GENE EXPRESSION

REGULATION of PROCARYOTES
& VIRUSES
Fera Ibrahim & T. Mirawati Sudiro
DEPARTEMEN MIKROBIOLOGI FKUI

Regulation of Gene Expression

-Cellular function is influenced by cellular environment.
Adaptation to specific environments is achieved by regulating
the expression of genes that encode the enzymes and proteins
needed for survival in a particular environment.
-Factors that influence gene expression include
nutrients, temperature, light, toxins, metals, chemicals,
and signals from other cells.
-Malfunctions in the regulation of gene expression can cause
various human disorders and diseases.

GENE EXPRESSION
GENE (DNA)
GENETIC MATERIAL
THE CHARACTER

RNA

GENOTYPE

PROTEIN

PHENOTYPE

GENE EXPRESSION ON OR OFF FOR ADAPTATION TO

SPECIFIC ENVIRONMENTS

Details of The process are

different in
eucaryotes/procaryotes

REGULATION OF GENE EXPRESSION IN CELLS

Transcription

DNA

(Initiation)

RNA TRANSCRIPT
RNA stability

RNA processing
mRNA

Translation

PROTEIN
Post-Translation
FUNCTION PERFORMED BY PROTEIN

GENE EXPRESSION
CONSTITUTIVE GENE EXPRESSION
genes that are always active
genes that are always "turned on"
genes are always needed
eg: genes that code for enzymes of glycolytic pathway
Constitutive genes
cellular housekeeping functions
tRNA, rRNA, ribosomal protein, RNA polymerase subunit
INDUCIBLE OR REPRESSIBLE GENE EXPRESSION
INDUCTION
enzymes for catabolic pathways (degradation)
synthesized only when needed (Expression occurs only
when substrate enzymes present ) induction
REPRESSION
enzymes from anabolic pathways (synthesis)
If product presents, gene expression turn off repression
( ADAPTIVE)

INDUCIBLE AND REPRESSIBLE GENE EXPRESSION

REGULATION OF GENE EXPRESSION

REGULATORY GENE
REGULATORY PROTEIN
EFFECTOR MOLECULES
Activator
Inducers

MECHANISME OF
POSITIVE CONTROL
TURN ON

Repressor

EFFECTOR MOLECULES
Co-repressor

MECHANISME OF
NEGATIVE CONTROL
TURN OFF

STRUCTURAL GENE EXPRESSION

INDUCTION REPRESSION

REGULATION OF GENE
EXPRESSION IN
PROKARYOTES

GENE ORGANIZATION
OF PROKARYOTES
REGULATORY GENE
OPERON :
The gene cluster and promoter,
plus additional sequences
that function together in regulation
Promotor
Operator
Structural Gene

Jacob and Monod proposed the Operon Model for gene regulation

Gene Regulation in Prokaryotes

Bacteria have a simple general mechanism for coordinating
the regulation of genes that encode products involved in a set
of related processes.

A. some genes not regulated

-constitutive genes = genes that are always active
-genes that are always "turned on"
-genes are always needed
-eg: genes that code for enzymes of glycolytic pathway
B. some genes are regulated
-some genes only needed under certain conditions
-transcription can be "turned on or off"
-enzymes produced from these genes vary greatly in
cytoplasmic concentration

Types of regulation
A. Inducible operon
- Induction is associated with catabolic pathways
-enzymes for a given catabolic pathway synthesized only when needed
-eg: catabolism of lactose
B. Represible operon
- Repression is associated with anabolic pathways
-focus on the "end products" of anabolic pathways
-amount enzyme varies inversely with amount of end product in cell
- This operon is normally in on mode, and will be turned off only when the end
product is no longer required
- Excess product plays a role as a corepressor, that slows the transcription of the
operon
-eg: synthesis of amino acids, purines and pyrimidines in cell

Types of regulation

a. Induction is associated with catabolic pathways

Catabolism of lactose
- Regulated by lactose operon
-lactose is not always present in the growth media
-lactose is not present in growth media
-bacteria do not make beta-galactosidase
-bacteria do not make permease
-lactose is present in growth media
-both galactosidase and permease are synthesized by cell
-galactosidase and permease regulated together
-enzyme synthesis "turned on" in presence lactose
-substrate induction = turning on enzyme synthesis
(transcription + translation) in the presence of the substrate
-inducible enzymes = enzymes whose synthesis requires
the presence of an inducer (usually a substrate)
-MOST catabolic pathways in bacteria are subject to
substrate induction

A. In the absence of lactose:

- A repressor ataches to the operator of
the operon --- locks the operator --suppress transcription of structural
proteins downstream of it
(OPERON OFF)
B. In the presence of lactose
- Lactose as genetic inducer --attaches to the receptor inactive
repressor released from the operator
- RNA polymerase bind to the
promoter and initiate transcription

Repressible operon
Example: regulation of arginine synthesis
A. Operon On
- A repressible operon remains on when its
nutrient pruduct (here: arginine) are in
great demand by the cells

B. Operon Off.
The operon is repressed when:
- Arginine builds up --- as a corepressor
--- activates the receptor
- The repressor complex binds to the
operator ---- block RNA polymerase --transcription blocked

ATTENUATION

Attenuation
- Attenuation regulates the termination of transcription as a
function of tryptophan concentration.
- At low levels of trp full length mRNA is made, at high levels
transcription of the trp operon is prematurely halted.
- Attenuation works by coupling transcription to translation.
- Prokaryotic mRNA does not require processing and since
prokaryotes have no nucleus, translation of mRNA can start
before transcription is complete.
- Consequently regulation of gene expression via attenuation is
unique to prokaryotes.

a. Attenuation is mediated by the formation of one of two

possible stem-loop structures in a 5' segment of the trp operon in
the mRNA.
b. If tryptophan concentrations are low then translation of the leader
peptide is slow and transcription of the trp operon outpaces translation.
This results in the formation of a nonterminating stem-loop structure
between regions 2 and 3 in the 5' segment of the mRNA. Transcription
of the trp operon is then completed.
c. If tryptophan concentrations are high the ribosome quickly translates
the mRNA leader peptide.
Because translation is occurring rapidly the ribosome covers region 2
so that it can not attach to region 3.
Consequently the formation of a stem-loop structure between regions
3 and 4 occurs and transcription is terminated.

EFFECTOR MOLECULES
-substrate induction uses effector molecules
-eg: lactose (the substrate) is the small molecule that
"turns on" the genes coding for the enzymes of the
pathway
-end-product repression uses effector molecules
-eg: tryptophan is the small molecule that "turns off"
the genes coding for the enzymes of the pathway
-effector molecules = small molecules that trigger the
activation or deactivation of a gene or group of genes
-effector molecules of catabolic pathways
-usually substrates
-act as inducers
-effector molecules of anabolic pathways
-usually end-products
-act as repressors

Dual Control of Inducible Operons

A. inducible operons often under dual control
(1) eg: lac operon
a. lac operon subject to negative control by a repressor
-allows the presence of lactose to "turn on" the system
b. but, lac operon also subject to positive control by CRP
-allows the "turn on" to only occur under appropriate
conditions

Mechanism of catabolite repression

a. cells have cyclic AMP Receptor Protein (CRP) chime
b. cAMP allosterically activates CRP
c. activated CRP binds to a specific DNA sequence (C)
just upstream from the lac promoter (Plac)
d. bound CRP greatly facilitates RNA polymerase binding
and transcription

Sigma Factors
A. bacterial RNA polymerase uses a sigma factor
(1) sigma factors help control initiation of transcription
-sigma factor binds to RNA polymerase
-sigma factor helps RNA polymerase find the promoter
-bacterial cells have different types sigma factor specific
for sets of genes
-sigma factor 70 (MW = 70 kDa) is most common form
-initiates transcription at most promoters
-sigma factor 32 (MW = 32 kDa) is produced after heat shock
-initiates transcription at promoters of genes needed for
responding to heat
-sigma factor 54 turns on genes for nitrogen utilization
-bacteriophage produces a powerful sigma factor that
preferentially transcribes the phage DNA instead of the
bacterial DNA

Genetic regulation and protein expression of viruses

Viruses are obligate intracellular parasites

- Bacteriophages
- Human and animal viruses

Phages reproduce using

lytic or lysogenic cycles
While phages are the best understood
of all viruses, some of them are also
among the most complex.
Research on phages led to the
discovery that some double-stranded
DNA viruses can reproduce by two
alternative mechanisms: the lytic
cycle and the lysogenic cycle.

 In the lytic cycle, the phage

reproductive cycle culminates in
the death of the host.
In the last stage, the bacterium
lyses (breaks open) and releases
the phages produced within the cell
to infect others.

Virulent phages reproduce only

by a lytic cycle.

 In the lysogenic cycle, the phage

genome replicates without destroying
the host cell.
Temperate phages, like phage
lambda, use both lytic and lysogenic
cycles.
Within the host, the virus circular
DNA engages in either the lytic or
lysogenic cycle.
During a lytic cycle, the viral genes
immediately turn the host cell into a
virus-producing factory, and the cell
soon lyses and releases its viral
products.

 During the lysogenic cycle, the viral

DNA molecule is incorporated by
genetic recombination into a specific
site on the host cells chromosome.
In this prophage stage, one of its
genes codes for a protein that
represses most other prophage genes.
Every time the host divides, it also
copies the viral DNA and passes the
copies to daughter cells.
Occasionally, the viral genome exits
the bacterial chromosome and initiates
a lytic cycle.
This switch from lysogenic to lytic may
be initiated by an environmental
trigger.

 The lambda phage which infects E.

coli demonstrates the cycles of a
temperate phage.

B
A
C
T
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R
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O
P
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A
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Genetic stages in the multiplication

of double stranded DNA viruses
(simplified)
- The virus penetrates the host cell
and release the DNA
1. DNA enters nucleus
2. DNA transcription
3. Viral RNA is translated into
protein in citoplasma, proteins
enter nucleus
4. Viral DNA replicate repeatedly
in nucleus
5. Viral DNA and proteins
assembled into a mature virus
6. Some DNA viruses integrated to
host chromosomes

Replication of positive-strand single

stranded RNA (simplified)
1. Penetration and uncoating of viral
RNA
2. Positive-strand RNA is tranlated into
proteins
3. A negative genome is synthetized
against the positive template to
produce large numbers of (+) strand
RNA
4. Synthesize (+) strand RNA
5. Assembly of RNA strands and proteins
into mature virus

Genetic recombination
produces new bacterial strains
In addition to mutations, genetic
recombination generates diversity within
bacterial populations.
Here, recombination is defined as the
combining of DNA from two individuals into
a single genome.
Transmission of genetic material in
bacteria occurs through three processes:
transformation
transduction
conjugation

 The impact of recombination can be

observed when two mutant E. coli
strains are combined.
If each is unable to synthesize one of its
required amino acids, neither can grow on
a minimal medium.
However, if they are combined, numerous
colonies will be created that started as cells
that acquired the missing genes for amino
acid synthesis
from the other
strain.
Some may have
resulted from
mutation.

 Transformation is the alteration of a

bacterial cells genotype by the uptake
of naked, foreign DNA from the
surrounding environment.
For example, harmless Streptococcus
pneumoniae bacteria can be transformed
to pneumonia-causing cells.
This occurs when a live nonpathogenic cell
takes up a piece of DNA that happens to
include the allele for pathogenicity from
dead, broken-open pathogenic cells.
The foreign allele replaces the native allele
in the bacterial chromosome by genetic
recombination.
The resulting cell is now recombinant with
DNA derived from two different cells.

 Many bacterial species have

surface proteins that are
specialized for the uptake of naked
DNA.
These proteins recognize and
transport only DNA from closely
related bacterial species.
While E. coli lacks this specialized
mechanism, it can be induced to take
up small pieces of DNA if cultured in a
medium with a relatively high
concentration of calcium ions.
In biotechnology, this technique has
been used to introduce foreign DNA
into E. coli.

 Transduction occurs when a phage

carries bacterial genes from one host
cell to another.
In generalized transduction, a small
piece of the host cells degraded DNA is
packaged within a capsid, rather than
the phage genome.
When this phage attaches to another
bacterium, it will inject this foreign DNA into
its new host.
Some of this DNA can subsequently replace
the homologous region of the second cell.
This type of transduction transfers bacterial
genes at random.

 Specialized transduction
occurs via a temperate phage.
When the prophage viral genome is
excised from the chromosome, it
sometimes takes with it a small
region of adjacent bacterial DNA.
These bacterial genes are injected
along with the phages genome into
the next host cell.
Specialized transduction only
transfers those genes near the
prophage site on the bacterial
chromosome.

 Both generalized and specialized

transduction use phage as a vector to
transfer genes between bacteria.

 CONJUGATION
Conjugation transfers genetic material
between two bacterial cells that are
temporarily joined.
One cell (male) donates DNA and its
mate (female) receives the genes.
A sex pilus from the male initially joins the
two cells and creates a cytoplasmic bridge
between cells.
Maleness, the ability to form
a sex pilus and donate DNA,
results from an F factor as a
section of the bacterial
chromosome or as a plasmid.

 Plasmids, including the F plasmid, are

small, circular, self-replicating DNA
molecules.
Episomes, like the F plasmid, can undergo
reversible incorporation into the cells
chromosome.
Temperate viruses also qualify as episomes.

Plasmids generally benefit the bacterial cell.

They usually have only a few genes that are
not required for normal survival and
reproduction.
Plasmid genes are advantageous in stressful
conditions.
The F plasmid facilitates genetic recombination when
environmental conditions no longer favor existing
strains.

F plasmid & Hfr transfer

The F factor or its F plasmid consists of about 25
genes, most required for the production of sex pili.
Cells with either the F factor or the F plasmid are called
F+ and they pass this condition to their offspring.
Cells lacking either form of the F factor, are called F-, and
they function as DNA recipients.

When an F+ and F- cell meet, the F+ cell passes a copy of

the F plasmid to the F- cell, converting it.

F plasmid & Hfr transfer

The plasmid form of the F factor can

become integrated into the bacterial
chromosome.
The resulting Hfr cell (high frequency
of recombination) functions as a male
during conjugation.

 The Hfr cell initiates DNA replication

at a point on the F factor DNA and
begins to transfer the DNA copy
from that point to its F- partner
Random movements almost always
disrupt conjugation long before an
entire copy of the Hfr chromosome
can be passed to the F- cell.

 In the partially diploid cell, the newly

acquired DNA aligns with the
homologous region of the Fchromosome.
Recombination exchanges segments
of DNA.
This recombinant bacteria has genes
from two different cells.

Antibiotic resistance transfer

In the 1950s, Japanese physicians began to
notice that some bacterial strains had
evolved antibiotic resistance.
The genes conferring resistance are carried by
plasmids, specifically the R plasmid (R for
resistance).
Some of these genes code for enzymes that
specifically destroy certain antibiotics, like
tetracycline or ampicillin.

When a bacterial population is exposed to

an antibiotic, individuals with the R plasmid
will survive and increase in the overall
population.
Because R plasmids also have genes that
encode for sex pili, they can be transferred
from one cell to another by conjugation.

TRANSPOSON
A transposon is a piece of DNA that can
move from one location to another in a cells
genome.
Transposon movement occurs as a type of
recombination between the transposon and
another DNA site, a target site.
In bacteria, the target site may be within the
chromosome, from a plasmid to chromosome (or
vice versa), or between plasmids.

Transposons can bring multiple copies for

antibiotic resistance into a single R plasmid
by moving genes to that location from
different plasmids.
This explains why some R plasmids convey
resistance to many antibiotics.

 Some transposons (so called jumping

genes) do jump from one location to
another (cut-and-paste translocation).
However, in replicative transposition,
the transposon replicates at its original
site, and a copy inserts elsewhere.
Most transposons can move to many
alternative locations in the DNA,
potentially moving genes to a site
where genes of that sort have never
before existed.

 The simplest bacterial transposon, an

insertion sequence, consists only of the DNA
necessary for
the act of transposition.
The insertion sequence consists of the
transposase gene, flanked by a pair of
inverted repeat sequences.
The 20 to 40 nucleotides of the inverted repeat on
one side are repeated in reverse along the opposite
DNA strand at the other end of the transposon.

 The transposase
enzyme recognizes
the inverted repeats
as the edges of the
transposon.
Transposase cuts
the transposon from
its initial site and
inserts it into the
target site.
Gaps in the DNA
strands are filled in
by DNA polymerase,
creating direct
repeats, and then
DNA ligase seals the
old and new material.

 Insertion sequences cause

mutations when they happen to
land within the coding sequence
of a gene or within a DNA region
that regulates gene expression.
Insertion sequences account for
1.5% of the E. coli genome, but a
mutation in a particular gene by
transposition is rare, about 1 in
every 10 million generations.
This is about the same rate as
spontaneous mutations from
external factors.

 Composite transposons (complex

transposons) include extra genes
sandwiched between two insertion
sequences.
It is as though two insertion sequences
happened to land relatively close together
and now travel together, along with all the
DNA between them, as a single transposon.

 While insertion sequences may not

benefit bacteria in any specific way,
composite transposons may help
bacteria adapt to new environments.
For example, repeated movements of
resistance genes by composite
transposition may concentrate several
genes for antibiotic resistance onto a
single R plasmid.
In an antibiotic-rich environment,
natural selection factors bacterial
clones that have built up composite R
plasmids through a series of
transpositions.

GENETIC EXCHANGE BETWEEN VIRAL PARTICLES

Rekombinasi
- ds DNA virus berekombinasi lebih efisien
- Virus RNA (misal coronavirus) : rekombinasi terjadi pada
proses transkripsi
- Terjadi antar virus dalam genus yang sama

Genome segment reassortment

- genetic reassortment
- reovirus, influenza virus, bunyavirus, arenavirus
- Pada virus influenza menyebabkan antigenic shift
subtipe baru

Influenza pandemics &

epidemics in man

Adapted from Mandell, Douglas and Bennetts Principles and Practice of Infectious Diseases, 5th
ed. 2000:1829. Modified from Kilbourne ED. Influenza. 1987:274

Influenza Virus: ss RNA, segmented

INFLUENZAVIRUS
REPLICATION

EMERGING OF NEW
INFLUENZA VIRUS
STRAINS
-ANTIGENIC DRIFT
-ANTIGENIC SHIFT

Postulated evolution of human influenza A viruses

from 1889 to 1977

Genetic reassortment between avian and human

influenza viruses in swine
1979

Complementation
- Dua mutan dengan kerusakan gen yang berbeda dapat
berbiak bila diinfeksi pada sel yang sama bila produk gen
yang diperlukan untuk multiplikasi tersebut dapat
terpenuhi

Phenotypic mixing dan phenotypic masking

- Salah satu contoh dari komplementasi

- Bila 2 virus yang berkerabat (misal poliovirus 1 dan poliovirus 3)

menginfeksi yang sama, genom virus yang terbentuk dapat
terkapsidasi oleh kapsidnya sendiri atau kapsid hibrid

PRION

Slow virus infection and prion diseases

Disease

Agent

Subacute sclerosing
measles virus variant
panencephalitis
Progressive multifocal Polyomavirus (JCV)
leukoencephalopathy
Creutzfeldt-Jakob disease Prion
Kuru

Prion

Visna
Retrovirus
Scrapie
Prion
Bovine spongiform
Prion
encephalopathy
Transmissible mink
Prion
encephalopathy
Chronic wasting disease Prion

Host

Human
Human
Human, chimpanzees,
monkeys
Human, chimpanzees,
monkeys
sheep
Sheep, goats, mice
cattle
mink, other animals
mule, deer, elk

PRION
STRUCTURE AND PHYSIOLOGY

Prion lacks detectable nucleic acid, consist of aggregates of proteaseresistant, hydrophobic glycoprotein (PrPSc).
Human and animals have a 33-35 kD protein (PrPc) that has identical
peptide sequence with PrPSc but different in other characters.
PrPSc : protease-resistant, in cytoplasma ( Mutated prion )
PrPc : protease-sensitive, in cell surface ( Normal protein )
Mutated prion : Resistant to a wide range of chemical
and physical treatment (e.g. formaldehyde, UV, heat to 80oC)
Sensitive to phenol 90%, ether, NaOH 2N,
10% sodium dodecyl sulphate,
5% hypochlorite solution, 1.0M sodium hydroxide and
autoclaving at 121oC 1 hour,

PRION (PRP) protein

245 aa - GPI linked glycoprotein
Highly expressed in NEURONS
Skeletal muscle, kidney, heart, lymphoid
tissue, leukocytes, intestinal tissues,
uterus , testis.

Cellular function

Conformational change = infectious

PRION Theory

PrPC

Sensitive to proteolysis
Soluble

PrPSC

Resistant to proteolysis
Insoluble

Associated w/cellular membrane

Located inside cells as aggregates

Mainly -Helical structure

Mainly -Sheet structure

Translation