Regulation of gene expression in prokaryotes and viruses

Gene Expression and Regulation
SUBMITTED BY – NOOR ARSHIA
2nd Sem M.Sc (Microbiology)
UNDER THE GUIDANCE OF – Dr. ANIL KUMAR M
Maharani cluster university
REGULATION OF GENE EXPRESSION IN
PROKARYOTES AND VIRUSES

KEY CONCEPT
Gene expression is carefully regulated in both
prokaryotic and eukaryotic cells.
PROKARYOTES

INTRODUCTION
• Prokaryotic organisms are single-celled
organisms that lack a cell nucleus, and their
DNA therefore floats freely in the cell
cytoplasm.
• To synthesize a protein, the processes of
transcription and translation occur almost
simultaneously.
• When the resulting protein is no longer
needed, transcription stops.

INTRODUCTION
• The primary method to control what type of
protein and how much of each protein is
expressed in a prokaryotic cell is the
regulation of DNA transcription.
• All of the subsequent steps occur
automatically. When more protein is
required, more transcription occurs.
• Therefore, in prokaryotic cells, the control
of gene expression is mostly at the
transcriptional level.

Prokaryotic organisms Eukaryotic organisms
Lack nucleus Contain nucleus
DNA is found in the cytoplasm
DNA is confined to the nuclear
compartment
RNA transcription and protein
formation occur almost
simultaneously
RNA transcription occurs prior to
protein formation, and it takes
place in the nucleus. Translation
of RNA to protein occurs in the
cytoplasm.
Gene expression is regulated
primarily at the transcriptional
level
Gene expression is regulated at
many levels (epigenetic,
transcriptional, nuclear shuttling,
post-transcriptional, translational,
and post-translational)
Differences in the Regulation of Gene Expression
of Prokaryotic and Eukaryotic Organisms

TYPES OF GENES

TYPES OF TEMPORAL
RESPONSES
• Biological systems exhibit 3 types of temporal
responses

NEED OF GENE REGULATION
• The cells need to regulate gene expression to
avoid wastage of energy in synthesis of
unnecessary RNAs (mRNA) and proteins
(enzymes), when not required.
• Prokaryotes are relatively simple than
complex multicellular eukaryotes, where
regulation is required for cell specialization,
with specialized function

NEED OF GENE REGULATION
• Regulation of gene expression in prokaryotes
and behaviour of a cell is determined not
only by what genes it possess, but also by
which of those genes are expressed at a
given time.
• Regulation of expression of gene is done by
alternate switching on and turning off of
genes as per the need of the cell.
• This type of regulation keeps order and
prevent wastes

• A gene is expressed through polypeptide synthesis. One
gene directs formation of one polypeptide chain.
• Concentration of particular protein varies even in a
simple bacterial cell ( E.coli) , with time and nature of
nutrients present in medium (environment) ,suggesting
regulation of gene expression.

PRINCIPLES OF GENE REGULATION
• 1) RNA polymerase binds to DNA at promoters.
2)Transcription initiation is regulated by proteins that
bind to or near promoters.

STEPS INVOLVING REGULATION 0F
GENE EXPRESSION
• POSTTRANSLATIONAL CONTROL
• TRANSCRIPTIONAL CONTROL
• TRANSLATIONAL CONTROL

Reproduce rapidly, short life span =
BACTERIA (Prokaryotes):
1 circular chromosome (dsDNA) plus accessory
genes carried on small circular plasmids
can affect evolution quickly
new mutations
Associated proteins cause the DNA to form a dense
“supercoil” region called the nucleoid

The basic concept for how
transcription is controlled in
bacteria was given by Jacob and
Monad (1961) by Operon model by
studying lactose metabolism.
According to this model, some
prokaryotic genes involved in the
operation of a metabolic sequence
are clustered.
GENE REGULATION IN
PROKARYOTES

Gene Expression: Prokaryotes
• Operon – grouped genes that are transcribed together
– code for functionally similar proteins
• Key Players
• Promoter – section of DNA where RNA polymerase
binds
• Operator – Controls activation of transcription
– on off switch
– between promoter and genes for proteins
– structural genes

• Repressor protein – binds to operator to block
RNA polymerase and shut down transcription
• Turns off the operon
• Corepressor – keeps the repressor protein on
the operator
• Trp operon
• Inducer – pulls repressor off the operator
• Turns on the operon – lactose on the lac
operon
• Regulatory gene – produces the repressor
protein
• Structural genes – code for proteins

Cistron: Smallest unit of gene expression
Polycistronic mRNA: A single mRNA that encodes more
than one separately translated protein. eg - lac operon

GENE REGULATION IN
PROKARYOTES
 In prokaryotes the primary control point is
the process of transcription initiation .
 Different ways for regulation of gene
expression in bacteria:
• PROMOTOR RECOGNITION
• TRANSCRIPTIONAL ELONGATION(Attenuation)
Regulation of gene expression can be done
by some operon pathways such as
1.lac operon. 2.tryptophan operon.

Prokaryotic cells turn genes on and
off by controlling transcription.
• A promotor is a DNA segment that allows
a gene to be transcribed.
• An operator is a part of DNA that turns a
gene “on” or ”off.”
• An operon includes a promoter, an
operator, and one or more structural
genes that code for all the proteins
needed to do a job.
• Operons are most common in prokaryotes.
• The lac operon was one of the first examples
of gene regulation to be discovered.

Lac Operon concept
• There are three gene sequences which code for the
enzymes which digest lactose –
• β-galactosidase,
• β-galactoside permease and
• thiogalactoside transacetylase
• they are located next to each other, and are referred to
as lacZ, lacY, and lacA.
• When these genes are transcribed by mRNA, a single
mRNA strand is produced, and this strand is referred to
as polycistronic, as it codes for more than one protein.
• The lac operon is said to be an inducible operon, as
gene transcription is induced by the presence of
lactose.

Lac operon
 Structural gene
• lacZ : β- galactosidase (lactase)
• lacY: galactoside permease
• lacA: thiogalactoside transacetylase
 Inhibitor gene
• lacI: LacI (lac operon repressor protein)
Promoter site
transcription of structural genes
 Operator site
binding of lac repressor
Total length – Regulator+ Promoter + Operator + Structural genes
+Terminator = 6100 bp of DNA , promoter-100 bp, Operator -45 bp

REGULATION OF LAC OPERON
• The lac operon is additionally regulated through binding of the
cAMP-receptor protein, CRP (also termed the catabolite
activator protein, CAP) to sequences near the promoter domain
of the operon.
• The result is a 50 fold enhancement of polymerase activity
• Actually lac operon also contains some elements of positive
control as well.
• Positive control means some effector is required for
transcription.
• In lac system , the Catabolic Activator Protein (CAP ) is an
effector.
• This protein when combined with cyclic Adenosine Mono
Phosphate (cAMP ), facilitates the attachment of RNA
Polymerase (Transcriptase ) to the operon.

• The cyclic AMP (cAMP ) has a hormone like action in
Prokaryote and Eukaryote, it is called the second
messenger, a term denoting its hormone type function.
• When CAP is attached to CAP site , the transcriptase
enzyme has a chance to anchored to position P
(promoter) site.
• The transcription is initiated at the Operator sequence
and continues through structural genes until terminator is
reached at the right end of the A gene.
• The Lac repressor and CAP can not be simultaneously
attached to the operon.
• Facilitating attachment of RNA Polymerase to Promoter
site Transcription begins at Operator, proceeds through
Structural genes Z,Y and A and then terminates.

Catabolite Repression of the lac
Operon
• Lactose is not the preferred
carbohydrate source for E. coli.
• If lactose and glucose are present, the
cell will use all of the glucose before the
lac operon is turned on.
• This type of control is termed catabolite
repression.
• To prevent lactose metabolism, a
second level of control of gene
expression exists.

Operon
• The promoter of the lac operon has two
binding sites.
• One site is the location where RNA
polymerase binds.
• The second location is the binding site for
a complex between the catabolite activator
protein (CAP) and cyclic AMP (cAMP).
• The binding of the CAP-cAMP complex to
the promoter site is required for
transcription of the lac operon.

Operon • As the concentration of
glucose cAMP
• As the cAMP
the amount of complex
• This decrease in the complex
inactivates the promoter, and
the lac operon is turned off.
• Because the CAP-cAMP complex
is needed for transcription, the
complex exerts a positive
control over the expression of
the lac operon.

1. When glucose is present and lactose is absent the
E. coli does not produce β-galactosidase.
2. When glucose is present and lactose is present
the E. coli does not produce β-galactosidase.
3. When glucose is absent and lactose is absent the
E. coli does not produce β-galactosidase.
4. When glucose is absent and lactose is present the
E. coli does produce β-galactosidase.
Four situations are possible

TRYTOPHAN OPERON
The trp operon works in a very similar way, except it
is referred to as a repressible operon, because gene
expression is repressed by the presence of high
levels of the amino acid tryptophan.
It works under the same principles as negative
feedback in eukaryotes.

TRYPTOPHAN OPERON
• Discovered in 1953 by Jacques Monod and
colleagues, the trp operon in E. coli was the first
repressible operon to be discovered.
• This operon contains five structural genes: trp E, trp
D, trp C, trp B, and trp A, which encodes
tryptophan synthetase.
• It also contains a promoter which binds to RNA
polymerase and an operator which blocks
transcription when bound to the protein
synthesized by the repressor gene (trp R) that binds
to the operator

TRYTOPHAN OPERON
When low levels of tryptophan are present, the
conformation of the repressor protein does not
allow it to bind to the operator region, therefore
the genes which code for tryptophan are
transcribed as RNA polymerase can bind to the
DNA.
When high levels of tryptophan are present, the
organism not longer needs to produce it, and two
molecules of tryptophan bind to the repressor
protein, and change it’s conformation in such a way
which means that it can now bind to the DNA and
prevent transcription.

TYPES OF GENE REGULATION

Positive and Negative Gene Regulation
Negative
• Repressible: usually on but
can be inhibited trp
operon, allosteric
inhibition, tryptophan
present prevents its own
production. (anabolic)
• Inducible: usually off, but
can be turned on, an
inducer (a specific small
molecule, allolactose in the
lac operon) inactivates the
repressor and allows
transcription (catabolic)
 Positive
• E. coli prefer to use glucose for
energy, they will only use lactose
when glucose is in short supply
glucose cAMP binds to
regulatory protein “CAP” &
stimulates gene transcription
Positive gene regulation!
• The cAMP & CAP combination
allow RNA polymerase to bind to
the promoter sequence more
efficiently.
• Remember cAMP is regulating
the gene expression in the
bacteria

CONCLUSION
Regulation of Gene Expression in Prokaryotes: Cells have
2 main ways of controlling metabolism.
Regulation of enzyme activity
(“feedback inhibition”): the end-product inhibits the
enzyme at the beginning of the pathway.
Good for: immediate, short-term response.
Regulation of gene expression: end-product
represses expression of genes for all the enzymes
needed for the pathway (longer-term response).

The Operon Model:
Many Bacterial genes are turned on or off by changes in the
metabolic status of the cell. In 1961, a basic mechanism for this
control of gene expression was first described using E.coli
bacteria.
E.coli bacteria require the amino acid tryptophan.
They can get it from their surroundings or they can
produce it using a multistep pathway requiring 5
enzymes.
The 5 genes that code for these enzymes are clustered
together on the bacterial chromosome and make
up one transcription unit.
A single promoter serves all 5 genes.

Negative Gene Regulation: Certain operons are switched OFF by
the active form of repressor proteins. There are two types:
Repressible operons are usually “on” but can be inhibited when a
small molecule binds allosterically to a regulatory protein:
tryptophan (corepressor) binds the trp repressor protein which
can only then bind the operator and block transcription.
Inducible operons are usually “off” but can be stimulated when a
small molecule interacts with a regulatory protein: lactose (lac)
operon contains genes needed for the digestion of lactose. These
genes are not transcribed unless lactose is present. It converts to
its isomer allolactose which binds directly to the lac repressor,
removing it from the operator. Allolactose is an
_________________
inducer.

Positive Gene Regulation: Some operons are switched
ON by the active form of activator proteins.
cyclic AMP (cAMP) accumulates when glucose is scarce
in E.coli cells. This binds to the repressor:
catabolite activator protein (CAP) which in turn attaches
upstream of the lac operon promoter and directly
stimulates transcription by increasing affinity for RNA
polymerase. Purpose – limit use of lactose for food unless
glucose is scarce.
Dual Control:
The lac operon has negative control by the
lac repressor and positive control by
CAP.

VIRUSES
KEY CONCEPT
VIRAL STRUCTURES
TYPES OF VIRUSES
GENERAL VIRAL REPLICATIVE CYCLES

VIRUSES:
First identified in tobacco plants
(Tobacco Mosaic Virus)
Tiny: 1000’s can fit in one cell.
Two main parts:
genome and protein capsid
Obligate intracellular parasites:
to reproduce
must be inside host cell
Host range varies: wide range = many host species,
narrow = few (can be only one host species).

Evolutionary debate: May have evolved from plasmids
(small circular DNA molecules in bacteria and yeasts)
or transposons (DNA segments which move within a
cell’s genome). Like viruses, these are both mobile
genetic elements.

Viral Structure: Capsids are made from protein subunits
called capsomeres. There is usually a large number of
proteins, but the number of different kinds of proteins is
usually small.
Helical Viruses: capsid is formed from a repeating single
type of protein with the overall shape of a rigid rod.
Icosahedral Viruses: capsid is formed from 252 identical
protein molecules arranged in a polyhedron with 20
triangular facets – an icosahedron. Example:
Adenoviruses which cause respiratory infections in
animals.

Influenza Viruses: capsid is surrounded by a
membranous envelope derived from the membranes
of the host (Such membranes prevent detection by
the immune system).
Bacteriophages: (Viruses which infect bacteria)
Complex capsids. The first seven studied were
nicknamed T1 – T7. They have an elongated
icosahedral head and an elaborate protein tail.

General Viral Replicative Cycles:
Virus identifies host cell: lock and key fit with
receptor molecules on host cell
Viral Genome: Enters host via membrane fusion,
endocytosis or injection (depending on type of
virus).
Viral Genome: Produces proteins which
reprogram the cell to copy the viral genome and
produce viral proteins using host:
_nucleotides, enzymes, ribosomes, tRNAs,
amino acids, ATP, etc. ___.

Replicative Cycles of Bacteriophages (the best
understood of all viruses):
Lytic: rapid destruction
of host cell
Phages that can only
replicate this way are
called virulent.

Lysogenic: Viral DNA integrates into host chromosomes
(via crossing over),
becomes a prophage
and can reproduce
(when cell divides)
without destroying
cell.
Lysogenic viruses can: become lytic in response
to environmental triggers
Phages that can replicate in both
ways are called ___ temperate ___.

Replicative Cycles of Animal Viruses:
Animal Viruses are classified by their type of nucleic acid: ssDNA,
dsDNA, ssRNA, dsRNA
Most have RNA genome and an envelope.
Viral envelopes:
Made of viral ___ glycoproteins ___ embedded in host
membrane ___ (plasma or nuclear) ___.
Help virus enter host cell.
Enveloped viruses can exit host cells without killing them .
Sometimes (as in herpes viruses) copies of the viral DNA can
remain latent in host cells until a physical or emotional stress
triggers active virus production.

RNA as Viral Genetic Material:
Three types of single-stranded RNA animal viruses:
Class IV viruses: Genome can directly serve as mRNA
(translated into viral protein immediately after infection).
Class V viruses: Genome serves as a template for
mRNA synthesis (RNA to RNA transcription requires a
special enzyme which is contained within the capsid).

Class VI Retroviruses (like HIV): can’t transcribe or translate their
genome directly.
Use reverse transcriptase (enzyme) to make DNA from RNA
(backwards). They contain 2 identical ssRNAs and 2 molecules of
reverse transcriptase within their enveloped capsids.
Newly made viral DNA integrates into the host DNA -- called a
___ provirus ___ (Unlike prophages, however, it never leaves
the host DNA.)
Then, the proviral DNA uses host RNA polymerase to transcribe
viral mRNA which can be translated into proteins or serve as new
viral genomes.
New viruses are assembled and released from the cell without

HIV Life Cycle

Virus components are toxic:
Three main ways viruses damage animal cells:
Release of hydrolytic enzymes from cell’s lysosomes
toxins
Viral activity causes cell to release:
especially envelope
proteins

Emerging Viruses seem to appear suddenly: 1918 Flu,
Ebola (1976), HIV (AIDS) in 1980’s, West Nile (1999),
H1N1 in 2009, can cause epidemics, or even
pandemics.
“New” epidemics are caused by: mutation of existing
viruses, dissemination from a small isolated group
(globalization, drug use…), spread from one species to
another (“swine flu”).
West Nile
Distribution

Vertical =
Plant Viruses: Cause agriculture damage. Most have RNA
Horizontal = plant is infected from external source
(often after epidermal damage)
plant inherits viral infection from parent
genomes and helical or icosahedral capsids.
Two routes for spreading viral diseases:
Once a virus enters a plant cell and replicates, it can
pass to other cells through plasmodesmata (some
virally encoded proteins aid this movement).

The Simplest Infectious Agents: Pathogens that
are even smaller and simpler than viruses include:
Viroids: Naked circular RNA strands which infect
plants, replicate, and cause errors in the regulatory
systems which control plant growth.

Prions: proteins that cause degenerative brain diseases
in animal hosts (such as mad cow disease): They are
most likely transmitted in food. They act very slowly
(incubation can lasts at least ten years) and are virtually
indestructible.
Current model on how they work: Prions are a
misfolded form of a protein normally found in brain
cells which triggers other proteins to convert to prions.
The prion aggregation then interferes with normal cell
functions.
Creutzfeldt-Jakob disease
(human variant of mad cow disease)

REFERENCES https://opentextbc.ca/biology2eopenstax/cha
pter/prokaryotic-gene-regulation

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