The Cellular Level of

Organization

Levels of Structural Organization

Six levels of organization

2
2 CELLULAR
LEVEL
1 CHEMICAL
1
LEVEL
3 TISSUE
3
LEVEL
Smooth muscle cell

Atoms (C, H, O, N, P)
Smooth muscle tissue
Molecule (DNA)

4 ORGAN
4
LEVEL

Serous
membrane

5
LEVEL
5 SYSTEM
Esophagus
Liver
Stomach
Pancreas
Gallbladder
Small intestine
Large intestine

Digestive system
6 ORGANISMAL LEVEL

Copyright 2009, John Wiley & Sons, Inc.

Smooth muscle
tissue layers
Stomach
Epithelial
tissue

Levels of structural organization

CHEMICAL LEVEL

Basic level

Subatomic particles: constituents of atoms

protons, neutrons & electrons
Atoms: the smallest unit of matter
Essential atoms for life include carbon (C), hydrogen (H),
oxygen (O), nitrogen (N), phosphorus (P), calcium (Ca),
and sulfur

Molecules: two or more atoms joined together

Carbon Dioxide
Glucose
DNA

Levels of structural organization

CELLULAR LEVEL

Molecules combine to form macromolecules

Macromolecules group to form organelles
Several organelles are associated in a cell

Cells are the basic structural and functional units of an

organism
Many kinds of cells in the body
Muscle cells, nerve cells, epithelial cells, etc.

Levels of structural organization

TISSUE LEVEL

Tissues are groups of cells and materials

surrounding them

Four basic types of tissues:

Epithelial
Connective
Muscular
Nervous

Levels of structural organization

ORGAN LEVEL

Tissues form organs

Organs are structures that are composed of two or

more different types of tissues
Specific functions and recognizable shapes

Heart, lungs, kidneys

Stomach is made of several tissues
Serous membrane (outer covering), layer of epithelial tissue and
connective tissue that reduces friction when the stomach moves and
rubs against other organs.
Underneath are the smooth muscle tissue layers, which contract to
churn and mix food and then push it into the next digestive organ, the
small intestine.
The innermost lining is an epithelial tissue layer that produces fluid and
chemicals responsible for digestion in the stomach.

Levels of structural organization

SYSTEM LEVEL

An organ system consists of related organs with

a common function
Organ-system level

Digestive system breaks down and absorbs food

It includes organs such as the mouth, small and large
intestines, liver, gallbladder, and pancreas
Eleven systems of the human body

Levels of structural organization

ORGANISMAL LEVEL

An organism or any living individual

All parts of the body functioning together

Levels of Organization

Subatomic particles - Chemical Level

Atoms - Chemical Level
Molecules - Chemical Level
Macromolecules Chemical Level
Organelles Cellular Level
Cells Cellular Level
Tissues Tissue Level
Organs Organ Level
Organ systems System Level
Organism Organismal level

Characteristics of Living Human

Organism

Basic Life Processes

Distinguish living from non-living things

Six important life process

Metabolism
Responsiveness
Movement
Growth
Differentiation
Reproduction

Metabolism and Responsiveness

Metabolism

Sum of all the chemical process that occur in the body

Catabolism or the breakdown of complex chemical

substances into simpler components
Anabolism or the building up of complex chemical
substances from smaller, simpler components
digestive processes catabolize (split) proteins-amino acids.
amino acids- anabolize (build) new proteins that make up
body structures such as muscles and bones.

Responsiveness

Bodys ability to detect and respond to changes

Decrease in body temperature

Responding to sound
Nerve (electrical signals) and muscle cells (contracting)

Movement and Growth

Movement

Motion of the whole body

Organs, cells, and tiny subcellular structures

Leg muscles move the body from one place to another
After you eat a meal that contains fats, your gallbladder contracts and
squirts bile into the gastrointestinal tract to aid in the digestion of fats

Growth

Increase in body size

Due to an increase in existing cells, number of cells, or both

In a growing bone, for example, mineral deposits accumulate
between bone cells, causing the bone to grow in length and width.

Differentiation and Reproduction

Differentiation

Development of a cell from an unspecialized to

specialized state

Cells have specialized structures and functions that differ from

precursor cells
red blood cells and several types of white blood cells all arise from
the same unspecialized precursor cells in red bone marrow.
Stem cells give rise to cells that undergo differentiation

Reproduction

Formation of new cells (growth, repair, or

replacement)
Production of a new individual

Homeostasis

A condition of equilibrium (balance) in the

bodys internal environment

Dynamic condition
Narrow range is compatible with maintaining life
Example

Blood glucose levels range between 70 and 110 mg of

glucose/dL of blood
Whole body contributes to maintain the internal
environment within normal limits

Control of Homeostasis

Homeostasis is constantly being disrupted

Physical insults

Changes in the internal environment

Drop in blood glucose due to lack of food

Physiological stress

Intense heat or lack of oxygen

Demands of work

Disruptions

Mild and temporary (balance is quickly restored)

Intense and Prolonged (poisoning or severe infections)

Feedback System

Cycle of events

Body is monitored and re-monitored

Each monitored variable is termed a controlled
condition eg blood glucose, body temp, blood
pressure
Stimulus: any disruption that changes controlled
condition

Three Basic components

Receptor
Control center
Effector

Feedback Systems

Receptor

Body structure that monitors changes in a

controlled condition
Sends input to the control center

Nerve ending of the skin in response to temperature

change

Feedback Systems

Control Center

Brain
Sets the range of values to be maintained
Evaluates input received from receptors and
generates output command
Nerve impulses, hormones

Brain acts as a control center receiving nerve impulses

from skin temperature receptors

Feedback Systems

Effector

Receives output from the control center

Produces a response or effect that changes the
controlled condition

Found in nearly every organ or tissue

Body temperature drops the brain sends and impulse to
the skeletal muscles to contract
Shivering to generate heat

Negative and Positive Feedback systems

Negative Feedback systems

Reverses a change in a controlled condition

Regulation of blood pressure (force exerted by blood as

it presses again the walls of the blood vessels)

Positive Feedback systems

Strengthen or reinforce a change in one of the

bodys controlled conditions

Normal child birth

Negative Feedback: Regulation of Blood

Pressure

External or internal stimulus

increase BP

Baroreceptors (pressure sensitive

receptors)

Detect higher BP
Send nerve impulses to brain for
interpretation
Response sent via nerve impulse
sent to heart and blood vessels
BP drops and homeostasis is
restored
Drop in BP negates the original
stimulus

Positive Feedback Systems: Normal

Childbirth

Uterine contractions cause

cervix to open
Stretch-sensitive receptors in
cervix send impulse to brain
Oxytocin is released into the
blood
Contractions enhanced and
baby pushes further down the
uterus
Cycle continues to the birth of
the baby

Homeostatic Imbalances

Normal equilibrium of body processes are

disrupted

Moderate imbalance

A disorder is any abnormality of structure or function.

Disease is a more specific term for an illness
characterized by a recognizable set of signs and
symptoms.
Signs are objective changes such as a fever or swelling
Symptoms are subjective changes such as headache

Severe imbalance

Death

The Cell

Cell

About 200 different types of specialized cells

carry out a multitude of functions that help
each system contribute to the homeostasis of
the entire body.
At the same time, all cells share key
structures and functions that support their
intense activity.
All cells arise from existing cells by the
process of cell division, in which one cell
divides into two identical cells.

A Generalized Cell
1. Plasma membrane
- forms the cells outer boundary
- separates the cells internal environment from the
outside environment
- is a selective barrier-regulates the flow of
materials into and out of cell
- Selectivity helps to maintain the appropriate
environment for normal cellular activities
- plays a role in cellular communication-among
cells, between cells and the external environment

A Generalized Cell
2. Cytoplasm
- all the cellular contents between the plasma
membrane and the nucleus
- cytosol - the fluid portion, mostly water
- organelles - subcellular structures having
characteristic shapes and specific functions
eg ribosomes, ER, Golgi complex,
Lysosomes, peroxisomes and mitochondria

A Generalized Cell
3. Nucleus
- large organelle that contains DNA
- contains chromosomes, each of which
consists of a single molecule of DNA and
associated proteins
- a chromosome contains thousands of
hereditary units called genes

Fig. 3.1 Generalized Body Cell

Plasma Membrane

Flexible yet sturdy barrier

The fluid mosaic model - the arrangement of
molecules within the membrane resembles a
sea of lipids containing many types of
proteins
The lipids act as a barrier to certain
substances
The proteins act as gatekeepers to certain
molecules and ions

Structure of a Membrane

Consists of a lipid bilayer - made up of

phospholipids (75%), cholesterol (20%) and
glycolipids (5%)
Integral proteins - extend into or through the
lipid bilayer
Transmembrane proteins - most integral
proteins, span the entire lipid bilayer
Peripheral proteins - attached to the inner
or outer surface of the membrane, do not
extend through it

Structure of the Plasma Membrane

Structure of a Membrane

Glycoproteins - membrane proteins with a

carbohydrate group attached that protrudes
into the extracellular fluid
Glycocalyx - the sugary coating
surrounding the membrane made up of the
carbohydrate portions of the glycolipids and
glycoproteins

Functions of Membrane Proteins

Some integral proteins are ion channels

Transporters - selectively move substances
through the membrane
Receptors - for cellular recognition; a ligand
is a molecule that binds with a receptor
Enzymes - catalyze chemical reactions
Others act as cell-identity markers

Figure 3.3

Membrane Permeability

The cell is either permeable or impermeable

to certain substances
The lipid bilayer is permeable to oxygen,
carbon dioxide, water and steroids, but
impermeable to glucose
Transmembrane proteins act as channels
and transporters to assist the entrance of
certain substances, for example, glucose and
ions

Passive vs. Active Transport

Passive transport - substances move across

cell membranes without the input of any
energy; use the kinetic energy of individual
molecules or ions
Active transport - a cell uses energy, primarily
from the breakdown of ATP, to move a
substance across the membrane, i.e., against
a concentration gradient

Simple Diffusion, Channel-mediated Facilitated Diffusion, and Carrier-mediated

Facilitated Diffusion

Channel-mediated Facilitated Diffusion of

Potassium ions through a Gated K + Channel

Carrier-mediated Facilitated Diffusion of Glucose across a Plasma Membrane

Extracellular fluid
Glucose
1

Plasma membrane

Glucose
transporter
Glucose
gradient

3
Glucose

Cytosol

Active Transport
Solutes are transported across plasma membranes with the use of energy, from an area
of lower concentration to an area of higher Concentration Sodium-potassium pump

Na+
gradient

Extracellular fluid
Na+/K+ ATPase

Cytosol
K+
gradient

3 Na+ expelled

3 Na+

2K+

ATP

ADP

+
4 2K

imported

Mechanism of action Digitalis

Regulation of cytosolic calcium concentration

Free cytosolic calcium concentrations at the end of

contraction must be lowered for cardiac muscle to relax.
The Na+/Ca2+ exchanger plays an important role in this
process by extruding Ca2+ from the myocyte in exchange
for Na+
The concentration gradient for both ions is a major
determinant of the net movement of ions.
By inhibiting the ability of the myocyte to actively pump Na+
from the cell, cardiac glycosides decrease the Na+
concentration gradient and, consequently, the ability of the
Na+/Ca2+ exchanger to move calcium out of the cell.

Mechanism of action of cardiac glycosides, or digitalis.

ATPase = adenosine triphosphatase.

Transport in Vesicles

Vesicle - a small spherical sac formed by budding off

from a membrane
Endocytosis - materials move into a cell in a vesicle
formed from the plasma membrane
three types: receptor-mediated endocytosis
phagocytosis
bulk-phase endocytosis (pinocytosis)
Exocytosis - vesicles fuse with the plasma
membrane, releasing their contents into the
extracellular fluid
Transcytosis - a combination of endocytosis and
exocytosis

1 Binding

Receptor-Mediated
Endocytosis

Receptor-LDL
complex

The cell uses cholesterol for

rebuilding its membranes and for
synthesis of steroids, such as
estrogen.
Fatty acids and amino acids can
be used for ATP production
or to build other molecules
needed by the cell.

Invaginated plasma
membrane

Clathrin-coated
pit

2 Vesicle formation

Cells take up cholesterol containing:

low-density lipoproteins (LDLs),
transferrin (an iron-transporting
protein in the blood), some vitamins,
antibodies, and certain hormones by
receptor-mediated Endocytosis.

Plasma
membrane

LDL particle
Receptor

Clathrin-coated
vesicle
3 Uncoating

10 Min
Transport
vesicle

Uncoated vesicle
4 Fusion with
endosome

5 Recycling
of receptors
to plasma
membrane

Endosome

Transport
vesicle

Digestive
enzymes
Lysosome

6 Degradation
in lysosome

Pathophysiology of HIV/AIDS
A retrovirus :
1. Cannot replicate outside of living host cells
2. Contains only RNA; no DNA
3. Destroys the bodys ability to fight infections and
certain cancers
4. Infects CD4 cells the primary target of HIV
infection
Patients infected with HIV are at risk for illness and death
from:
1. Opportunistic infections
2. Complications

Pathogenesis
Retrovirus RNA plus reverse transcriptase,
integrase and protease
Attachment: Binds to CD4 receptors (TH)
and chemokine receptors gp 120 or gp 41
Internalization RNA enters the cell
Reverse transcriptase converts RNA DNA
Integrase inserts viral DNA into Host DNA

Viral DNA is transcribed into mRNA

mRNA is translated into protein polyprotein
Cleavage of polyprotein into usable proteins
Viruses are assembled
Host cell is killed as viruses are released
CD4 cells decrease over time.

Phagocytosis

Bulk-phase Endocytosis
Most body cells carry out bulk-phase
endocytosis, the nonselective uptake of
tiny droplets of extracellular fluid.
No receptor proteins are involved;
all solutes dissolved in the
extracellular fluid are brought into
the cell.

EXOCYTOSIS

Exocytosis releases materials from a cell.

All cells carry out exocytosis, but it is
especially important in two types of cells:

secretory cells that liberate digestive enzymes,

hormones, mucus, or other secretions;
nerve cells that release substances called
neurotransmitters

TRANSCYTOSIS

Transport in vesicles may also be used to successively move a

substance into, across, and out of a cell.
In this active process, called transcytosis, vesicles undergo
endocytosis on one side of a cell, move across the cell, and
then undergo exocytosis on the opposite side.
As the vesicles fuse with the plasma membrane, the vesicular
contents are released into the extracellular fluid.
Transcytosis occurs most often across the endothelial cells
that line blood vessels and is a means for materials to move
between blood plasma and interstitial fluid.

Cytoplasm - 2 components
1. Cytosol (55% total cell volume)- intracellular fluid, surrounds the
organelles
- the site of many chemical reactions eg glycolysis ( 10 reactions)
- energy is usually released by these reactions (2 ATP)
- reactions provide the building blocks for cell maintenance,
structure, function and growth
2. Organelles
Specialized structures within the cell
The cytoskeleton - network of protein filaments throughout the cytosol
-provides structural support for the cell
-three types according to increasing size: microfilaments (muscle
contraction, cell division, and cell locomotion), intermediate filaments
(helps to stabilize the position of organelles such as the nucleus), and
microtubules (helps to determine cell shape).

Organelles

Ribosomes - sites of protein synthesis

high content of one type of ribonucleic acid, ribosomal RNA

(rRNA), but each one also includes more than 50 proteins.
Structurally, a ribosome consists of two subunits, one about
half the size of the other

Ribosomes
The large and small subunits
are made separately in the
nucleolus, a spherical body inside
the nucleus.
Once produced, the large and
small subunits exit the
nucleus separately, then come
together in the cytoplasm.

Ribosomes

Some ribosomes are attached to the outer surface of the nuclear

membrane and to an extensively folded membrane called the
endoplasmic reticulum.
These ribosomes synthesize proteins destined for specific
organelles, for insertion in the plasma membrane, or for export from
the cell.
Other ribosomes are free or unattached to other cytoplasmic
structures.
Free ribosomes synthesize proteins used in the cytosol.
Ribosomes are also located within mitochondria, where they
synthesize mitochondrial proteins.

Endoplasmic reticulum

Is a network of membranes in the shape of flattened sacs or

tubules.
The ER extends from the nuclear envelope (membrane
around the nucleus), to which it is connected, throughout the
cytoplasm.
The ER is so extensive that it constitutes more than half of the
membranous surfaces within the cytoplasm of most cells.

Endoplasmic
Reticulum

Rough ER

Rough ER connected to the nuclear envelope, a series of flattened

sacs, surface is studded with ribosomes, produces various proteins
Proteins synthesized by ribosomes attached to rough ER enter
spaces within the ER for processing and sorting.
In some cases, enzymes attach the proteins to carbohydrates to
form glycoproteins.
In other cases, enzymes attach the proteins to phospholipids, also
synthesized by rough ER.
These molecules may be incorporated into the membranes of
organelles, inserted into the plasma membrane, or secreted via
exocytosis.
Thus rough ER produces secretary proteins, membrane proteins,
and many organellar proteins.

Smooth ER

Smooth ER - a network of membrane tubules, does not have

ribosomes,
Smooth ER contains unique enzymes that make it functionally more
diverse than rough ER.
It does synthesize of fatty acids and steroids, such as estrogens and
testosterone.
In liver cells, enzymes of the smooth ER helps to release glucose
into the bloodstream and inactivate or detoxify lipid-soluble drugs or
potentially harmful substances, such as alcohol, pesticides, and
carcinogens
In liver, kidney, and intestinal cells a smooth ER enzyme removes
the phosphate group from glucose-6-phosphate, which allows the
free glucose to enter the bloodstream.
In muscle cells, the calcium ions that trigger contraction are released
from the sarcoplasmic reticulum, a form of smooth ER.

Smooth ER and drug tolerance

Individuals who repeatedly take such drugs, such as the

sedative phenobarbital, develop changes in the smooth ER in
their liver cells.
Prolonged administration of phenobarbital results in
increased tolerance to the drug; the same dose no longer
produces the same degree of sedation.
With repeated exposure to the drug, the amount of smooth
ER and its enzymes increases to protect the cell from its toxic
effects.
As the amount of smooth ER increases, higher and higher
dosages of the drug are needed to achieve the original effect.

Organelles
Golgi apparatus - consists of 3-20 flattened,
membranous sacs called cisternae
The cisternae are often curved, giving the Golgi complex a cuplike
shape.
Most cells have several Golgi complexes, and Golgi complexes are
more extensive in cells that secrete proteins, a clue to the organelles
role in the cell.

- modify, sort, and package proteins for

transport to different destinations
- proteins are transported by various
vesicles

Golgi Complex

Functions

Modifies, sorts, packages, and transports

proteins received from the rough ER.
Forms secretory vesicles that discharge
processed proteins via exocytosis into
extracellular fluid; forms membrane vesicles that
ferry new molecules to the plasma membrane;
forms transport vesicles that carry molecules to
other organelles, such as lysosomes.

Processing and packaging of proteins by the Golgi

complex
1.

2.

3.

Proteins synthesized by ribosomes on the rough ER

are surrounded by a piece of the ER membrane,
which eventually buds from the membrane surface
to form transport vesicles.
Transport vesicles move toward the entry face of
the Golgi complex.
Fusion of several transport vesicles creates the
entry face of the Golgi complex and releases
proteins into its lumen (space).

Processing and packaging of proteins by the Golgi

complex
The proteins move from the entry face into one or
more medial cisternae.

4.

Enzymes in the medial cisternae modify the proteins to

form glycoproteins, glycolipids, and lipoproteins.
Transfer vesicles that bud from the edges of the cisternae
move specific enzymes back toward the entry face and
move some partially modified proteins toward the exit
face.

Processing and packaging of proteins by the Golgi

complex
5.

6.

7.

The products of the medial cisternae move into the

lumen of the exit face.
Within the exit face cisterna, the products are
further modified and are sorted and packaged.
Some of the processed proteins leave the exit face
and are stored in secretory vesicles. These vesicles
deliver the proteins to the plasma membrane,
where they are discharged by exocytosis into the
extracellular fluid. For example, certain pancreatic
cells release the hormone insulin in this way.

Processing and packaging of proteins by the Golgi

complex
8.

Other processed proteins leave the exit face in

membrane vesicles that deliver their contents to
the plasma membrane for incorporation into the
membrane. In doing so, the Golgi complex adds
new segments of plasma membrane as existing
segments are lost and modifies the number and
distribution of membrane molecules

Processing and packaging of proteins by the Golgi

complex
9.

Finally, some processed proteins leave the exit face

in transport vesicles that will carry the proteins to
another cellular destination. For instance, transport
vesicles carry digestive enzymes to lysosomes

Processing and Packaging of Proteins

Synthesized
protein

Ribosome

Transport vesicle

Entry face cisterna

2
Medial cisterna
3
Exit face cisterna
9

Transport vesicle
(to lysosome)

4
8
Rough ER
6

Transfer vesicle
4

7
5

Transfer vesicle
Secretory
vesicle

Membrane
vesicle

Proteins in vesicle
membrane merge
with plasma
membrane
Proteins exported
from cell by exocytosis
Plasma
membrane

Lysosomes
vesicles that form from the Golgi complex,
contain powerful digestive enzymes
60 kinds of enzymes- digestive and hydrolytic
eznymes- breakdown of molecule
Acidic ph of 5100 times greater than cytosol
Active transport pump- H+ Ions movement
The lysosomal membrane also includes
transporters that move the final products of
digestion, such as glucose, fatty acids, and
amino acids, into the cytosol

Lysosomes

Lysosomal enzymes also help recycle worn-out cell

structures.
A lysosome can engulf another organelle, digest it,
and return the digested components to the cytosol
for reuse. In this way, old organelles are continually
replaced.
The process by which entire worn-out organelles are
digested is called autophagy

Lysosomes

In autophagy, the organelle to be digested is enclosed by

a membrane derived from the ER to create a vesicle
called an autophagosome; the vesicle then fuses with a
lysosome.
In this way, a human liver cell, for example, recycles
about half of its cytoplasmic contents every week.
Autophagy is also involved in cellular differentiation,
control of growth, tissue remodeling, adaptation to
adverse environments, and cell defense.

Lysosomes

Lysosomal enzymes may also destroy the entire cell that

contains them, a process known as autolysis
Autolysis occurs in some pathological conditions and also
is responsible for the tissue deterioration that occurs
immediately after death.
However, some operate in extracellular digestion. One
example occurs during fertilization.
The head of a sperm cell releases lysosomal enzymes
that aid its penetration of the oocyte by dissolving its
protective coating in a process called the acrosomal
reaction

Tay-Sachs Disease

Some disorders are caused by faulty or absent lysosomal enzymes.

It is an inherited condition characterized by the absence of a single
lysosomal enzyme called Hex A.
This enzyme normally breaks down a membrane glycolipid called
ganglioside GM2 that is especially prevalent in nerve cells.
As the excess ganglioside GM2 accumulates, the nerve cells function
less efficiently.
Children with Tay-Sachs disease typically experience seizures and
muscle rigidity.
They gradually become blind, demented, and uncoordinated and
usually die before the age of 5.
Tests can now reveal whether an adult is a carrier of the defective
gene

Organelles

Proteasomes

continuously destroy unnecessary, damaged, or faulty

proteins, found in the cytosol and the nucleus
proteins that are part of metabolic pathways are degraded
after they have accomplished their function.
Such protein destruction plays a part in negative feedback by
halting a pathway once the appropriate response has been
achieved.

Proteasomes

Proteasomes were so named because they contain proteases,

enzymes that cut proteins into small peptides.
Once the enzymes of a proteasome have chopped up a protein into
smaller chunks, other enzymes then break down the peptides into
amino acids, which can be recycled into new proteins.
Some diseases could result from failure of proteasomes to degrade
abnormal proteins.
For example, clumps of misfolded proteins accumulate in brain cells
of people with Parkinson disease and Alzheimer disease.
Discovering why the proteasomes fail to clear these abnormal
proteins is a goal of ongoing research.

Peroxisomes

Smaller than lysosomes, detoxify several toxic substances

such as alcohol, abundant in the liver
By product of the oxidation reactions is hydrogen peroxide
(H2O2), a potentially toxic compound.
Peroxisomes also contain the enzyme catalase, which
decomposes H2O2.
Because production and degradation of H2O2 occur within
the same organelle, peroxisomes protect other parts of the
cell from the toxic effects of H2O2.

Organelles
Mitochondria - the powerhouses of the cell

Generate ATP
More prevalent in physiologically active cells: muscles, liver and
kidneys- liver cell 1700
Inner and outer mitochondrial membranes
Cristae - the series of folds of the inner membrane
Matrix - the large central fluid-filled cavity
Self-replicate during times of increased cellular demand or before
cell division

Mitochondria

Mitochondria

Like peroxisomes, mitochondria self-replicate, a process that occurs

during times of increased cellular energy demand or before cell
division.
Synthesis of some of the proteins needed for mitochondrial
functions occurs on the ribosomes that are present in the
mitochondria matrix.
Mitochondria even have their own DNA, in the form of multiple
copies of a circular DNA molecule that contains 37 genes.
These mitochondrial genes control the synthesis of 2 ribosomal
RNAs, 22 transfer RNAs, and 13 proteins that build mitochondrial
components

Nucleus

Spherical or oval shaped structure

Usually most prominent feature of a cell
Nuclear envelope - a double membrane that separates
the nucleus from the cytoplasm
Nuclear pores - numerous openings in the nuclear
envelope, control movement of substances between
nucleus and cytoplasm

Nucleolus

Inside the nucleus are one or more spherical bodies called

nucleoli; singular is nucleolus that function in producing
ribosomes.
Each nucleolus is simply a cluster of protein, DNA, and RNA; it
is not enclosed by a membrane.
Nucleoli are the sites of synthesis of rRNA and assembly of
rRNA and proteins into ribosomal subunits.
Nucleoli are quite prominent in cells that synthesize large
amounts of protein, such as muscle and liver cells.

Chromatin

Within the nucleus are most of the cells hereditary units, called
genes, which control cellular structure and direct cellular activities.
Genes are arranged along chromosomes. Human somatic (body)
cells have 46 chromosomes, 23 inherited from each parent.
Each chromosome is a long molecule of DNA that is coiled together
with several proteins
This complex of DNA, proteins, and some RNA is called chromatin.
The total genetic information carried in a cell or an organism is its
genome.

Histones
(proteins)
DNA double helix
Nucleosome
Chromatin
Chromatin fiber

Chromatid

Chromosome

Histones

Histones are highly conserved basic proteins which

associate with DNA with a definite stoichiometry to
constitute the 'nucleosome' that are essential for the
packaging of the genomic DNA into compact structures.
There are 5 types of histones namely H2A, H2B, H3, H4
and H1 linker histone.
Within a nucleosome, they exist as two dimers of (H2AH2B) and a complex of (H32-H42) ultimately forming an
octamer.
H1 linker histone associates externally with the
nucleosome and helps in further compaction of the
chromatin structure.
Copyright 2009 John Wiley & Sons, Inc.

Histones

Histone proteins have a characteristic 'histone fold

domain' which consists of structural motif called helixturn-helix' that consists of 3 alpha helices connected by
loops.
Each histone perfectly fits with the counterpart to form a
heterodimer that structurally gives an appearance of a
hand-shake.
Histones are buried inside the core structure of the
nucleosome.
N-terminal tails of all histones are particularly of interest
since they protrude out of the compact structure.

Copyright 2009 John Wiley & Sons, Inc.

Histones

These N-terminal tails are often subjected to a variety of

post-translational modifications such acetylation,
methylation, phosphorylation, ubiquitination etc.
The combinations of these marks on histones determine
which factors bind to that region of DNA and eventually
regulate the expression status of the particular locus.
Histone genes are typically transcribed from multiple loci
which occur as distinct clusters on different
chromosomes.

Copyright 2009 John Wiley & Sons, Inc.

Histones

In all, histones make five types of interactions with DNA:

Helix-dipoles form alpha-helixes in H2B, H3, and H4 cause a net

positive charge to accumulate at the point of interaction with
negatively charged phosphate groups on DNA
Hydrogen bonds between the DNA backbone and the amide
group on the main chain of histone proteins
Nonpolar interactions between the histone and deoxyribose
sugars on DNA
Salt bridges and hydrogen bonds between side chains of basic
amino acids (especially lysine and arginine) and phosphate
oxygens on DNA
Non-specific minor groove insertions of the H3 and H2B Nterminal tails into two minor grooves each on the DNA molecule

The highly basic nature of histones, aside from facilitating DNA-histone

interactions, contributes to their water solubility.
Copyright 2009 John Wiley & Sons, Inc.

Post-translational Modification of Histone Nterminal Tails

94

Amino Acid

Lysine (k)

Modifications
Accetylation
SUMOylation
Ubiquitation
Biotinylation
Methylation
Citrullination

Glutamic acid (E)

ADP-ribosylation

Threonine (T)

Phosphorylation

Proline (P)

Amino Acid

Arginine (R)

Serine (S)

Cis-trans
isomerization

95

Histone proteins on 14% SDS-PAGE

H3
H2A
H2B
H4

Protein Estimation

Principle

The principle behind the Lowry method of determining

protein concentrations lies in the reactivity of the peptide
nitrogen[s] with the copper [II] ions under alkaline
conditions.
The subsequent reduction of the Folin-Ciocalteay
phosphomolybdic phosphotungstic acid to
heteropolymolybdenum blue by the copper-catalyzed
oxidation of aromatic acids.
The Lowry method is sensitive to pH changes and
therefore the pH of assay solution should be maintained
at 10 - 10.5

Principle

The Folin reagent is added under alkaline

conditions, and the Folin reagent is subsequently
reduced by the Copper ions as well as Tyr, Trp
and polar amino acid side chains
Folin- Ciocalteau reagent which consists of
sodium tungstate molybdate and phosphate.
Thus the intensity of color depends on the
amount of these aromatic amino acids present
and will thus vary for different proteins.

Principle

Reagents
1.
2.
3.

Alkaline Sodium Bicarconate solution

CuSO4 solution
Potassium Sodium tartrate
Biuret reagent: 1+2+3 (100:1:1)
Folin Reagent: 1:1
BSA standard

SDS PAGE

Introduction
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique used in biochemistry,
genetics and molecular biology to separate proteins
according to their molecular weight
The electrophoretic mobility of proteins depends upon
their size
The purpose of SDS-PAGE is to separate proteins
according to their size
As proteins are amphoteric compounds, their net
charge can therefore be determined by the pH of the
medium in which they are suspended

Introduction
At a given pH and under non-denaturing conditions,
the electrophoretic separation of proteins is
determined by both size and charge of molecules.
As proteins are high molecular weight molecules, it
needs porous gels to get separated.
Polyacrylamide gels are those which provide a means
of separating proteins by size as they are porous.
Polyacrylamide is the best gel recommended to
provide such an environment.

Principle
To separate different protein molecules of different
shapes and sizes, they first have to be denatured so
that the proteins no longer have any secondary,
tertiary or quaternary structure.
Sodium dodecyl sulphate (SDS) is an anionic detergent
which denatures proteins by wrapping around the
polypeptide backbone.
SDS denatures all the proteins to their respective
primary structure.
SDS confers a negative charge to the polypeptide in
proportion to its length.

Principle

SDS treatment has two important features:

All proteins retain only their primary structure.
All proteins have a large amount of negative
charge.

Principle
Polyacrylamide is a synthetic gel which is thermo-stable,
transparent, strong and relatively chemically inert and
can be prepared with a wide range of average pore sizes.
It can withstand high voltage gradients and is feasible to
various staining and destaining procedures and can be
digested to extract separated fractions or dried for
autoradiography and permanent recording.
A polymer gel is formed of acrylamide monomers and
the proteins are run through this gel by electrophoresis,
hence this entire process is called Polyacrylamide Gel
Electrophoresis (PAGE).

SDS PAGE
There are two layers of gel, namely Stacking or spacer
gel, and Separating or resolving gel.
Stacking gel - The stacking gel contains large pores of
polyacrylamide gel (generally 5%).
This gel is prepared with Tris buffer of pH 6.8 which is of
about 2 pH units lower than that of the electrophoresis
buffer.
This gel is formed over the separating gel.
Separating Gel - The separating gel contains small pores
of polyacrylamide gel (5-30%).
The Tris buffer used is of pH 8.8. In this gel, macro
molecules separate according to their size.

Materials- SDS PAGE

Tris:
It is used as a buffer because it is an innocuous substance to
most proteins. Its pKa is 8.3 at 20 degree C and reasonably a
very satisfactory buffer in the pH range 7.0 9.0.
Acrylamide:
This is a white crystalline powder and while dissolving in water,
autopolymerisation takes place.
It is a slow spontaneous process by which acrylamide molecules
join together by head on tail fashion.
But in presence of free radicals generating system, acrylamide
monomers are activated into a free-radical state.
These activated monomers polymerise quickly and form long
chain of polymers.
This kind of reaction is known as Vinyl addition polymerisation.

Materials- SDS PAGE

Bisacrylamide (N,N-Methylenebisacrylamide):
Bisacrylamide is the most frequently used cross linking agent
for polyacrylamide gels.
Chemically it has two acrylamide molecules coupled head to
head fashion at their non-reactive ends.
Ammonium Persulphate (APS):
APS is an initiator for gel formation.
N, N, N, N-tetramethylethylenediamine (TEMED):
Chemical polymerisation of acrylamide gel is used for SDSPAGE.
It can be initiated by ammonium persulfate and the
quaternary amine, N,N,N,N- tetramethylethylenediamine
(TEMED).

Materials- SDS PAGE

Sodium Dodecyl Sulphate (SDS):

SDS is the most common denaturing agent used to denature

native proteins to individual polypeptides.
When a protein mixture is heated to 100C in presence of
SDS, the detergent wraps around the polypeptide backbone.
It binds to polypeptides in a constant weight ratio of 1.4 g/g
of polypeptide.
In this process, the intrinsic charges of polypeptides become
negligible when compared to the negative charges
contributed by SDS.
Thus, polypeptides after treatment become a rod like
structure possessing a uniform charge density, that is same
net negative charge per unit length.

Preparation
Preparation of 12% Separating Gel30% Acrylamide-bisacrylamide Solution - 6 ml
Distilled water - 3 ml
2.5X Tris-SDS Buffer (pH 8.8) - 6 ml
10% APS Solution - 125 l
TEMED - 7.5 l
Pour the gel in-between the plates and allow it to
solidify for an hour.
Immediately after the gel is poured, add distilled
water to level the gel.

Preparation
Preparation of 5% Stacking Gel

30% Acrylamide-bisacrylamide Solution - 1.3 ml

Distilled water* - 5.1 ml
5X Tris-SDS Buffer (pH 6.8) - 1.6 ml
10% APS Solution - 75 l
TEMED - 15 l

After addition of TEMED gently mix all the components

by swirling the beaker.
Pour the stacking gel on top of the separating gel and
immediately place the comb avoiding air bubbles.
Allow it to solidify for 30 minutes

Flow Chart

Protein Separation

H3
H2A
H2B
H4

Problems

Problems

Electrophoretic transfer
Electrophoretic transfer proteins separated in
gels (for example, following polyacrylamide gel
electrophoresis, or PAGE), are transferred onto
membranes by electrophoretic transfer
Microfiltration (dot blotting) proteins in
solution are bound to membranes by vacuum
driven microfiltration. This rapid approach is
useful for determining working conditions for a
new blotting assay or any other situation where
the resolving power of gel electrophoresis is not
needed

Electrophoretic transfer

Electrophoretic Transfer Principle

Proteins migrate to the membrane following a current (I) that is generated
by applying a voltage (V) across the electrodes* following Ohm's law:
V=IxR
R is the resistance generated by the materials placed between the
electrodes (that is, the transfer buffer, gel, membrane, and filter papers).
The electric field strength (E, measured in V/cm) that is generated
between the electrodes is the driving force for transfer.
Both the applied voltage and the distance between the electrodes then
play a major role in governing the rate of elution of the proteins form the
gel.
A number of other factors, including the size, shape, and charge of the
protein, the pH, viscosity, and ionic strength of the transfer buffer, and the
composition of the gel, also influence the elution of particular proteins
from gels.
Proteins denatured with sodium dodecyl sulfate (SDS) carry a net negative
charge and migrate toward the anode.

Electrophoretic Transfer Systems

Tank transfer systems
gels and membranes are submerged in transfer
buffer in tanks; these systems are useful for most
routine protein work, for efficient and quantitative
protein transfers, and for transfers of proteins of
all sizes.
Tank transfer systems offer the most flexibility in
choosing voltage settings, blotting times, and
cooling options

Electrophoretic Transfer Systems

Semi-dry systems
gels and membranes are sandwiched between
buffer-saturated filter papers that are in direct
contact with plate electrodes; these systems are
typically easier to set up than tank systems and
are useful when high-throughput is necessary and
extended transfer times are not required, or when
discontinuous buffer systems are used.
Active cooling options are limited with semi-dry
blotting

Membranes
Selecting the appropriate membrane, transfer
buffer, and power conditions is critical to
successful protein transfer.
The size and charge of the proteins, the transfer
method, and the binding properties of the
membrane must all be considered
Though nitrocellulose was once the only choice
for protein blotting, advances in membrane
chemistries have made a variety of membrane
types available, each offering key attributes to
suit particular experimental conditions

Membranes
Membranes are commonly available in two
pore sizes: the 0.45 m pore size membranes
are recommended for most analytical blotting
experiments, while the 0.2 m pore size
membranes are most suitable for transfer of
low molecular weight (<15,000) proteins that
might move through larger membrane pores.

Nitrocellulose
Nitrocellulose was one of the first membranes
used for western blotting and is still a popular
membrane for this procedure.
Protein binding to nitrocellulose is
instantaneous, nearly irreversible, and
quantitative up to 80 to 100 g/cm2.
Nitrocellulose is easily wetted in water or
transfer buffer and is compatible with a wide
range of protein detection systems.

Nitrocellulose

Polyvinylidene Difluoride
(PVDF) Membrane
PVDF membrane is an ideal support for N-terminal
sequencing, amino acid analysis, and immunoassay of
blotted proteins.
PVDF retains proteins during exposure to acidic or
basic conditions and in the presence of organic
solvents.
PVDF membrane exhibits better binding efficiency of
electro blotted material in the presence of SDS in the
transfer buffer.
PVDF membrane must be wetted in 100% methanol
prior to use but may be used with a transfer buffer that
contains no methanol

Transfer Buffer
Sr.
No.
1.
2.
3.
4.

Ingredients
Tris

Gycine

Methanol / SDS
Distilled Water

Quantity
1.5 g
7.2 g

50 mL / 50 mg
Upto 500 mL

Always store under refrigeration

Protein Detection

Detection Methods
Once proteins have been transferred to a
membrane, they can be visualized using a
variety of specialized detection reagents
Total protein stains allow visualization of the
protein pattern on the blot and immunological
detection methods, employing antibody or
ligand conjugates, allow visualization of
specific proteins of interest

Total Protein Staining

Total protein staining of a membrane provides
a visual image of the complete protein pattern

Anionic Dyes
The first techniques developed for total protein
staining for staining proteins in polyacrylamide gels.
Dyes that became popular for detecting proteins
bound to membranes include Amido Black, Coomassie
Brilliant Blue R-250, Ponceau S, and Fast Green FCF
Of the anionic dyes used, Amido Black and Ponceau S
are the best choices because they destain rapidly in
distilled water or low concentrations of methanol and
produce very little background staining.

Anionic Dyes
These dyes are easy to prepare and they stain
proteins quickly, but they are relatively insensitive
when compared to immunological detection
assays.
The stains that require alcohol-containing
solutions (for example, Amido Black, Coomassie
Brilliant Blue, Fast Green FCF) for solubility can
shrink nitrocellulose membranes, making direct
comparison of an immunologically detected
antigen to the total protein on the stained
membrane difficult.

Colloidal Gold
An alternative to anionic dyes that provides
detection sensitivities that rival those of
immunological detection methods is colloidal
gold
When a solution of colloidal gold particles is
incubated with proteins bound to a nitrocellulose
or PVDF membrane, the gold binds to the
proteins through electrostatic adsorption.
The resulting gold-protein complex produces a
transient, reddish-pink color due to the optical
properties of colloidal gold.

Fluorescence
Fluorescent protein stains like SYPRO Ruby,
SYPRO Red, SYPRO Orange, and Deep Purple
provide highly sensitive detection of proteins on
blots as well as in gels.
SYPRO Ruby protein blot stain allows detection as
low as 28 ng.
After staining, target proteins can be detected by
colorimetric or chemiluminescent
immunodetection methods, or analyzed by micro
sequencing or mass spectrometry with no
interference from the protein stain.

Immunological Detection Systems

Immunological detection
systems are used to
identify specific proteins
blotted to membranes.
Though there are
numerous systems
available for
immunological
detection, the basic
procedure for these
assays varies very little.

Immunological Detection Systems

The primary antibody is specific for the protein of interest,
and the secondary antibody enables its detection
The secondary antibody can be radiolabeled, labeled with a
fluorescent compound or gold particles, or conjugated to
an enzyme like AP or HRP.
For many years, radiolabeled secondary antibodies were
the method of choice for detection, but newer methods
have evolved that are less hazardous and easier to use than
radioactivity, yet maintain the same degree of sensitivity.
Available detection methods now include in addition to
autoradiography of radiolabeled probes colorimetric,
chemiluminescent, bioluminescent, chemifluorescent,
fluorescent, and immunogold detection

Blocking Reagents
Following transfer, unoccupied binding sites on the
membranes must be blocked to prevent nonspecific binding
of probes; failure to adequately saturate the membrane can
lead to high background, since many probes are also proteins,
and can also bind to the membrane.

Antibody Incubations
A typical experimental system utilizes two layers of
antibody in the detection procedure.
The primary antibody is directed against the target
antigen; the antigen may be a ligand on a protein, the
protein itself, a specific epitope on a protein, or a
carbohydrate group.
The secondary antibody is specific for the primary
antibody; it is usually conjugated to an enzyme such as
alkaline phosphatase (AP) or horseradish peroxidase
(HRP), and an enzyme-substrate reaction is part of the
detection process

Primary Antibodies
The primary antibody recognizes and binds to the target
antigen on the membrane.
For incubations with primary antibody, the entire blot
must be covered with antibody containing solution.
The appropriate concentration or dilution (titer) of the
primary antibody must be determined empirically for
each new lot of primary antibody
The optimal antibody concentration is usually considered
to be the greatest dilution of antibody still resulting in a
strong positive signal without background or nonspecific
reactions.

Species-Specific Secondary
Antibodies
Blotting-grade species-specific secondary antibodies are
the detection reagents of choice.
Secondary antibodies are specific for the isotype (class)
and the species of the primary antibody (for instance, a
goat anti rabbit secondary antibody is an antibody
generated in goat for detection of a rabbit primary
antibody).
Secondary antibodies bind to multiple sites on primary
antibodies to increase detection sensitivity.

Washes
Between the two antibody incubations and prior to
detection, the blot must be washed to remove excess
antibody to prevent nonspecific binding.
Though the washing solutions and times may vary,
depending on the antibodies and detection systems
used, washes generally utilize tris-buffered saline
(TBS) or TBS with additional detergent (Tween 20;
TTBS).

Detection Methods
Blotted proteins are generally detected using
secondary antibodies that are labeled with
radioisotopes or colloidal gold, or conjugated to
fluorophores, biotin, or an enzyme like HRP or AP.
Early blotting systems used 125I-labeled reagents similar
to those used in radioimmunoassay.
These systems provide sensitive results, but the special
handling and disposal problems of 125I reagents have
discouraged continued use of this technique.
Instead, a number of enzyme systems and detection
reagents evolved

Detection Methods

H2O2 induced activation of ERK, p38 and Akt in a time dependent manner.

Protein Synthesis
Cell
DNA

Transcription

mRNA
Translation
Ribosome

Polypeptide
(protein)

Protein Structure

Made up of amino acids

Polypeptide- string of amino acids
20 amino acids are arranged in different
orders to make a variety of proteins
Assembled on a ribosome

Questions to be answered

How do we get from the bases found in DNA

to amino acids?
How do we get from a bunch of amino acids
to proteins?

DNA and RNA

Nucleic acids are huge organic molecules that contain

carbon, hydrogen, oxygen, nitrogen, and phosphorus.
Deoxyribonucleic acid (DNA) forms the genetic code
inside each cell and thereby regulates most of the
activities that take place in our cells throughout a
lifetime.
Ribonucleic acid (RNA) relays instructions from the
genes in the cells nucleus to guide each cells assembly
of amino acids into proteins by the ribosomes.
The basic units of nucleic acids are nucleotides,
composed of a nitrogenous base, a pentose, sugar, and
a phosphate group.

In 1953 three papers appeared in a single issue of Nature

providing support for a double helical structure. Two of these,
one by Maurice Wilkins and colleagues and the other by
Rosalind Franklin and Raymond Gosling, presented impressive
evidence based on X-ray diffraction studies.
In the third paper, James Watson and Francis Crick succinctly
proposed that DNA is made up of two helical chains held
together by hydrogen bonds between complementary purine
and pyrimidine bases pointing inwards from a phosphate
sugar backbone.
They observed that the most plausible three-dimensional
structure would involve exclusive pairing of adenine with
thymine and cytosine with guanine.

They also observed, in a masterpiece of understatement, that

their proposed structure suggests a possible copying
mechanism for the genetic material
The message of this manuscript was contained within less
than a single published page and contained only a single
illustration, consisting of a simple diagrammatic
representation of the double helix as drawn by Francis Cricks
wife
In recognition of their achievements, Watson and Crick,
together with Wilkins, were awarded the Nobel prize for
medicine in 1962.

RNA Structure

Differs from DNA

single stranded
ribose sugar not deoxyribose sugar
uracil nitrogenous base replaces thymine

Types of RNA within the cell, each with

a specific function

messenger RNA
ribosomal RNA
transfer RNA

Central Dogma
Flow of Information:
DNA

RNA
Transcription

Proteins
Translation

1. Transcription is the process by which a molecule of DNA

is copied into a complementary strand of RNA.
2. This is called messenger RNA (mRNA) because it acts as
a messenger between DNA and the ribosomes where
protein synthesis is carried out

The regulation of transcription

Transcription is initiated when proteins known as
transcription factors bind to the promoter region and to
other upstream regulatory regions such as the enhancers.
It can also be initiated more autonomously in selected genes,
such as - and -globin, by activity at an upstream locus
control region (LCR).
The relevant transcription factors contain specific structural
domains, such as leucine zippers and zinc fingers, which bind
to the regulatory regions that become exposed in surface
folds of the double helix through the process of chromatin
remodeling.

The regulation of transcription

This involves a change in the structure of the basic unit of DNA
packaging, known as a nucleosome, resulting in strands of DNA
which had previously been concealed becoming unfolded.
There is increasing evidence that changes in chromatin
structure play a key role in gene expression.
Chromatin exists in two forms, euchromatin and
heterochromatin, which differ in that euchromatin is loosely
packed whereas heterochromatin is tightly condensed.
Euchromatin is transcriptionally active and Heterochromatin is
transcriptionally inactive

RNA Polymerase II
RNA polymerase II (also called RNAP II and Pol II) is an
enzyme found in eukaryotic cells.
It catalyzes the transcription of DNA to synthesize
precursors of mRNA and most snRNA and microRNA.
A 550 kDa complex of 12 subunits, RNAP II is the most
studied type of RNA polymerase.
A wide range of transcription factors are required for
it to bind to upstream gene promoters and begin
transcription.

RNA Polymerase II
Small nuclear ribonucleic acid (snRNA), also commonly
referred to as U-RNA, is a class of small RNA molecules that are
found within the nucleus of eukaryotic cells.
The length of the an average snRNA is approximately 150
nucleotides. They are transcribed by either RNA polymerase II
or RNA polymerase III, and studies have shown that their
primary function is in the processing of pre-mRNA (hnRNA) in
the nucleus.
A microRNA (abbr. miRNA) is a small non-coding RNA molecule
(ca. 22 nucleotides) found in plants and animals, which
functions in transcriptional and post-transcriptional regulation
of gene expression

Transcription
Transcription commences at a transcriptional initiation site and
involves the synthesis of a single strand of messenger RNA
(mRNA) by RNA polymerase, proceeding in a 5 to 3 direction
This 5 to 3 orientation refers to the coding or sense strand.
The DNA used as a template for this synthesis is the non-coding
or antisense strand. This is transcribed in a 3 to 5 direction.
The net effect is the synthesis of a primary RNA transcript
consisting of a strand of mRNA which is an exact replica of the
sense strand, with the exception that uracil replaces thymine.
The primary transcript is then processed to produce the mature
mRNA for transportation into the cytoplasm.

Transcription
Processing involves:
the splicing out of the noncoding introns, and the addition of
a protective 5 guanine nucleotide to form a 5 cap (the 5
cap is a specially altered nucleotide on the 5 end of
precursor messenger RNA and some other primary RNA
transcripts as found in eukaryotes. The process of 5 capping
is vital to creating mature messenger RNA, which is then
able to undergo translation)
and of adenine residues at the 3 end to form a poly-A tail
(The poly-A tail is a long chain of adenine nucleotides that is
added to a messenger RNA (mRNA) molecule during RNA
processing to increase the stability of the molecule).

Transcription
The splicing out of the introns requires recognition of the
GT and AG dinucleotides which mark the beginning and
end of most intron boundaries.
These are known as the splice donor and splice acceptor
sites respectively.
Mutations which involve these dinucleotides, so-called
splice-site mutations, can have a serious effect on
protein structure and function.

Transcription is donewhat
now?
Now we have mature mRNA transcribed from
the cells DNA. It is leaving the nucleus
through a nuclear pore. Once in the
cytoplasm, it finds a ribosome so that
translation can begin.
We know how mRNA is made, but how
do we read the code?

TranslationmRNA to protein
The mRNA passes through the nuclear membrane to the ribosomes
in the cytoplasm, where it provides a template for the synthesis of a
polypeptide chain.
Ribosomes are made up of a complex of protein and ribosomal RNA
(rRNA) which catalyses the synthesis of the polypeptide.
This is achieved by binding of mRNA to transfer RNA (tRNA) on the
surface of the ribosome.
Each tRNA contains a set of three nucleotide bases, referred to as an
anticodon, which complement a set of three bases in mRNA known
as a codon.
Each codon is specific for an amino acid. Thus each tRNA conveys a
specific amino acid residue to its position on the mRNA template,
resulting in the sequential synthesis of the encoded polypeptide
chain.

The degenerate genetic code

A codon consists of three bases which code for a specific amino acid.
As there are four bases, there are 4 4 4 = 64 possible codons.
These represent the genetic code. The code is described as being
degenerate because there are 64 codons but only 20 amino acids,
so several different codons specify the same amino acid.
Translation always begins at a codon for methionine (AUG), which is
often then removed before the completion of synthesis of the
polypeptide.
This is referred to as the initiator codon, and it determines the
reading frame of the mRNA.
Three combinations (UAA, UAG, UGA) specify stop codons, also
known as nonsense or termination codons. These signal the end of
translation.