
fragility of red blood cells.
 9. Vitamin K- It plays an important role in blood clotting. Deficiency of vitamin K increases
the time taken by blood to clot. Severe deficiency may cause death due to excessive
blood loss in case of a cut or an injury.

Although these compounds are required in very small quantities by our body to perform several
biological functions, and their deficiency may lead to severe diseases.

Vitamin A (retinol):
 Vitamin B1 (thiamine):

Vitamin B2 (riboflavin):
Vitamin B6 (pyridoxine):

Vitamin C (ascorbic acid):

Folic acid:

Biotin (vitamin H, vitamin B7):

Niacin/nicotinic acid:
Pantothenic acid/ vitamin B5:

Lipoic acid:
Co-factors and Co-enzymes:

Proteins
• Proteins are major constituents of all living organisms, it can be defined as, and Protein
contains mainly, the carbon, hydrogen, nitrogen, and sulphur.
• The protein rich foods are called as bogy building foods.
Peptide bond formation:
Classification of Proteins:

Fibrous Protein
They contain atoms having one or more polypeptide chains as fibrils.

They are water insoluble and form tendons, muscle fibers, connective tissues and bone matrix.

Secondary structure is of great importance in them. They are water insoluble, non-crystalline
and elastic. They form tendons, muscle fibers, connective tissues and bone matrix. Myosin,
silk fibers, fibrin, and keratin are examples.
 Globular Protein
 Globular proteins are ellipsoidal and spherical because of numerous wrinkling of
polypeptide chains.
 Tertiary structure is most essential in them. They are soluble in water and can be
crystallized.
 They adjust themselves with changes in the physical environment.
 Cases are enzymes, hormones, antibiotics and haemoglobin.
Structural organization of Proteins
Primary structure
 The sequence of the amino acids combined in a peptide chain is referred to as the
primary structure. Peptide bond held together the primary structure which is made during
the course of protein biosynthesis.

 In primary structure the amino acid sequence that make up a polypeptide chain.

Secondary structure

 Is a regular coiling or zigzagging of polypeptide chains produced by hydrogen bonding

between NH and C=O groups of amino acids near each other in the chains. They typically
curl into a helix.

 To observe the secondary structure of proteins, it’s a regular, repeated pattern of folding
namely alpha helix and beta sheet of the protein backbone.

Tertiary structure

 The three dimensional twisting and folding of the polypeptide chain results in the tertiary
structure of proteins.

 Polypeptide chain twists and folds upon itself shaping a globular shape.

 The proteins' tertiary conformity is held by three kinds of bonds, i.e. Hydrogen, ionic and
disulfide (-S-S-).
 In the aqueous environment, the steadiest tertiary conformity is that in which
hydrophobic amino acids are covered inside while the hydrophilic amino acids are on the
surface of the Atom.

Quaternary structure

 In complex proteins, polypeptide tertiary sequences are coiled in multi-complex subunits.

 Quaternary proteins are made up of the arrangement of two or more tertiary chains and
are held together by hydrophobic interactions, ionic and hydrogen bonds.

 Haemoglobin, the oxygen transport protein has such a structure.

Properties of Proteins

Physical Properties:

 Colloidal Properties: Visible turbidity formation

 Denaturation: Native structure of protein disorganized.
 Renaturation: attainment of original 3D structure and formed native confirmation.
Chemical Properties:

 Hydrolysis: Hydrochloride formation

 Color Reactions with Biuret, Xanthoproteic reaction, Hopkins-cole reactions.
  Biological Functions of Proteins
 Acts as a Biocatalyst -Enzymes
 Hormonal function –Insulin growth hormone
 Transport functions –Hemoglobin.
 Involved in Blood clotting process – Fibrinogen, Thrombin
 Involved in defense mechanism –Immunoglobin
 Genetic proteins- Nuceloproteins
 Contractile Protein – Myosin and Actin
 Storage Proteins- Casein, egg white.
Structural Importance –Collagen, Keratin
ENZYMES AND HOW ENZYMES FUNCTION WITH ONE TYPICAL EXAMPLE

The human body is composed of different types of cells, tissues, and other complex organs. In
order to function efficiently, there are certain chemicals released by our body to speed up the
biological processes like digestion, respiration, excretion, and other metabolic activities in order
to maintain a healthy life. Thus, enzymes play an important role in all living organisms by
regulating all the biological processes.

There are three kinds of cofactors present in enzymes:

 Prosthetic groups: These are cofactors tightly bound to an enzyme at all times. A fad is
a prosthetic group present in many enzymes.
 Coenzyme: A coenzyme is bound to an enzyme only during catalysis. At all other times,
it is detached from the enzyme. NAD+ is a common coenzyme.
 Metal ions: For the catalysis of certain enzymes, a metal ion is required at the active site
to form coordinate bonds. Zn2+ is a metal ion cofactor used by a number of enzymes.
Classification of Enzyme

Oxidoreductases

These catalyze oxidation and reduction reactions, e.g. pyruvate dehydrogenase, which catalyzes
the oxidation of pyruvate to acetyl coenzyme A.
Transferases

These catalyze the transfer of a chemical group from one compound to another. An example is a
transaminase, which transfers an amino group from one molecule to another.

Hydrolases

They catalyze the hydrolysis of a bond. For example, the enzyme pepsin hydrolyzes peptide
bonds in proteins.

Lyases

These catalyze the breakage of bonds without catalysis, e.g. aldolase (an enzyme in glycolysis)
catalyzes the splitting of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate.

Isomerases

They catalyze the formation of an isomer of a compound. Example: phosphoglucomutase

catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate (transfer of a phosphate
group from one position to another in the same compound) in glycogenolysis (conversion of
glycogen to glucose for quick release of energy.

Ligases

Ligases catalyze the joining of two molecules. For example, DNA ligase catalyzes the joining of
two fragments of DNA by forming a phosphodiester bond.

Cofactors

Cofactors are non-proteinous substances that associate with enzymes. A cofactor is essential for
the functioning of an enzyme. An enzyme without a cofactor is called an apoenzyme. An enzyme
and its cofactor together constitute the holoenzyme.

Industrial applications

Beverages
Alcoholic beverages generated by fermentation vary a lot based on many factors. Based on the
type of the plant’s product, which is to be used and the type of the enzyme applied, the fermented
product varies.

For example, grapes, honey, hops, wheat, cassava roots, and potatoes depending upon the
materials available. Beers, wines and other drinks are produced from plant fermentation.

Food Products

Bread can be considered as the finest example of fermentation in our everyday life.

A small proportion of yeast and sugar is mixed with the batter for making bread. Then one can
observe that the bread gets puffed up as a result of fermentation of the sugar by the enzyme
action in yeast, which leads to the formation of carbon dioxide gas. This process gives the texture
to the bread, which would be missing in the absence of the fermentation process.

Drug Action

Enzyme action can be inhibited or promoted by the use of drugs which tend to work around the
active sites of enzymes.

Nucleic acids: Genes, Genome and Chromosome Nucleic acids

Nucleic acids are long-chain polymeric molecules, the monomer (the repeating unit) is known as
the nucleotides and hence sometimes nucleic acids are referred to as polynucleotides.

1. Nucleotides: Nucleotides consist of 5-carbon sugar + nitrogenous base + 1, 3-phosphate

groups.

2. Pentose sugar: It is either ribose or deoxy ribose (not having oxygen at C2).

3. Nitrogenous base: Derived from purines having two rings in their structure e.g., Adenine (A)
and Guanine (G) and derived from pyrimidines having one ring in their structure e.g., Thymine
(T), Uracil (U) and Cytosine (C). Two H-bonds are present between A and T (A = T) while three
H-bonds are present between C and G (C ≡ G).
4. Ribonucleotide: Phosphate unit + Ribose + one base unit from A, G, C, or U. 5. Deoxyribo
nucleotide: Phosphate unit + Deoxyribose + one base from A, G, C or T. 6. Nucleoside: Ribose /
deoxyribose + one base unit from A, G, C, Tor U. Nucleic acids are composed of nucleotide
monomers linked together.

Nucleotides contain three parts:

• A Nitrogenous Base

• A Five-Carbon Sugar

• A Phosphate Group

• Nucleotides are linked together to form polynucleotide chains. Nucleotides are joined to one
another by covalent bonds between the phosphate of one and the sugar of another. These
linkages are called phosphodiester linkages. Phosphodiester linkages form the sugar-phosphate
backbone of both DNA and RNA.

• Similar to what happens with protein and carbohydrate monomers, nucleotides are linked
together through dehydration synthesis. In nucleic acid dehydration synthesis, nitrogenous bases
are joined together and a water molecule is lost in the process.Interestingly, some nucleotides
perform important cellular functions as "individual" molecules, the most common example being
ATP.

Structure and function of DNA and RNA

DNA • Deoxyribonucleic acid contains information for the cell and genetic material. It has a
phosphate group/phosphoric acid, deoxyribose sugar and nitrogen containing bases (adenine,
thymine, cytosine, and guanine).

• It is composed of polynucleotides -- have a phosphate group, deoxyribose sugar and a nitrogen

containing bases (adenine, thymine, cytosine and guanine). Has a double helix that is formed by
hydrogen bonds between polynucleotides.

• Purine: Bases that have a double ring structure (adenine and guanine)

• Pyrimidines: Bases that have a single ring structure (thymine and cytosine)
Functions of DNA

• Stores an organisms genetic material in the nuclei.

• Provides code or template for the particular sequencing of amino acids that bond together and
make a protein.

RNA

• RNA is essential for the synthesis of proteins. Information contained within the genetic code is
typically passed from DNA to RNA to the resulting proteins.

• There are several different types of RNA:

• Messenger RNA (mRNA) is the RNA transcript or RNA copy of the DNA message produced
during DNA transcription. Messenger RNA is translated to form proteins.

• Transfer RNA (tRNA) has a three dimensional shape and is necessary for the translation of
mRNA in protein synthesis

• Ribosomal RNA (rRNA) is a component of ribosomes and is also involved in protein synthesis.
• MicroRNAs (miRNAs) are small RNAs that help to regulate gene expression.

• RNA most commonly exists as a single stranded molecule.

RNA is composed of a phosphate-ribose sugar backbone and the nitrogenous bases adenine,
guanine, cytosine and uracil (U). When DNA is transcribed into an RNA transcript during DNA
transcription, guanine pairs with cytosine (G-C) and adenine pairs with uracil (A-U).

Differences between DNA and RNA Composition

The nucleic acids DNA and RNA differ in composition. The differences are listed as follows:
DNA Nitrogenous Bases: Adenine, Guanine, Cytosine, and Thymine

Five-Carbon Sugar: Deoxyribose

RNA Nitrogenous Bases: Adenine, Guanine, Cytosine, and Uracil

Five-Carbon Sugar: Ribose

What is a gene?

A gene is the basic physical and functional unit of heredity. Genes are made up of DNA. Some
genes act as instructions to make molecules called proteins. However, many genes do not code
for proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2
million bases. An international research effort called the Human Genome Project, which worked
to determine the sequence of the human genome and identify the genes that it contains, estimated
that humans have between 20,000 and 25,000 genes. Every person has two copies of each gene,
one inherited from each parent. Most genes are the same in all people, but a small number of
genes (less than 1 percent of the total) are slightly different between people. Alleles are forms of
the same gene with small differences in their sequence of DNA bases. These small differences
contribute to each person’s unique physical features. Genes are made up of DNA. Each
chromosome contains many genes.

What is a chromosome?

In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called
chromosomes. Each chromosome is made up of DNA tightly coiled many times around proteins
called histones that support its structure. Chromosomes are not visible in the cell’s nucleus—not
even under a microscope—when the cell is not dividing. However, the DNA that makes up
chromosomes becomes more tightly packed during cell division and is then visible under a
microscope. Most of what researchers know about chromosomes was learned by observing
chromosomes during cell division. Each chromosome has a constriction point called the
centromere, which divides the chromosome into two sections, or “arms.” The short arm of the
chromosome is labelled the “p arm.” The long arm of the chromosome is labelled the “q arm.”
The location of the centromere on each chromosome gives the chromosome its characteristic
shape, and can be used to help describe the location of specific genes.

Genome

An organism's complete set of DNA is called its genome. Virtually every single cell in the body

contains a complete copy of the approximately 3 billion DNA base pairs, or letters, that make up

the human genome.

DNA as a Genetic Material

In early 20 th century scientist did not know what the genetic material was in an organism. By
doing many experiments finally it was evident that DNA is a genetic material. What is genetic
material? Genetic material means, any material of an organism (bacteria, plant, animal or viruses)
that carries the genetic information and that passes from one generation to next generation. For
example, a mango tree will produce many mangoes with seeds and from the seeds new mango
tree will develop. What is transferred to the seeds and what determines in the seed to become
another mango tree? Many experiments were carried out and finally it was concluded that DNA
is the genetic material.
In 1928, experiments were carried out by using two different types of bacterial strains of
Streptococcus pneumoniae. One of the S. pneumoniae strains was referred as Smooth (S) and
highly virulent. This S strain can kill mice if it was injected into its body. Another strain, S.
pneumoniae, referred as Rough (R) is not virulent, not capable of killing mice, when it was
injected to its body.
Frederick Griffith Experiment (1928)

Griffith did an interesting experiment with heat-killed virulent (S) strain of S. pneumoniae. Now
the mice were alive. However, when he injected mouse with the mixture of heat-killed virulent
and live non-virulent strain of (R) of S. pneumoniae, the mouse were dead. These bacteria infect
the lung cells. Griffith found the smooth strain (lethal-virulent strain) in the lungs of dead mice.
He concluded that there is something transformed from the heat-killed S strain into live non
virulent strain of S. pneumoniae. The genetic material was transformed from one bacterium to
another bacterium. But it was not clear whether it was DNA or something else.

Later in year 1944, Scientists Avery, MacLeod, and McCarty did well plan experiment with
many controls and determined that DNA is the transformation material from the heat-killed S
strain to live R strain. The experiment they designed is as given below as a diagram
In all 5 different treatments, the non-virulent R strain was added with 5 different molecules. In
first case, the polysaccharides in the S strain were destroyed after the extract was prepared from
S strain culture. In 2nd case, lipids from the heat killed S strain extract were destroyed by the
enzymes lipases. In 3rd case, RNA was destroyed from the heat killed S strain extract and
injected along with non-virulent R strain. In 4th case all proteins were destroyed by the enzyme
protease and injected with live R strain. In all the above-mentioned 4 cases, the mice were dead.
It showed that polysaccharides, lipid, RNA or protein are not the genetic material. In the 5th case,
DNA was destroyed by enzymes DNases in the S strain extract and mixed with non-virulent R
strain of S. pneumoniae. Now the mice were alive and clearly indicated that DNA is the genetic
material.

Convincing final evidence came in year 1952, when scientists, Hershey and Chase did an
experiment with bacteriophage and bacteria. Bacteriphages are viruses that infect only bacteria
and multiply inside the bacteria. These scientists used T2 bacteriophage which contains double
stranded DNA inside the protein coat. This bacteriophage has only two components. One is

protein coat and double stranded DNA inside the protein coat.
These viruses can replicate or multiply only inside another bacterium. Protein can be
radioactively labeled with sulfur (35S) and DNA can be labeled with radioactive 32P. By
instrument one can estimate the radioactivity in a sample. Phages were multiplied in the medium

containing 35S or 32P separately. It means, one flask the proteins are labeled in the
bacteriophages and in another flask, DNA molecules are labeled. Now the cultures are stirred in
the blender to separate the empty protein shell (without DNA inside). These samples are
centrifuged to pellet the bacteria and estimated the radioactivity both in the supernatant and the
pellet. They identified the radioactivity in the pellet (bacterial cells) where they had bacteria and
DNA of bacteriophage. It clearly indicated that DNA is sent inside the bacterium cell and gets
multiplied and form new bacteriophages. Hence, forming new bacteriophages is determined by
the DNA and not by the protein. Hence it clearly proved that DNA is the genetic material.

DNA Replication:

∙ DNA replication is semiconservative, meaning that each strand in the DNA double helix acts
as a template for the synthesis of a new, complementary strand.

∙ This process takes us from one starting molecule to two "daughter" molecules, with each newly
formed double helix containing one new and one old strand.

∙ It is a biological polymerization which proceeds in the sequence of initiation, elongation, and

termination.

∙ It is an enzyme-catalysed reaction.

∙ DNA Polymerase is the main enzyme in the replication process.

Role of Enzymes in DNA Replication:

DNA replication is a highly enzyme-dependent process. There are many enzymes involved
inDNA replication, which includes the enzymes, DNA-dependent DNA polymerase, helicase,

ligase, etc. Among them, DNA-dependent DNA polymerase is the main enzyme.

Topoisomerase

∙ This is the enzyme that solves the problem of the topological stress caused during unwinding.

∙ They cut one or both strands of the DNA allowing the strand to move around each other to
release tension before it rejoins the ends.

∙ And therefore, the enzyme catalysts the reversible breakage it causes by joining the broken
strands.

∙ Topoisomerase is also known as DNA gyrase in E. coli.

Helicase

Helicase is the enzyme, which unzips the DNA strands by breaking the hydrogen bonds between
them. Thus, it helps in the formation of the replication fork.

DNA-dependent DNA polymerase

It helps in the polymerisation, catalyses and regularises the whole process of DNA replication

with the support of other enzymes. Deoxyribonucleoside triphosphates are the substrate as well

as the energy provider for the replication process.

Primase

This enzyme helps in the synthesis of RNA primer complementary to the DNA template strand.

Ligase

Ligase is the enzyme which joins together the Okazaki fragments of the discontinuous DNA

strands.

Single-stranded Binding Proteins

It binds to single-stranded DNA and protects it from forming secondary structures.

Initiation:

∙This is the stage where DNA replication is initiated.

∙ DNA synthesis is initiated within the template strand at a specific coding region site known as
origins.

∙ The origin sites are targeted by the initiator proteins, which recruit additional proteins that help
in the replication process to form a replication complex around the DNA origin. ∙

There are several origin sites on which DNA replication is initiated and they are all known as

replication forks.
∙ The formed replication complex contains the DNA helicase enzyme whose function is to
unwind the double helix, exposing the two strands, which act as templates for replication.

The mechanism of DNA helicase enzyme is by hydrolyzing the ATP that is used to form the
bonds between the nucleobases, thus breaking the bond that holds the two strands. Additionally,
during initiation DNA primase enzyme synthesizes small RNA primers that kick-start the
function of DNA polymerase.

∙ DNA polymerase enzyme functions by growing the new DNA daughter strand.

Elongation

∙ This is the phase where the DNA polymerase grows the new DNA daughter strand by

attaching to the original unzipped template strand and the initiating short RNA primer. The DNA
polymerase is able to synthesize a new strand that matches the template, by extending the primer
via the addition of free nucleotides to the 3′ end.

∙ One of the templates reads in the 3′ to 5′ direction, and therefore, the DNA
polymerasesynthesizes the new strand in the 5′ to 3′ direction, which is known as the leading
strand. Along the template strand, DNA primase synthesizes a short RNA primer at the
beginning of the template in the 5′ to 3′ direction, which initiates the DNA polymerase to
continue synthesizing new nucleotides, extending the new DNA strand.

∙ The other template (5′ to 3′) is elongated in an antiparallel direction, by the addition of short
RNA primers which are filled with other joining fragments, forming the newly formed lagging
strand. These short fragments are known as the Okazaki fragments.

∙ The synthesis of the lagging strand is discontinuous since the newly formed strand is disjointed.

∙ The RNA nucleotides from the short RNA primers must be removed and replaced by DNA
nucleotides, which are then joined by the DNA ligase enzyme

Leading Strand
∙ The leading strand is the new DNA strand that is continuously synthesized by the DNA

polymerase enzyme.

∙ It is the simplest strand that is synthesized during replication.

∙ The synthesis starts after the DNA strand has unzipped and separated. This generates a short

piece of RNA known as a primer, by the DNA primase enzyme.

∙ The primer binds to the 3′ end (start) of the strand, thus initiating the synthesize of the new

strand (leading strand).

∙ The synthesis of the leading strand is a continuous process.

The Lagging Strand

∙ This is the template strand (5′ to 3′) that is synthesized in a discontinuous manner by RNA

primers.

∙ During the synthesis of the leading strand, it exposes small, short strands, or templates that

are then used for the synthesis of the Okasaki fragments.

∙ The Okasaki fragments synthesize the lagging strand by the activity of DNA polymerase which
adds the pieces of DNA (the Okasaki fragments) to the strand between the primers. The
formation of the lagging strand is a discontinuous process because the newly formed strand
(lagging strand) is the fragmentation of short DNA strands.

Termination

∙ After the synthesis and extension of both the continuous and discontinued stands, an enzyme
knows as exonuclease removes all RNA primers from the original strands. ∙ The primers are
replaced with the right nucleotide bases.

∙ While removing the primers, another type of exonuclease proofread the new stands, checking,
removing, and replacing any errors formed during synthesis.
∙ DNA ligase enzyme joins the Okazaki fragments to form a single unified strand. ∙ The ends of
the parent strand consist of a repetition of DNA sequences known as telomeres which act as
protective caps at the ends of chromosomes preventing the fusion of nearby chromosomes. The
telomeres are synthesized by a special type of DNA polymerase enzyme known as telomerase. It
catalyzes the telomere sequences at the end of the DNA. On completion, the parent and
complementary strand coil into a double helical shape, producing two DNA molecules each
passing one strand from the parent molecule and one new strand.

DNA Transcription:

∙ Transcription is the synthesis of RNA using DNA as a template.

∙ RNA is the intermediate between DNA and proteins∙ DNA was in the nucleus but proteins were
made in the cytoplasm

∙ RNA synthesized in the nucleus was exported to the cytoplasm

Properties of RNA – Similar to DNA except:

• Contains ribose instead of deoxyribose

• Contains Uracil instead of Thymine

• Single stranded instead of double stranded

4 stages of Transcription:

• (1) promoter recognition and identification

• (2) the initiation of transcript synthesis

• (3) transcript elongation

• (4) transcription termination.

Promoters:

• A promoter is a double-stranded DNA sequence that is the binding site for RNA polymerase

• RNA polymerase is attracted to promoters by the presence of consensus sequences, short

regions of DNA sequences that are highly similar.

Initiation:

• RNA polymerase scans the DNA looking for promoters.

• σ factor of RNA polymerase binds the corresponding σ factor recognition sequence in the

promoter.

• Recent evidence suggests that at some promoters, the α subunit may bind to AT rich regions
upstream of the sigma binding sites.

• RNA polymerase is bound covering approx. 60 base pairs. The DNA is still is a double helix
(closed complex).

• RNA polymerase unwinds the DNA resulting in open complex formation.

• First nucleotides are added to start RNA chain. Transcriptional initiation has occurred!
• Accessory transcription factors may aid in all of the above listed steps

Elongation:

• Elongation is 5’ to 3’

• σ factor is ejected from RNA polymerase after first 2-10 nucleotides are added.

• It was once believed that elongation occurred at a constant rate; however, recent work suggests
that RNA polymerase may pause during elongation. In fact, pausing is important in termination

Termination:

Rho independent: A specific sequence at the end of the gene signals termination.The sequence
is transcribed into RNA and it is the RNA sequence that is important. This sequence contains
numerous Gs and Cs, which forms a hairpin structure, followed by a string of Us. • The hairpin
destabilizes the DNA:RNA hybrid leading to dissociation of the RNA from the DNA

Rho dependent:

• Rho protein binds to a sequence in the RNA

• Rho moves along the RNA in the 3’ direction until in eventually unwinds the DNA:RNA
hybrid in the active site, thereby pulling the RNA away from the DNA and RNA polymerase.

• Rut sites are located 5’ to sites in the DNA that cause RNA polymerase to pause. It is thought
that this allows Rho to catch up to RNAP and the RNA-DNA hybrid.
RNA processing:

• 5’ capping: Occurs early in transcription. Guanosyl transferase adds 5’methylguanosine (Cap)

to 5’ end of mRNA. The Cap is important for translation initiation and for export from the
nucleus.

3’ poly(A) tail: AAUAAA sequence in the RNA signals a cleavage event in the RNA. Poly(A)
polymerase then adds 150-200 A residues are added to the 3’end of the mRNA. The poly(A) tail
increases the stability of the mRNA in eukaryotes.

Splicing

• The primary transcripts often contain intervening sequences (introns) that are removed from the
RNA prior to translation by a cleavage reaction catalyzed by snRNPs (small nuclear ribonuclear
proteins which contain RNA and protein). Frequently, the splicing site in the intron has a GU at
the 5’end and an AG at the 3’ end. The snRNP aligns these ends in a lariat formation to allow
precise splicing. Complexes containing the snRNP, mRNA, and associated proteins are called
Spliceosomes.

Translation:

∙ Translation is the production of a polypeptide (protein) using RNA as a template and tRNA
molecules as “adapters” that convert the nucleic acid code to protein code. ∙The nucleotides
(letters) of RNA formed codons (words) that specify a particular amino acid.

∙ The tRNA contains an anticodon that is complementary to the codon and carries a specific
amino acid.

Important elements of the genetic code:

• The code is a triplet code: Each mRNA codon (word) that specifies a particular amino acid in
a polypeptide chain consists of three nucleotides (letters).For example, AAG = lysine

• The code is non-overlapping: The mRNA encoding one protein is read in successive groups
of three nucleotides.

• The code is degenerate: More than one mRNA codon (word) occurs for some amino acids (ie.
AAG and AAA are read as both read as lysine)

• There is more than one tRNA type (therefore more than one anticodon) for some amino acids

• The code has start signals (AUG and rarely GUG) and stop signals (UAA,UAG, and UGA).
Stop signals are also called nonsense codons because they donot designate an amino acid.

Role of mRNA

• carries codons (3-nucleotide sequences) arranged in linear fashion that code for amino acids

• also carries signals needed to tell how to recognize ribosomes, start and stop signals for
decoding protein.
Role of ribosome

• two parts: a small subunit and a large subunit . These are separated except when attached to
m-RNA• ribosomes contain a set of ribosomal proteins and several types of ribosomal RNA (r-
RNA)

• ribosomes catalyze the formation of peptide bonds;

Role of tRNA

• structure: 4 loops, anticodon, AA binding site

• only 73-93 nucleotides long

• extensive hairpin loops

• anticodon site: recognizes codon on mRNA

• Activation of tRNA: adding amino acids

• requires special enzyme: AA-tRNA activating enzymes

Translation Process

INITIATION

• Binding of small ribosomal subunit to mRNA

• Formation of initiation complex

. Initiation factors, 2.GTP, 3.mRNA, 4. 30S Subunit of ribosome

ELONGATION

• Binding of incoming aminoacyl tRNA at a site• Formation of peptide bond

• Translocation

TERMINATION

• Termination occurs when one of the three termination codons moves into the A site

• No tRNA molecules that recognize these termination codons.

• Releasing factors are needed for chain termination