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The Operon Model
 Bacteria adapt to changes in environmental conditions
 Adaptation requires the capacity to quickly express the
genes necessary to cope with specific environmental
stimuli
 Advantage: saving energy, faster growth and better
use of available resources
Essential genes are always expressed in CONSTITUTIVE
the
cell
(rRNAs, tRNAs, ribosomal proteins, RNA
polimerases, etc)
GENES
Whose activity is regulated depending upon
specific requirements
REGOLATED
To regulate gene expression
1. Bacterias must recognize the environmental conditions
in which activate or repress specific genes.
2. Bacterias must be able able to activate or repress
specific genes or set of genes coordinately.
Control of proteins to use sugars
 Bacterias can use different sugars as carbon and energy
sources
 (glucose, lactose, arabinose, xylose, etc.)
 The proteins required for sugar metabolism include
 Those favouring sugars uptake in the cell
 Those catalyzing the sugars degradation.
Regulation of lactose catabolism in E.
coli
 Lactose metabolism was studied in details in the 1950s
by François Jacob and Jacques Monod
 The description of the transcriptional control system
had an enormous scientific value (Nobel prize in 1965)
 E. coli grows on minimal medium containing glucose
 The genes of glucose metabolism are constitutive,
glycolysis is a fundamental process
 If we add lactose to a minimal medium, instead of
glucose, E. coli syntetizes enzymes necessary to
metabolize this sugar
Enzymes induced by lactose
 b-galactosidase (gene lacZ)
 Divides lactose in galactose and glucose
 Catalizes isomerization of lactose to allolactose
 Lactose permease (gene lacY)
 Enhance cellular lactose uptake
 b-galactoside transacetylase (gene lacA)
 trasfers an acetyl group to b-galactosides.
These are structural genes
 Mutations in the 3 structural genes (lacZ, lacY e
lacA)
 mutations in lacZ−, lacY−, lacA− were mapped with classic
techniques;
 The 3 genes are strictly linked:
lacZ−lacY−lacA
 The 3 genes are transcribed in one mRNA (polycistronic
or polygenic).
 Mutations affecting regulation of all 3 structural
genes
 Constitutive Mutants
 The structural genes are always expressed, in the presence
or absence of lactose
 Mutants blocking the expression of structural genes
even in the presence of lactose
Mapping of constitutive mutants
Two classes:
1a class:
mapping on a small region upstream
of lacZ called Operator (lacO)
2a class:
mapping upstream of Operator in a
gene called lacI, coding for a
repressor
Structure of the genomic region
The term OPERON indicates a cluster of genes
with related functions and regulated in a
coordinated manner
Regulation
Catabolism/degradation (lac)
INDUCIBLE
ANABOLISMS/biosynthesis (trp)
REPRESSIBLE
REGULATORS
ACTIVATORS
REPRESSORS
Binds a regulatory regionin presence of
EFFECTOR MOLECULES
INDUCERS
CO-REPRESSORS
Influencing the three dimensional structure of regolators
Inducible systems:
POSITIVE REGULATION
INDUCIBLE SYSTEMS:
POSITIVE REGULATION
INDUCER ABSENT
INDUCER PRESENT
INDUTTORE
INDUCIBLE SYSTEMS:
NEGATIVE REGULATION
INDUCIBLE SYSTEMS:
NEGATIVE REGULATION
INDUCER
operatore
To define the role of each component
of the Operon, Jacob and Monod used
partially diploid strains
They used F’ strains carrying operon genes on
the F factor
They could define dominant and recessive
mutations
They made hypothesis on the role of each
operon region
Partial diploid for mutations of lacOc
GENOTYPE:
lacI+ P O+ Z- Y+
F’ lacI+ P Oc Z+ Y−
lacI+ P O+ Z− Y+
PLASMID F’
BACTERIAL Chromosome
lacI+ P O+ Z− Y+
F’ lacI+ P Oc Z+ Y−
NO INDUCER
CON INDUTTORE
b-galactosidase
+
+
permease
−
+
(mutated form)
 Lac Z is expressed constitutively
 Lac Y is subject to inducible control
A lacOc mutation alters genes downstream on the SAME
DNA molecule
These MUTATIONS are CIS-DOMINANT
The operator DOES NOT CODE FOR A DIFFUSIBLE PRODUCT or
one of the two alleles would control all genes of the lactose
pathway
Partial diploid for mutations lacI−
GENOTYPE:
lacI+ P O+ Z− Y+
F’
lacI− P O+ Z+ Y−
lacI+ P O+ Z− Y+
PLASMID F’
BACTERIAL CHROMOSOME
lacI+ P O+ Z− Y+
lacI− P O+ Z+ Y−
F’
NO INDUCER
b-galactosidase
−
permease
−
 The expression of both genes is inducible
 lacI+ is dominant on lacI−
BECAUSE lacI GENES ARE ON DIFFERENT DNA
MOLECULES (configuration in trans)
THE MUTATION lacI+ IS TRANS-DOMINANT on lacI−
Jacob e Monod hypothesized that the lacI gene codes for a
DIFFUSIBLE REPRESSOR
NEGATIVE REGULATION MODEL
NO LACTOSE
WITH LACTOSE
Does the model explain the mutants?
MUTANTS lacOc in the absence of LACTOSE
CONSTITUTIVE MUTANTS lacI-
The model with partial diploids
lacI+ P O+ Z- Y+ A+
GENOTYPE
F’ lacI+ P Oc Z+ Y- A+
NO INDUCER
b-galactosidase
permease
+
−
(mutated)
NO LACTOSE
lacI+ P O+ Z- Y+ A+
GENOTYPE
F’ lacI+ P Oc Z+ Y- A+
WITH INDUCER
b-galactosidase
+
permease
+
WITH LACTOSE
The second partial diploid analyzed
GENOTYPE
lacI+ P O+ Z− Y+ A+
F’ lacI− P O+ Z+ Y− A+
SENZA
INDUTTORE
b-galactosidase
−
permease
−
NO LACTOSE
lacI+ P O+ Z− Y+ A+
GENOTYPE
F’ lacI− P O+ Z+ Y− A+
CON
INDUTTORE
b-galactosidase
+
permease
+
WITH LACTOSE
Regulatory mutants identified
GENE
MUTATION
PHENOTYPE
lacI
lacI-
synthesis constitutive of 3 enzymes
lacO
lacOc
synthesis constitutive of 3 enzymes
lacI
lacIs
No synthesis even with lactose
lacP
lacP-
No synthesis even with lactose
La mutazione lacIs (super-repressor)
In the partial diploids (lacI+/lacIs) lacIs is TRANS-DOMINANT
blocking the synthesis of structural genes on both copies of the operon
The lactose operon has also a positive
regulatory system
 This enables that lactose operon genes are expressed
at high levles ONLY if lactose is the ONLY carbon
source and in the absence of glucose
 Glucose is preferred because it can be directly
available for glycolysis
 The other sugars must be converted into glucose to be
used
 These conversions require energy
The positive regulatory model
CAP
cAMP (AMPcyclic)
The regulatory protein CAP “feels” the
presence of glucose in the cell binding to cAMP
whose concentration is inversely correlated to
the amount of glucose
(Catabolite Activator Protein)
cAMP-CAP binding increases
the affinity of CAP for a site
adjacent to lacP
RNA polymerase
The binding of the CAPcAMP complex to DNA
favors RNA polymerase
recruitment to the promoter
CAP and cAMP are involved in
operons of arabinose and
galactose
Operons are very common in
prokaryotes
Allowing:
 Regulation of multiple genes involved in the same
metabolism at the same time
 Maintenance of the correct ratios of transcripts
 Quick response to environmental stimuli
Other examples:
 tryptophan
 arabinose
The Tryptophan operon
Repressible operon
trpR
P
O
trpE
trpD
trpC
trpB
trpB
repressor
active
repressor
inactive
trp
Corismic acid ->Tryptophan
The operon is under negative control of the repressor coded by the trpR
gene
Tryptophan acts as a corepressor activating the repressor and inhibiting
transcription
Transcriptional attenuation
trpR
P
O
trpE
trpD
trpC
trpB
trpB
leader
162 nt
codon trp
1
2
3
4
mRNA
Leader peptide (14AA)
attenuator
When deleted, the leader sequence determines increase of trp operon
With no effects on repression of the operator.
Transcriptional attenuation
trpR
P
O
trpE
trpD
trpC
trpB
trpB
leader
162 nt
codon trp
1
leader (14AA)
2
3
4
mRNA
Attenuator
Palindromic seq. rich in G:C followed by A:T
Second level of regulation -> attenuation
The presence of the tRNA-trp loaded causes premature termination
of operon transcription -> truncated transcript (140nt)
1
1
2
2
3
4
3
4
mRNA
Nascent RNA forms
stem-loop structures
followed by uraciles
Attenuator
(terminator of
transcription)
UUUUUUU
This cause a change in a RNA Pol conformation
with termination of transcription
HOWEVER…..if Segment 1 is not allowed
to pair with Segment 2, the latter pairs with
Segment 3. Segment 1 is single and the
terminator is not formed
ACTIVE TRANSCRIPTION
How does trp influence attenuation?
2
1
3
4
The ribosome behaviour during translation of the leader peptide
dictates the activity of the RNA polymerase
Leader peptide
1
AUG
UGA
2
3
4
mRNA
With enough trp is present, the ribosome synthesizes the leader peptide and will
reach the stop codon. The ribosome will stay on Segment 2 preventing it from
forming a pairing with Segment 3
3
AUG
1
UGA
4
2
WITH TRYPTOPHAN -> Termination stem-loop->OPERON TRP NOT TRANSCRIBED
Leader peptide
AUG
1
UGA
2
3
4
mRNA
If tryptophan is insufficient, the ribosome will stop
in front of the two Trp codons preventing Segment
1 to pair with Segment 2.
Hence Segment 2 pair with Segment 3
2
AUG
1
UGA
3
4
WITH TRYPTOPHAN -> ATTENUATION ->OPERON trp ATTENUATED
RNA Polymerase terminates
transcription
2
3
4
3-4 STEM-LOOP TERMINATION
ABSENCE OF TRYPTOPHAN -> 2-3 LOOP ->OPERON trp NOT ATTENUATED
RNA Polymerase moves on
2
3
Acting together, repression and attenuation coordinates the speed of
synthesis of aminoacids biosynthetic enzymes with aminoacids availability
and the global protein synthesis speed.
When trp is present at high concentrations, RNA polymerases not inhibited
by the repressor are unlikely to move beyond the attenuator sequence.
Repression reduces transcription about 70-fold and attenuation reduces it
further 8-10-fold: when both operates together, transcription can be reduced
some 600-fold. SYNERGISTIC EFFECT
Attenuation has a role in the regulation of biosynthesis of many aminoacids