Download The Operon Model

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
—  β-galactosidase (gene lacZ)
—  Divides lactose in galactose and glucose
—  Catalizes isomerization of lactose to allolactose
—  Lactose permease (gene lacY)
—  Enhance cellular lactose uptake
—  β-galactoside transacetylase (gene lacA)
—  trasfers an acetyl group to β-galactosides.
These are structural genes
—  Mutations in the 3 struttural 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+
BACTERIAL Chromosome
PLASMID F’
lacI+ P O+ Z− Y+
F’ lacI+ P Oc Z+ Y−
NO INDUCER
CON INDUTTORE
β-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+
BACTERIAL CHROMOSOME
PLASMID F’
lacI+ P O+ Z− Y+
F’
lacI− P O+ Z+ Y−
NO INDUCER
β-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
β-galactosidase
permease
+
−
(mutated)
NO LACTOSE
lacI+ P O+ Z- Y+ A+
GENOTYPE
F’ lacI+ P Oc Z+ Y- A+
WITH INDUCER
β-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
β-galactosidase
−
permease
−
NO LACTOSE
lacI+ P O+ Z− Y+ A+
GENOTYPE
F’ lacI− P O+ Z+ Y− A+
CON
INDUTTORE
β-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
Leader peptide (14AA)
3
4
attenuator
When deleted, the leader sequence determines increase of trp operon
With no effects on repression of the operator.
mRNA
Transcriptional attenuation
trpR
P
O
leader
trpE
trpD
trpC
trpB
trpB
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