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

DNA
Transcription
(Initiation)

RNA TRANSCRIPT
RNA processing
RNA stability 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 EFFECTOR MOLECULES
Inducers Activator Repressor Co-repressor

MECHANISME OF MECHANISME OF
POSITIVE CONTROL NEGATIVE CONTROL
TURN ON 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
E
R
I
O
P
H
A
G
E
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 F- chromosome.
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 Host
Subacute sclerosing measles virus variant Human
panencephalitis
Progressive multifocal Polyomavirus (JCV) Human
leukoencephalopathy
Creutzfeldt-Jakob disease Prion Human, chimpanzees,
monkeys
Kuru Prion Human, chimpanzees,
monkeys
Visna Retrovirus sheep
Scrapie Prion Sheep, goats, mice
Bovine spongiform Prion cattle
encephalopathy
Transmissible mink Prion mink, other animals
encephalopathy
Chronic wasting disease Prion mule, deer, elk
PRION
STRUCTURE AND PHYSIOLOGY

Prion lacks detectable nucleic acid, consist of aggregates of protease-

resistant, 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 PrPSC

Sensitive to proteolysis Resistant to proteolysis

Soluble Insoluble

Associated w/cellular membrane Located inside cells as aggregates

Mainly -Helical structure Mainly -Sheet structure