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!

H2 BIOLOGY
CORE SYLLABUS I

Topics covered:
1
...
Eukaryotic gene expression

2
...
Eukaryotic control/org

3
...
Mutations

4
...
Cancer

5
...
Genetics of viruses

6
...
Genetics of bacteria

!

Chapter 1: Cell Structure and Function
INTRODUCTION
Eukaryotic cells have a plasma membrane on their outer surface, and extensive and elaborately
arranged internal membranes that compartmentalize the cell
Cell

Cytoplasm

Cytosol

Membranous

Nucleus

Organelles
Nonmembranous

Cell
membrane

Cell wall

Membranous Organelles
Endoplasmic reticulum, Golgi apparatus, Lysosomes,
Vesicles, Mitochondria, Chloroplasts, Nucleus
Non-membranous Organelles
Centrioles (animals only)

Cytoplasm:
Cytosol is an aqueous solute rich matrix that contains:
• Various essential ions and soluble organic molecules (sugars and aa)
• Soluble proteins (enzymes)
• Cytoskeleton
MEMBRANOUS ORGANELLES
Nucleus:
Contains most of the genetic material in a cell and helps regulate all cellular activities
...

It is also the site of peptide bond formation
Found in
• Attached to rough ER
• Free in the cytosol
• Mitochondrial matrix
• Chloroplast stroma
Centrioles:
Only found in eukaryotic cells, in a region known as the centrosome
• Found in pairs at right angles to each other
• Consist of nine triplets of microtubule arranged in a ring
• Act as MTOC: microtubule organizing centers for spindle apparatus

CELL FRACTIONATION
Homogenization:
First step to cell fractionation to break tissues intro small fragments to release organelles to isolate
them for studies
It is done in:
• Isotonic medium containing sucrose, mannitol or sorbitol
• Buffer solution ! maintain suitable pH
• Temperature of 4oC ! inhibit protease activity
Differential centrifugation:
Centrifugal force is used where spinning an object about a point generates it
• Particles spun down to the bottom of the tube forms the pellet
• Remaining fluid called the supernatant
o Contains smaller and more soluble particles
1st level
Nuclei

2nd level
Mitochondria
Chloroplasts
Lysosomes
Peroxisomes

3rd level
Plasma
membrane
Fragments of ER

4th level
Ribosomal
subunit
Small
polyribosomes

5th level
Cytosol

AUTORADIOGRAPHY
Used to identify the synthesis and cellular distribution sites of metabolic products by tagging
specimen molecule with radioisotopes
~ END ~

Chapter 2: Biomolecules
SECTION 1: LIPIDS
Water:
• Universal solvent
• H-bond: bond formed between H atom and electronegative atom (N, O, F)
• Dissolves through electrostatic interaction with H2O molecules
Introduction:
Hydrophilic – affinity for water, readily dissolve in it
Hydrophobic – non-polar and are not very soluble in water
Lips are generally soluble in non-polar solvents and insoluble in water
SIMPLE LIPIDS (TRIGLYCERIDES)
Simple lipids usually contain an alcohol (usually glycerol) linked to one or more fatty acids via an
ester linkage
Structure of glycerol:
Contains three-carbon alcohol, with each carbon bearing a hydroxyl (OH) group
Structure of fatty acid:
Contains carboxylic acids (COOH) attached to long hydrocarbon tail
Lengths of fatty acids differ in:
• Length of hydrocarbon – length proportional to hydrophobicity
• Number and locations of double bonds
o Saturated: no double bonds
o Unsaturated: One or more double C=C bonds, contains a kink
Formation of triglycerides:
Formed when glycerol is linked to one or more fatty acids, resulting in the formation of an ESTER
linkage
...
Melting point of fats increases with hydrocarbon chain length
• Longer hydrocarbon chains, greater HI, thus higher mp
2
...
Micelles: Small spherical droplet
2
...
Liposome / Vesicle: Lipid bilayer folds back on itself, creating a separate aqueous compartment
Structure of glycolipids:
Contains one glycerol, two fatty acids and one polar, short carbohydrate chain
• Carbohydrate group joined by glycosidic bond
LIPID DERIVATIVES (CHOLESTEROL)
Serves to modulate membrane fluidity and is also a precursor for synthesis of bile acids, steroid
hormones and vitamin D
STRUCTURE TO FUNCTION OF LIPIDS
Structure
High proportion of C and H to O per
unit mass
• Making it highly reduced
C-H bonds are non polar and
hydrophobic
• No associated water molecules
stored along with them
• No extra weight due to water of
hydration
• Lack of free electrons
Weak HI between fat molecules,
thus can slide aside under pressure
Lower in molecular weight than
water per unit volume

Function
Triglycerides
Upon oxidation, release large amounts of energy ! efficient
energy store
Release water when oxidized during cellular respiration,
known as metabolic water ! important to desert animals
Do not affect water potential of cells when stored in large
amounts
Allow motile organisms to keep mass to minimum
Good thermal insulator as subcutaneous fat insulates body
Adipose tissue (containing fat) around vital organs help to
cushion and protect vital organs against impact
Less dense than water, aids buoyancy of aquatic animals

Structure
Amphipathic molecule with 2 nonpolar, hydrophobic fatty acids tails
and a changed, hydrophilic
phosphate head

HI between fatty acid tails

Phospholipids contain choline

Carbohydrate chain attached to
lipids
Hydrocarbon tails

Function
Phospholipids
Forms selectively permeable membrane
Forms liposome
• Vesicles for storage and transport of cellular products
and digestion of waste
• Vesicles for drug delivery in humans
Forms micelle for transport of fats between gut and body
tissues
Maintain integrity of membrane bilayer due to large number
of interactions
Individual HI are weak, allowing lateral movement of
phospholipids, thus membrane fluidity
Choline is important for synthesis of acetylcholine, a
neurotransmitter
Glycolipids
Found at exterior cell surface membrane
• Marker for cell-cell recognition
• Involved in cell-cell adhesion
HI between fatty acid tails serve to anchor glycolipids at cell
surface membrane
~ END ~

!
SECTION 2: CARBOHYDRATES
!
INTRODUCTION
Carbohydrates essentially contain Carbon, Hydrogen and Oxygen
• Condensation – loss of water molecule
• Hydrolysis – addition of water molecule
Uses of monosaccharide:
1
...
Building blocks to synthesize polysaccharides
3
...
The bond
that is formed is known as a GLYGOSIDIC bond and is defined as the bond formed when the
ANOMERIC hydroxyl group of one sugar unit and any hydroxyl group on the other sugar react
together
...
Both of them consist of α-glucose monomers joined by
one of two types of bonds:
1
...
α (1,6) glycosidic bond: formation of branches
• Found in amylopectin and glycogen
Starch:
Commonly found in plant tissue, serves as carbon and energy sources
• Consists unbranched AMYLOSE (10% - 30%) and branched AMYLOPECTIN (70 - 90%)
Amylose
Consists of hundred to thousands of α-glucose residues joined by α (1,4) glycosidic bond
• Allow chains to COIL helically (S) into more compact shape for storage (F)
• Its bulky size (S) makes it poorly soluble (F)
Amylopectin
Also contains α (1,6) glycosidic bond
• Making it branched (S) thus allowing for large number of enzymes to act on it and easily
hydrolysed (F)
• Also highly compact (F)

Glycogen:
It is a macromolecule that is a major form of storage in animals
• Consist of only α-glucose monomers
• Similar structure to amylopectin, but more branched with both α (1,4) and α (1,6) bonds
o α (1,6) glycosidic bond occur every 8-12 glucose units thus more compact
Structure and Function of Starch and Glycogen:
Structure
Large molecule
Highly branched due to presence of α (1,6)
glycosidic bond
Composed of several hundreds to thousands of
α-glucose monomers
Glucose units linked by α (1,4) glycosidic bond

Function
Insoluble ! ideal for storage as it does not
affect water potential
Compact shape allows for easy storage
Supply large number of free ends for hydrolysis
at any one time
Act as large store of carbon and energy
Can be hydrolysed by glycogen phosphorylase

STRUCTURAL POLYSACCHARIDES
Cellulose: It is a polysaccharide / carbohydrate
Provides structural support, made up of β-glucose monomers, thus forming β (1,4) glycosidic bond
• Alternate monomers are rotated 180o relative to one another
Forms long, unbranched straight chain
• Their hydroxyl groups project outwards thus allowing formation of extensive H-bonds and
establishment of rigid cross links
• Many chains run parallel to each other, allowing them to associate to forms microfibrils !
macrofibrils
Properties of cellulose:
1
...
Full permeability – to water and solutes, important for proper functioning of plant cells
• Also important food source for animals with enzyme cellulose
3
...
This process is known as a condensation reaction
where the resulting covalent bond formed is a carbon-nitrogen bond (–CN) and is known as a
peptide bond
...
Effective in small amounts
2
...
Speed up rate at which chemical equilibrium is reached
4
...
ROR is affected by [enzymes]
6
...
ROR is affected by temperature
Cofactors: non-protein components to function
MODE OF ACTION OF ENZYMES
Enzyme active site:
Type of aa residue
Catalytic
Binding
Structural
Non-essential

Properties
Catalytic activity by making / breaking bonds ! act as acceptors and
donators of H+
Hold substrate in position by associating transiently with substrate
Maintain specific 3D conformation of active site and enzyme
No specific function

Formation of ES complex:
Close proximity and correct orientation
They lower the AE by:
1
...
Strain critical bonds, attain unstable transition state configuration
3
...
They are chemically
unchanged at the end of the reaction and can be reused
Induced-fit
• Active site is flexible, not exact same conformation as substrate
• When substrate enters active site, it binds through temporary / non-covalent bonds
o Ionic / H-bonds form between substrate and R groups of binding aa
o Induces change in the shape of the active site, fits more snugly around substrate as it is
molded into a precise conformation
• Forms ES complex, strain critical bonds / bring reacting groups in substrate to close proximity /
active site provides microenvironment
• Stabilize transition state, lowers AE
• Shows group specificity of enzymes
Measuring rate of enzyme reaction:
1
...
Substrate used
• Note: Initial rate = Initial amount of product / time
FACTORS AFFECTING RATE OF REACTION
Limiting factor: factor in shortest supply that limits rate of reaction
Concentration of substrate:
Low [S]
• It is the limiting factor as not all active sites are used up
• Increase in [S] causes an increase in the frequency of effective collision and formation of the ES
complex
• Increases rate of product form and hence ROR
High [S]
• Enzymes active site fully saturated
• Need to wait till ES complex dissociates, release product and free E to form new ES
• Increasing [S] does not increase ROR
• Need to increase [E]

Concentration of enzyme:
Low [E]
• Increase in [E] provides more active sites
• Increase frequency of effective collision and formation of ES complex and thus the RORHigh [E]
• [E] no longer limiting as there is insufficient S to compete for active sites
• [S] limiting so increasing [S] increases ROR
Temperature:
Increases to optimum
• Kinetic energy of S and E increases
• Increases frequency of effective collision and formation of ES complex
• Raises energy level of E and S, more likely to overcome AE barrier in order to form products
• Increases ROR
At optimum
• Maximum rate of ES complex formation
Above optimum
• ROR decreases drastically
• Thermal agitation disrupts H-, ionic and other weak interactions that stabilize the precise 3D
conformation
• 3D conformation of E and active site altered
• No longer complementary to S
• Enzyme is denatured, loses its catalytic function
Note: ROR doubles for every 10oC increase in temperature from 10-40oC
Changes in pH:
Structural aa
• Ionic charge of acidic and basic R groups are altered
• Disrupts ionic / h-bonds that maintain the 3D conformation
• Denatures enzyme
Binding aa
• Alters ionic charge at active site
• Substrate no longer held in the correct orientation
Catalytic aa
• Alters ionic charge
• R groups no longer possess the correct charge needed to catalyze the reaction between E and S

Vmax and Km:
Helps determine the effectiveness of a reaction
Vmax – maximum rate that a reaction can proceed at with specific concentration of enzyme and
excess substrate
Km – substrate concentration that allows an enzyme catalyzed reaction to proceed at half the
maximum velocity, ½ Vmax
• Low Km: Refers to high affinity for substrate, enzyme binds efficiently
Inhibitors:
Two main types of inhibitors
1
...
Non-competitive – binds to non-active site and induces change in conformation
Comparison of inhibitors:
Competitive
3D conformation / shape of inhibitor resembles
substrate
Binds to active site
Blocks the active site and prevents substrate
from binding to it
Could be overcome by an increase in [S]
Vmax is the same as that of the reaction in the
absence of inhibition, but at higher [S]
Km is higher than that of reaction in the absence
of inhibition
Affinity of enzyme for substrate is reduced

Non-competitive
3D conformation / shape does not resemble
substrate
Binds to the enzyme at a region other than the
active site
Induces a change in the 3D conformation of
active site to prevent substrate from binding
Could be overcome by an increase in [E]
Vmax is less than that of the reaction in the
absence of inhibition
Km is the same as the reaction in the absence of
inhibition
Affinity of enzyme for substrate is unchanged

Allosteric regulation:
Involves binding of molecules at an allosteric site
• Molecules are known as activators and inhibitors

Presence of
allosteric
activators

Activators – stabilizes the active form and increase the affinity of the enzyme for its substrate
Inhibitors – stabilizes the inactive form and decreases the affinity of the enzyme for its substrate

Reversible / Irreversible inhibition:
Reversible – Binds by weak non-covalent bonds, effect is temporary, no permanent damage to E
Irreversible – binds by strong covalent bonds, effect is permanent, permanent damage to E
End-product inhibition:
End product of a metabolic pathway accumulates, acts as an inhibitor to control the preceding steps
of the pathway (example of negative feedback)

Advantages:
1
...
Permits build up of high local concentrations of substrate and thus a higher rate of reaction
3
...
Reactants may be modified in a series of small steps thus energy is released in controlled
amounts
5
...
Allows for regulation and control of metabolism as each step in an enzyme-controlled pathway
is a point of control for the entire pathway
7
...
Fluid layer is asymmetrical
• Lipid layers may differ in composition of proteins and lipids
• Membrane lipids: Phospholipids, cholesterol and glycolipids
2
...
Acting as a hydrophobic barrier preventing polar molecules and ions from passing through the
cell membrane
2
...
Hydrophobic interactions between hydrophobic amino acids and hydrocarbon tails
2
...
g
...
g
...
Concentration gradient
2
...
Area of diffusion
4
...
Size and type of diffusing molecule: Smaller molecule faster
6
...
Phagocytosis
• Large solid particles are taken in (usually for digestion)
2
...
Receptor-mediated endocytosis
• Coated pits (with clathrin) form vesicles when specific molecules bind to receptor proteins
Exocytosis
• Cells secrete macromolecules by fusion of vesicles with plasma membrane
• Vesicles usually bud off from ER or GA (secretary vesicles from the trans face of the GA)
~ END ~

Chapter 5: DNA & Genomics
STRUCTURE OF DNA
Properties:
1
...
Able to replicate accurately – due to (1) semi-conservative replication, (2) complementary base
paring and (3) proofreading
3
...
Consists of two polypeptide chains where each strand forms right-handed helix coiling around
each other to form double helix
2
...
Strands are anti-parallel
4
...
Bases of opposite strands held together by relatively weak H-bonds
6
...
4A apart, where complete 360o turn comprises 10
base pairs, spanning 34A
7
...

•
•
•

Steric restrictions
Sugar-phosphate backbone has regular helical structure and uniform diameter of 2nm
T and C are pyrimidine, with single ring while A and G are purines, which is twice as wide
Thus purine is paired with pyrimidine

2
...
Allows genome to be compact so that relatively large size is tightly packed within small
volume of the nucleus
2
...
Allows it to be compact to prevent access of transcription machinery to allow for control of
gene expression
4
...
coli was cultured for several generations in medium containing 15N to label all DNA strands
with 15N
• E
...
coli cells were extracted after one and two generations of growth in the 14Nmedium respectively
• DNA molecules were then separated based on different densities / mass by ultracentrifugation

Results for first generation
• First generation produced a single DNA band of intermediate density where each daughter DNA
molecule contains 1 15N parental DNA strand & 1 newly synthesized 14N daughter DNA strand
• This is consistent with semi-conservative model of DNA replication: 15N strands of parental
DNA separate & function as templates for synthesis of new complementary daughter strand
• This result eliminated the conservative model as two classes of DNA, 15N parental DNA and
newly synthesized 14N DNA would have been produced

Results for second generation
• Second generation of bacterial growth in 14N-medium produced one band of low density and
one band of intermediate density
• As replication of each hybrid 15N14N DNA molecule produces one daughter DNA molecule
containing 14N14N and another DNA molecule 15N14N hybrid
• The dispersive model can be eliminated as only a single DNA band would have been expected
Mechanism of DNA Replication:
Stage
Location of
Replication origins
Separation of
Parental DNA
strands

Synthesis of RNA
primer
Synthesis of
Daughter DNA
strands

Explanation
• DNA replication is semi-conservative
• Begins at the origin of replication
• 2 parental strands unwind and separate by breaking the H-bonds,
facilitated by helicase
• Topoisomerase helps to relieve tension ahead of the replication fork by
creating transient nicks in DNA
• Single-stranded DNA becomes templates for synthesis of new strands
• Kept single-stranded by single-stranded DNA-binding proteins
• Primase catalyzes synthesis of RNA primer
• In order to provide free 3’OH end for DNA polymerase to initiate
synthesis of DNA strand
• Free deoxyribonucleoside triphosphate (dNTPs) are incorporated via
complementary base pairing with parental strands, A to T, G to C
• DNA polymerase catalyzes phosphodiester bond formation between free
3’OH end of terminal nucleotide of growing daughter DNA strand and 5’
phosphates of incoming dNTPs
• The above happens through formation of CBP with the DNA template –
bond breakage in pyrophosphate lost from dNTP releases free energy for
phosphoester bond formation
• DNA synthesis occurs in the 5’ ! 3’ direction
• In replication fork, leading strand is synthesized continuously
• Lagging strand is discontinuously synthesized as Okazaki fragments
• Okazaki fragments are ligated together by DNA ligase

DNA double helix:

~ END ~

Chapter 6: Cell and Nuclear Division
CELL AND NUCLEAR DIVISION
Cell Division: Contains both Nuclear Division and Cytoplasmic Division
• Unicellular – produce genetically identical cells (Asexual reproduction)
• Multicellular – growth and development of sexually reproducing organisms, renewal and repair
Ploidy level: Number of sets of chromosomes
• Haploid (n) – pairs of chromosomes (homologous pairs) and
• Diploid (2n) – number of chromosomes
Chromosome number: Number of chromosomes
• 22 pairs of autosomes, 1 pair of sex chromosomes
Human Life cycle:
Somatic cells – generated by mitosis (all diploid)
Reproductive cells – gametes, by meiosis (all haploid to maintain constant number of chromosomes
and prevent chromosomal doubling in the next generation)
Definitions:
Term
Chromatin
Chromosome
Homologous
Chromosomes
Sister
chromatids
Centromere
Kinetochore
Centrosome
Spindle fibers

Definition
Uncoiled and diffused state of DNA in interphase / non-dividing phase
Condensed form, with scaffolding proteins – most visible in mitosis / meiosis
Appears as a double arm: During S-phase interphase, DNA undergoes semi
conservative replication to form 2 identical chromatids joined at the centromere
Structurally identical: same size, shape, centromere position and sequence of
gene loci
Allele is an alternative form of a gene; each chromosome called a homolog
Replicated forms of a single chromosome
Structurally and genetically identical: same alleles at gene locus
Region where sister chromatids join
Associated with kinetochore for attachment of spindle fibers
Allow chromosomes to align properly at metaphase plate during metaphase
Form by proteins in centromere where microtubule of spindle attach
Plays active part in movement of chromosomes
Contains pair of centrioles and surrounding cytoplasm
Assembly of spindle microtubules, known as microtubule organizing center
Astral: from centriole to peripheral regions (acts as brace)
Kinetochore: attached to kinetochore, pull sister chromatids to opposite poles
Polar: from pole to pole, overlapping at equator to elongate cell during anaphase

CELL CYCLE
Consists of Interphase (G1, S and G2), Mitosis and Cytokinesis
• Cells may enter G0 phase (quiescent non-dividing state)
• Checkpoints present at end of G1, end of G2 and end of Prophase
G1: Check for presence of growth factors to stimulate cell division
G2: Allows cells with successful DNA replication without damage to proceed to mitosis
M: Ensures successful formation and attachment of spindle fibers to kinetochores of chromosomes
INTERPHASE
Known to be the longest phase of the cell cycle
3 main stages:

G1 PHASE

S PHASE
G2 PHASE

Cells are small in size and low in ATP
Thus they increase in size and acquire ATP in this phase
Intensive cellular gene expression and synthesis of appropriate organelles
(mitochondria, ER) and proteins (enzymes, DNA polymerase, helicase) occur
DNA Replication occurs
Each chromosome now has two genetically identical sister chromatids
Synthesis of histone proteins
Second growth and energy acquisition (as formation of DNA is energy consuming)
Further synthesis of appropriate organelles and proteins
Centrioles replicate and mitotic spindle begins to form

Comparing cells in diving and non-dividing forms
Non-dividing cells
DNA exists in loosely-coiled and uncondensed
threads called chromatin
• Euchromatin (loosely coiled)
• Heterochromatin (tightly coiled)
Nuclear envelope intact and nucleolus present
Cell is transcriptionally active (gene expression)

Dividing cells
DNA exists in highly condensed form known as
chromosomes

Nuclear envelope disintegrates, nucleolus absent
Cell is transcriptionally inactive

NUCLEAR DIVISION
Similarities between mitosis and meiosis:
1
...
During telophase, chromosomes uncoil and decondense
3
...
of units
One polynucleotide chain
Two polynucleotide chain
Almost always single stranded
Always double stranded
3D structure
• Folded into complex tertiary
• Coiled around histone proteins,
structure
organized into chromosomes
Ration of bases
A:U =/= G:C =/= 1:1
A:T = G:C = 1:1
Monomers
Ribonucleotides
Deoxyribonucleotides
Pentose sugar
OH group at C2
H group at C2
Less stable – due to additional
More stable – lacking 2’OH group
Chemical stability
reactive 2’OH group
Nitrogenous base A, U, G, C
A, T, G, C
mRNA, tRNA, rRNA, snRNA,
Only one basic form
Basic forms
siRNA
Throughout the cell
Exclusively in nucleus (as well as
Location
mitochondria and chloroplast)
Amount per cell Varies
Constant

1
...

3
...

5
...
Messenger RNA (mRNA) – carries information about DNA, codes for aa sequence of proteins
2
...
Ribosomal RNA (rRNA) – plays catalytic and structural roles in ribosomes (peptidyl transferase
activity)

TRANSCRIPTION (DNA ! RNA):
Definition: Process in which complementary RNA copy is made under the direction of the template
strand of a specific region of the DNA molecule, catalyzed by the enzyme RNA polymerase
...
Gene
2
...
General / basal transcription factors
4
...
Addition of 5’methylguanosine cap
2
...
RNA Splicing
Functions of 5’methylguanosine cap:
• Protects mRNA from degradation by nucleases and phosphatases that degrade RNA during
transport from nucleus to cytoplasm
• Signals the 5’end of mRNA – assembly point to recruit small subunit of ribosome for translation
to begin
• Distinguish mRNA from other RNA
Functions of 3’Poly(A) tail: Addition of a series of ~200 adenine (A) nucleotides, catalyzed by
enzyme poly(A)-polymerase
• Protects mRNA from degradation by nucleases
• Make mRNA a more stable template for translation in cytoplasm
• Required to facilitate export of mRNA
Messenger RNA splicing: Exons – protein-coding sequences; Introns – non-coding sequence
Occurs after release of pre-mRNA from RNA polymerase
• Defined as removal of introns and ligation of remaining exons
• Requires hydrolysis of ATP
• Carried out by spliceosome, where splice sites are sits on mRNA where splicing occurs
Steps
1
...
Cleavage at 3’ splice site and simultaneous ligation of exons, causing exition of intron as a
lariat-like structure

GENETIC CODE
General features:
• List of codons on mRNA
• Also sequence of triplet bases in the non-template strand of DNA
• Total of 43 = 64 possible codons
o Since there are 3 nucleotides in each codon, but 4 possible bases
• 61 code for amino acids and include a start signal
• 3 code for termination signals of polypeptide synthesis (stop codon)
Key features:
1
...
Genetic code is universal where a particular codon represents same amino acid in all species of
organisms
3
...
Genetic code is degenerate, more than 1 codon may code for a single amino acid
5
...
Stop codons are UAG, UAA, UGA – do not code for amino acids and signal end of translation
7
...
Mature messenger RNA
2
...
Amino acids
4
...
Ribosome
6
...
Attaching to biochemical functional groups such as acetate, methyl, phosphate, various lipids
and carbohydrates
• Glycosylation – addition of specific short chain carbohydrate
2
...
Removing sequence of amino acids from proteins (Insulin)
PROTEIN SORTING
Definition: Mechanism where cell transports proteins to the appropriate positions in the cell or
outside of it
Free ribosomes: Suspended in the cytoplasm, mostly synthesize proteins that function within the
cytosol or are destined for nucleus, chloroplast, and mitochondria
Bound ribosomes: Attached to cytosolic side of rough ER are responsible for synthesizing both
membrane-bound and soluble proteins for endomembrane system
~ END ~

Chapter 8: Org & Control of E
...
Telomerase RNA template binds complementarily to 3’ overhang of parental DNA strand
2
...
Synthesis of daughter strand can be extended during next round of DNA replication ! longer
telomere
STEP 1:
• Telomerase binds to 3’ overhang on parental DNA strand
• Through CBP using the bound RNA template
STEP 2:
• It elongates the 3’ overhang in a 5’ to 3’ direction, using the bound telomerase RNA as a
template for addition of dNTPs via CBP
• Catalytic residues at active site catalyze formation of phosphoester bond between 3’OH of 3’
overhang and 4’ phosphate of incoming nucleotide
STEP 3:
• Telomerase continues extension of 3’ overhang by repeated translocation and addition of repeat
sequence to the 3’ overhang
STEP 4:
• Replication of incomplete lagging daughter strand is completed by using these extensions as
template for synthesis of complementary strand by DNA polymerase
• Leaves 3’ overhang
CENTROMERE
Structure:
• Position of centromere is unique for each chromosome
• Consists of short AT-rich sequences
• Embedded in a centric heterochromatin, where centromeric DNA is bound by specialized
nucleosomes with a centromere-specific histone
• Folding of DNA facilitates assembly of other centromere-binding proteins to form kinetochore
Function of centromere:
1
...
Site of assembly of kinetochore, protein complex that attaches to microtubules of mitotic
spindle, where sister chromatids are joined via centromere to spindle microtubules
3
...
Allow chromosomes to align properly at metaphase plate during metaphase

SECTION II: CONTROL OF EUKARYOTIC GENOME
Gene expression: process by which information from a gene is used in synthesis of functional gene
product by transcription and translation
Control: determines the
• Amount (quantity)
• Types (quality)
• Timing of appearance (temporal control)
• Cell type in which proteins are expressed (spatial control)
GENE AMPLIFICATION
Definition: Production of multiple copies of a specific gene to amplify the quantity of gene product
• Gene expression increased significantly, more gene copies available to act as DNA templates
for transcription
• Leads to increase in gene product
Case Study: Ribosomal RNA gene amplification in frog Xenopus laevis
Observation
During development of oocyte, original 500 copies of genes are amplified
• Mature oocyte contains 2 million copies of genes for rRNA by repeated rounds of replication
• Many copies of circular DNA molecules called minichromosomes are formed, each with 1-20
copies of the RNA genes
Significance
Increase in transcription of rRNA genes due to large amount of templates
• Accommodate enormous amount of ribosome biosynthesis needed for oogenesis
• Required to sustain high rate of protein synthesis
• Amplification is developmentally regulated: only occurs during development of oocyte
TRANSCRIPTIONAL CONTROL
Chromosome Remodeling: Chemical modification of histones and DNA, thus modify chromatin
structure and gene expression
• Histone modification and DNA methylation
Chromatin within a nucleus can be organized into
• Euchromatin – diffused (transcriptionally active)
• Heterochromatin – highly condensed (transcriptionally inactive)
o Prevents transcription factors and RNA polymerase to gain access to promoter of
specific genes and activate transcription

Histone Modification
N-terminus of each histone molecule protrudes outwards (tail)
• Histone tails are rich in lysine residues, which are positively charged
• Interact strongly with negatively-charged phosphate groups of DNA, increases affinity of DNA
for nucleosome surface
Acetylation
• Histone acetyltransferase acetylates lysine
residues
• Positive charge on lysine neutralized and
becomes uncharged
• Reduction in affinity of histone complex for
DNA molecule
• Chromatin structure becomes less compact,
exposing DNA regions to transcription
factors and RNA polymerase

Deacetylation
• Histone deacetylases catalyze deacetylation
of acetylated lysine residues
• Lysine residues become positively charged
again
• Increase in affinity of histone complex for
DNA molecule
• Chromatin structure becomes more compact,
prevents access of transcription factors and
RNA polymerase

DNA Methylation
Done through the addition of methyl groups to specific nucleotides
• Restricted to cytosine in the sequence 5’-CG’-3’ (CpG dinucleotides)
• Usually found in promoters
Why does it repress gene expression?:
1
...
Methylated DNA serves as recognition signals for proteins that recruit histone deacetylases,
causing chromatin structure to be more compact and prevents binding of transcription
machinery
Significance: genes are more heavily methylated in cells which they are not expressed
Initiation of transcription: Most important control point
• Interaction of general transcription factors and RNA polymerase often initiates transcription at
only a low basal rate
• Requires interaction of distal control elements and specific transcription factors to increase the
rate of transcription
Regulatory Sequences (Control Elements)
1
...
Promoter-proximal elements
• Essential for maximum rate of transcription
• Serves as binding sites for general transcription factors
3
...
Germline mutation – occurs in germline cells (germ cells + gametes), which may be transmitted
to the offspring and future generations
2
...
Point mutations are gene mutations that
involve changes at specific sites in a gene, causing change in one of a few bases:
• Nucleotide substitution
• Nucleotide insertion or deletion (frameshift mutation)
NUCLEOTIDE SUBSTITUTION
Definition: replacement of one deoxyribonucleotide with another
• May or may not affect the aa sequence of a protein and also the function
1
...
Nonsense mutation:
• Changes a codon for an aa into a STOP CODON, leading to premature termination of
translation
• Resulting polypeptide is shorter than normal (truncated)
• Nearly all nonsense mutation leads to non-functional proteins
Results in change in the overall three-dimensional conformation of the protein and hence alters the
function of the protein
3
...
Neutral mutation:
• Resulting aa substitution produces no detectable change in function
• Substitution of aa with similar physical and chemical properties OR non-essential aa
• Aa sequence of polypeptide is changed
Both will not result in a change in the overall 3D confirmation of protein and hence function of
protein is not altered

DNA BASE INSERTIONS AND DELETIONS
Definition:
• Base insertion is defined as the addition of one or more nucleotide pairs in a gene
• Base deletion is defined as the removal of one or more nucleotide pairs in a gene
Effects: dependent on the location/site in the DNA sequence and number of deoxyribonucleotides
added or deleted
• Since mRNA is read as series of non-overlapping codons, it results in frameshift mutation
• Might also cause non-functional protein (nonsense mutation)
Deletion mutation
Insertion mutation
• Single base deletion leads to frameshift
• Single base insertion leads to frameshift
• Hence this results in an incorrect amino acid • Hence this results in an incorrect amino acid
sequence of the polypeptide chain
sequence of the polypeptide chain
• Results in a change in the three-dimensional • Results in a change in the three-dimensional
conformation
conformation
• Results in a change in function
• Results in a change in function
Note: However, insertion of nucleotides in multiples of three does not lead to frameshift, though the
3D conformation might be changed
Results in change in the overall three-dimensional conformation of the protein and hence alters the
function of the protein
Gene Amplification:
Also a form of mutation – occurs as a result of DNA slippage leading to increase in copies of proto
oncogene and proteins encoded are thus over produced
GENE MUTATION CASE STUDIES
Sickle-Cell Anemia: Involves a mutation in the β-globin gene which codes for one polypeptide
subunit in haemoglobin
Genetic and molecular basis
• Substitution of T for A resulting in missense mutation
• Change in the 6th aa residue from GLUTAMATE (hydrophilic) to VALINE (hydrophobic)
• Specific 3D conformation and function of haemoglobin is altered
• Results in a hydrophobic spot on the outside of the protein structure which sticks to the
hydrophobic region of an adjacent haemoglobin molecule’s beta chain
• Mutant haemoglobin subunits tend to stick to one another when oxygen concentration is low
• Aggregated proteins form fiber-like structures within RBC
Psychological effects
• Causes RBC to lose their normal morphology and become sickle-shaped, thus lowering oxygen
carrying capacity
• They are less able to move through capillaries and can block blood flow
• They are also fragile and easily destroyed, reducing oxygen carrying capacity of blood

Cystic Fibrosis (CF): Involves mutation in the CFTR (cystic fibrosis transmembrane regulator)
gene found on chromosome 7, codes for chloride channel protein found in membranes of cells
lining lungs, liver and pancreas
Genetic and molecular basis
• Deletion of triplet base TTT in the template strand of CFTR results in the absence of the 508th
aa residue, phenylalanine (Phe)
• Primary sequence of protein is changed, leading to change in 3D conformation and function of
chloride channel protein
• Results in synthesis of non-functional CFTR channel, does not allow Cl- to diffuse out of
epithelial cells
• Thus water does not leave the epithelial cells due to absence of steep water potential, resulting
in production of thick mucus:
o Narrowing of air passages, reducing air flow and making breathing difficult
o Blockage of ducts (e
...
pancreatic ducts ! affecting digestion)
o Increased diffusion distance for gas exchange in lungs, insufficient oxygen supplied to
CF patient
o Bacterial infection – thick mucus serves as breeding ground for bacteria
o Inability of cilia to sweep away bacteria, fungi spores and other particles
CAUSES OF GENE MUTATIONS
Spontaneous mutations: Mutations that occur naturally (without use of chemical/physical
mutagenic agents)
DNA Replication and Repair
• DNA polymerase sometimes insert the wrong, too many or too few nucleotide
• Some of the mistakes are corrected immediately through proofreading
o DNA polymerase recognize mistakes and replace incorrectly inserted nucleotide
• Some are corrected after replication through mismatch repair
o Enzymes recognize and fix deformities in the secondary structure of the DNA molecule
• Those that fail to be recognized are then passed down from one cellular generation to the other
• Those that occur in germline cells cause it to be transmitted to the next generation
• Purine-purine / pyramidine-pyramidine ! transitions
• Else known as transversion
DNA Slippage
• Daughter/parental DNA strand slips during DNA replication, followed by folding back of strand
• Mispairing between daughter DNA strand and parental template strand resulting in gene
duplication or deletion
Slippage of daughter strand
• Results in insertion of repeats in daughter strand
Slippage of parental strand
• Results in deletion of repeats in daughter strand

Induced Mutation:
• Result od deliberate application of mutagens (chemical or physical)
• Increased rate of mutagenesis
MUTAGEN AND EFFECT ON DNA
Physical Agents (Radiation)
X-Rays
• Results in production of free radicals (OH)
• Interact with DNA to produce double stranded breaks leads to chromosomal
rearrangements and deletions
UV Rays
• Adsorbed by bases of DNA
• Results in production of covalent attachment between adjacent pyrimidines,
usually thymine dimers (T-T)
• Or base pair substitutions, insertions and deletions
• Blocks transcription and DNA replication
Chemical Agents
Base Analogues
• Similar to bases normal found in DNA, and incorporated in place of normal
bases ! base substitution
• E
...
Bromouracil (5-BU): can exist in alternate states
Base-Modifying
• Modify chemical structures and properties of bases
Agents
• Mispairing during DNA replication ! base substitution
• E
...
Ethylmethylsulfonate (EMS) and mustard gas: transfers alkyl to
structure
Intercalating
• Flat molecules with multiple ring structures
Agents
• Insert themselves between adjacent bases ! insertion or deletion !
frameshift mutation
• E
...
Proflavin, ethidium bromide (used in gel electrophoresis)
CHROMOSOMAL ABERRATIONS
CHANGES IN CHROMOSOMAL NUMBER
Variation in chromosome number can be due to:
1
...
Addition/Loss of one or more haploid sets of chromosomes
Addition/Loss of One or More Chromosomes (Aneuploidy):
Originates as a random error during production of gametes
• Nondisjunction
• Failure of chromosomes or chromatid to separate and move to opposite poles
• Loss of single chromosome called monosomy (45, X Turner Syndrome)
• Gain of one chromosome called trisomy (47, XXY Klinefelter syndrome and Down syndrome)
Can happen during Meiosis I or Meiosis II
Meiosis I
Results in trisomic gamete x2
Monosomic gamete x2
More deleterious

Meiosis II
Results in normal gamete x2
Results in trisomic gamete x2
-

Addition/Loss of One or More Haploid Sets of Chromosomes (Polyploidy):
Refers to the instance where more than two copies of haploid chromosome set are found
• More common in plants
o Autopolyploidy: addition of sets of chromosomes of same species
o Allopolyploidy: addition of sets of chromosomes of different species due to interspecific
mating
Autopolyploidy
Occurs in a few ways:
• Failure of all chromosomes to segregate
(complete non-disjunction)
• Two sperms fertilize an ovum, resulting in
triploid zygote
• Produced under experimental conditions by
crossing diploids with tetraploid

Allopolyploidy
• Haploid ovum fertilized by haploid sperm,
resulting diploid hybrid is usually sterile
• If new genetic recombinant undergoes
natural or induced chromosomal doubling
(through non-disjunction), a fertile hybrid
can be produced

CHANGES IN CHROMOSOME STURCTURE
Includes deletion and duplications of genes or part of chromosome
• Exchanges and transfers are called translocations
Deletion
• Deletion can occur near one end or from interior of chromosome and causes genotype to be
altered
• If deletion affects same gene loci on both homologous chromosomes, effect is lethal
• If only one is affected, alleles on non-deficient homologue will be expressed, even if recessive
Duplication
• Arise as a result of unequal crossing over between synapsed chromosomes during meiosis
• Or through replication error prior to meiosis
Inversion (normally better tolerated)
• Segment of chromosome is turned around 180o
• Does not involve loss of genetic information, but simply rearranges linear sequence
Translocation (normally better tolerated)
• Movement of a chromosomal segment to a new location in the genome
• Rearrangement of genetic material
~ END ~

Chapter 10: Cancer
INTRODUCTION TO CANCER
Results from uncontrolled cell division which might be as a result of dysregulation of cellular
checkpoints
• Rate of cell division exceeds cell death, causing net uncontrolled proliferation of new cells
• Gives rise to a clone of altered cells
• Develops via a multi-step model with accumulation of important mutations eventually allowing
it to METASTASIZE to distant sites
• Benign tumors: Few genetic mutations, do not cause serious health problems
• Malignant: Invasive and impair functions of organs (metastasized)
Important characteristics of cancer
Characteristic
Metastasis
M

Inducing
angiogenesis
I

N

No contact
inhibition

I

Independent of
anchorage

O

Omnipotent –
never dies

N
S
E

Number of
mutations increase
Still grows without
factors
Evades immune
detection

Elaboration
Process where primary tumor cells invade local tissues and blood
vessels, and establish second tumors called metastases at distant sites
1
...
Transported by circulatory system through the body
3
...
It is
small and localized, and angiogenesis and metastasis has yet to occur
It deals less damage to other organs and most cell division checkpoints are not lost – hence less
uncontrolled cell division and fewer mutations
CELL CYCLE AND CHECKPOINTS
In order for tissues and organs to grow to an appropriate size and develop in a coordinated manner,
previse control of cell cycle is required ! hence presence of cell cycle checkpoints/control systems
Checkpoints: Help to ensure the orderly progression of cell cycle

Mutations in genes controlling cell cycle:
There are two main classes of cancer-critical genes that exert their effects by acting on cell-cycle
control machinery and affecting variety of normal cellular functions
1
...
Proto-oncogene – normal gene that encodes protein which stimulates normal cell division [ras]

Tumor suppressor gene (TSG)
Loss of such proteins allows cells to divide through absence of suppression in cell division
Function:
• Take part in cell-signaling pathway
• Halt cell division if DNA is damaged
• Trigger DNA repair mechanisms ! prevent cells from accumulating DNA damage
• Initiate apoptosis if DNA damage cannot be repaired
• Maintain cell adhesion (metastasis suppressors)
Loss of function mutation: Results in abolished protein function
...
DNA damage is an intracellular signal passed via protein kinases and leads to activation of p53
2
...
In a proto-oncogene, a mutation of EITHER copy results in abnormal cell proliferation
• Oncogene acts in a dominant manner
Ras, a proto-oncogene
Ras gene encodes a small protein which is a G-protein involved in signal transduction:
Mode of Action
• Last protein kinase of the signal transduction pathway activates transcription of genes encoding
proteins that stimulate cell division
• Pathway normally activated when growth factor binds to receptor
Mutation
1
...
Results in GTP being bonded to ras protein as ras-GTP complex, thus constantly active, even in
the absence of growth factors and leads to constant stimulation of phosphorylation cascade
3
...
Lifestyle and diet
(a) Cigarette and tobacco smoking
• Chemicals such as polycyclic aromatic hydrocarbons (PAHs), an important class of carcinogens
are found in smoke inhaled
• They bind to DNA to form an adduct, causing damage to DNA
• If not repaired, it could cause mistakes in DNA synthesis, thus causing mutations
• PAHs tend to form adducts at several sites on the p53 gene in lung cells, results in loss of
normal cell growth and uncontrolled proliferation
(b) Exposure to chemical carcinogens
• Meat which is charred or grilled at high temperatures contains heterocyclic amines (HCAs) and
polycyclic aromatic hydrocarbons (PAHs)
• Similarly, they form adducts on p53 genes and thus loss of normal cell growth
• Derivatives (nitrosamines) are dangerous at high concentrations and can act directly on cellular
DNA, leading to gene mutations ! gene critical genes
2
...
Age
Cancer results from accumulation of mutations, thus the longer we live, the greater the risk of
developing cancer
4
...
g
...
Loss of Immunity
Immune system is suppressed by drugs, viruses such as HIV or mental states of depression, causes
it to be unable to detect and destroy cancerous cells
6
...
Genome comprising DNA/RNA
2
...
Envelop comprising phospholipids from host cell
4
...

•
•
•

Viral genome (ALL viruses)
Contains either DNA/RNA
Either circular or linear, single stranded or double stranded
Carry genes needed for synthesizing viral capsid and genetic material and genes for regulating
action of host genes for packaging of mature virus

2
...
Envelop (Enveloped viruses)
• Usually found in viruses that infect animals, and is derived from the host cell
• When released via budding, they take with them the host’s cell surface membrane and insert
proteins of viral origins
• These include glycoproteins: essential for attachment of virus to next cell and protects virions
nucleic acid from effects of various enzymes and chemicals

4
...
They identify their host cells by
• A complementary fit between proteins on outside of virus and
• Specific receptor molecules on surface of host cell
Types of viruses:
1
...
Animal viruses
3
...
Genomic – nucleic acid: linear double stranded DNA
2
...
Head contains DNA of the virus
2
...

2
...

2
...
T4) tail fiber allowing phage to adsorb to surface of bacterial cell by binding to
specific receptors site

Reproductive cycle – Lysogenic cycle (replication of phage genome without destroying host)
Stage
Process
Adsorption Single tail fiber of lambda phage attaches to specific receptor sites
Base plate settles down on host cell’s surface
Penetration Conformational changes causes tail sheath to contract and allow DNA to be extruded
from head, capsid is left outside bacterial cell wall
Prophage formation
• Phage genome circularizes and inserts into a prophage insertion site by genetic
recombination
• Viral DNA is then replicated along with the chromosome each time host cell
divides, thus passed on to generations
• Eventually gives rise to large population of bacteria
Spontaneous induction
• Environment cause virus to switch from lysogenic to lytic
• Lysis genes repressed during lysogeny are activated, allowing lambda phage to
exit from bacterial chromosome and give rise to new active phages

Comparing between Lytic and Lysogenic cycle:
Similarities
1
...
Adsorption occurs when tail fiber(s) of phages attach to specific receptor sites on surface of
bacterial host cells
3
...
Bacterial host cells are infected
Differences
LYTIC
LYSOGENIC
Results in lysis / cell death of host cell Allows for replication of phage genome without lysis of /
destroying host cell
Viral genome is not integrated into
Viral genome integrated into host genome via genetic
host genome
recombination, resulting in formation of prophage
Viral genome replication independent Viral DNA replicated along with chromosome each time
of host cell division
host cell divides, and passed on to generations of daughter
cells
Advantages of lysogenic cycle:
1
...
When virus to bacteria ratio is high
3
...
Genome – nucleic acid: Eight different segments of negative (-) sense single strand RNA & (-)
sense strands must be copied to complementary (+) sense RNA before proteins can be
synthesized
2
...
Viral Envelope: phospholipid bilayer obtained from host upon budding
2
...
Protein envelope: Matrix protein forms second layer of envelope, enclosing the nucleocapsids
• M1: monomers of matrix protein
• M2: acts as ion channel to lower or maintain pH of endosome
4
...

2
...

2
...

4
...
Genetic material in bacteria is also stored on either
circular bacterial chromosome or plasmids; they also lack introns
Bacterial chromosome:
Typically single, circular, double-stranded DNA containing essential genes for survival –
• Compacted about 1000 times through association of DNA with positively charged histone-like
proteins to compact DNA into looped domains
• Further compaction through super coiling
• Genes are grouped as operons where multiple genes are under the control of 1 promoter and
same regulatory elements

Plasmids:
Exist as small, circular, double-stranded extrachromosomal DNA molecule –
• Passed on to cells of the same generation (conjugation, transformation, transduction) and to
offspring (binary fission)
• Can replicate independently of bacterial chromosome as they contain their own origin of
replication
• Contains genes which confer advantageous and / or protective traits such as antibiotic
resistance, toxin synthesis and enzyme production – thus giving them selective advantage
Comparing structure of prokaryotes and eukaryotes:
Prokaryotic Genome
Structure of chromosomes
Circular DNA
Degree of compaction
High degree
No
...
of Usually one per cell
sets of chromosomes
One set of chromosome and
bacterial cells are haploid
Location within cell
Not enclosed by nuclear
membrane, aggregated in
nucleoid
Association with proteins
Wound around basic proteins
that are similar to histone
proteins
Number of origin of replication Single origin of replication
Size of chromosome
Small (4000kb in E
...
Transformation
2
...
Conjugation
DNA must be integrated into chromosome of recipient cell through homologous recombination
Transformation:
Process by which a recipient cell takes up small fragments of naked DNA from the surrounding
environment, where DNA originates from:
• Donor bacterial cell which lysed and releases DNA to surrounding environment
• Artificially constructed plasmids
Only competent bacterial cells can undergo transformation, which require presence of competence
factors – cell surface proteins that bind to DNA fragments and aid in their uptake
Process
1
Competent recipient cell takes up one or more donor DNA fragments into its cytoplasm via
competence factors
2
Homologous recombination of donor DNA fragment with homologous section of recipient
cell’s chromosome
3
Homologous segment of donor cell’s DNA incorporated into recipient cell’s chromosome
and that of recipient cell is excised and degraded
• Recipient cell now known as recombinant cell
*Transformation can be induced through heat shock or electroporation

Transduction:
Bacteriophages carry bacterial genes from first host cell to second host cell due to aberrations in
phage reproductive cycle
...
Antibiotic drug resistance
2
...
Ability to use new metabolite
Process
1
F+ donor cell uses sec pilus to attach to F- recipient cell
2
Temporary cytoplasmic mating bridge is formed, providing route for DNA transfer
3
Sugar phosphate backbone of one strand of F plasmid in F+ cell is nicked by endonuclease
• It then separates from its complementary strand and moves to F- recipient cell through
the cytoplasmic mating bridge
4
Each parental strand becomes template for DNA synthesis of complementary daughter
strand
• Occurs be semi-conservative replication, catalyzed by DNA polymerase
• DNA ligase then catalyzes synthesis of phosphodiester bond
5
Cells move apart and sex pilus breaks, forming two bacterial cells that are both F+, hence
converting an F- to and F+ cell
DNA
molecule
transferred
Donor cells
Recipient
cells
Mode of
transfer

Transformation
Degraded naked
DNA fragments
from surrounding
Dead / lysed cell
Competent cells
Competence factors
required for uptake
of DNA

Transduction
Degraded fragments of excised
portions of bacterial DNA

Conjugation
F plasmid / F factor

Bacterial cells infected by
bacteriophages
Cells infected by same
bacteriophages
Infecting phages carry either
random bacterial DNA (general)
or specific DNA adjacent to
prophage insertion site (specific)

F+ bacteria cells with F
plasmid
F- cell
Formation of sex pilus
followed by cytoplasmic
mating bridge

GENE EXPRESSION IN PROKARYOTIC CELLS
Prokaryotes do not contain nuclear membrane, thus process of gene expression is much simpler
than a eukaryotic cell
• Cannot separate transcription and translation
• Simultaneous transcription and translation possible
• Post-transcriptional modification in eukaryotic cells do not occur in prokaryotic cells
Structure and organization of prokaryotic genes:

Components of a prokaryotic structural gene:
1
...
Coding region not interrupted by introns
3
...
Fewer types of tRNA and initiator tRNA carries N-formyl-methionine thus known as N-formylmethionyl tRNA (tRNAfMet)
2
...
Prokaryotic mRNAs are polycistronic – different segments in the same mRNA are translated to
give rise to different proteins
Stage
Amino Acid
activation

Process
• tRNA molecules bind with specific amino acids as determined by anticodon
• Aminoacyl-tRNA synthetase catalyzes formation of aminoacyl-tRNA complex
• Process driven by ATP hydrolysis
Initiation
• Small ribosomal subunit binds to both mRNA and initiator tRNA, tRNAfMet
• Eukaryotic initiation factors (eIFs) mediate process, driven by GTP hydrolysis
• Small ribosomal subunit bind to mRNA, moves along in searching for start
codon
• tRNAfMet binds start codon
• Large ribosomal subunit binds to complete translation initiation complex
• tRNAfMet situated at P site of ribosome, A site ready for next aminoacyl-tRNA
Elongation & • Anticodon of incoming aminoacyl-tRNA form CBP with mRNA codon in A
Translocation
site of ribosome (Elongation factors mediate process)
• Peptidyl transferase catalyzes peptide bond formation between initial
methionine at P site and 2nd amino acid at A site
• Covalent bond between initiator tRNA and methionine is broken
• Growing peptidyl-tRNA in A site is translocated to P site
• Ribosome shifts mRNA one codon in the 5’ to 3’ direction
• Process driven by GTP hydrolysis
Termination • Elongation repeated until ribosome reaches stop codon
• Release factor binds directly to the stop codon in the A site and causes addition
of water molecule
• Bond between polypeptide and tRNA at P site is hydrolysed

CONTROL OF GENE EXPRESSION IN PROKARYOTES
Control in prokaryotes is much less complex and since no post-transcriptional modification is
possible, translational regulation is not common utilized
Coordinated control – Operon:
A transcription unit where structural genes with related functions are generally located adjacent to
each other and placed under control of the same promoter and regulator regions
• Major mode of control exists at transcriptional control
• Controls amount of a protein and regulation of this occurs mainly ar level of transcription (how
much mRNA is produced)

An operon typically consists of:
1
...
Operator – regulates rate of transcription of structural genes by interacting with specific
repressor protein
3
...
Regulatory genes – code for specific protein that regulates expression of structural gene
(repressor proteins)
Structural genes
Codes for functional RNA, structural protein,
enzyme, or any functional protein within cell
Usually found as part of operon

Regulatory genes
Codes for regulatory proteins that regulate
transcription of structural genes
Lie outside the operon

Why operons are necessary:
• Allows for coordinated regulation of functionally related genes using a single on-off switch
• Only produces enzymes when required
• Prevents inefficient use of resources
• Presents a selective advantage – use variety of sugars

Inducible operons:
Normally inactive and are turned on (induced) by substrate of the enzyme for which structural
genes code, which acts an inducer
• Ensures enzymes are only produced when substrate is present
• Usually code for enzymes involved in catabolic pathways – degradation of substrate
Example: LAC OPERON
Lac operon contains the following structural genes
1
...
lacY gene – codes for transport protein galactoside permease, enables bacteria to take up lactose
3

---