1. The Operon Model

2. 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
CONSTITUTIVE
Essential genes are always expressed in the cell (rRNAs, tRNAs, ribosomal proteins, RNA polimerases, etc)
GENES
REGOLATED
Whose activity is regulated depending upon specific requirements

3. To regulate gene expression
Bacterias must recognize the environmental conditions in which activate or repress specific genes.
Bacterias must be able able to activate or repress specific genes or set of genes coordinately.

4. 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.

5. 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)

6. 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

7. 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

8. 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

9. 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

10. Structure of the genomic region
The term OPERON indicates a cluster of genes with related functions and regulated in a coordinated manner

11. 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

12. Inducible systems: POSITIVE REGULATION

13. INDUCIBLE SYSTEMS: POSITIVE REGULATION
INDUCER ABSENT
INDUCER PRESENT
INDUTTORE

14. INDUCIBLE SYSTEMS: NEGATIVE REGULATION

15. INDUCIBLE SYSTEMS: NEGATIVE REGULATION
INDUCER
operatore

16. 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

17. Partial diploid for mutations of lacOc
lacI+ P O+ Z- Y+
F’ lacI+ P Oc Z+ Y−
GENOTYPE:
lacI+ P Oc Z+ Y-
lacI+ P O+ Z− Y+
PLASMID F’
BACTERIAL Chromosome

18. + − (mutated form) 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

19. Partial diploid for mutations lacI−
lacI+ P O+ Z− Y+
F’ lacI− P O+ Z+ Y−
GENOTYPE:
lacI− P O+ Z+ Y−
lacI+ P O+ Z− Y+
PLASMID F’
BACTERIAL CHROMOSOME

20. THE MUTATION lacI+ IS TRANS-DOMINANT on lacI−
lacI+ P O+ Z− Y+
F’ lacI− P O+ Z+ Y−
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

21. NEGATIVE REGULATION MODEL
NO LACTOSE

22. WITH LACTOSE

23. Does the model explain the mutants?
MUTANTS lacOc in the absence of LACTOSE

24. CONSTITUTIVE MUTANTS lacI-

25. The model with partial diploids
lacI+ P O+ Z- Y+ A+
F’ lacI+ P Oc Z+ Y- A+
GENOTYPE
NO LACTOSE
NO INDUCER
b-galactosidase +
permease
− (mutated)

26. lacI+ P O+ Z- Y+ A+ F’ lacI+ P Oc Z+ Y- A+ GENOTYPE WITH LACTOSE
WITH INDUCER
b-galactosidase +
permease

27. The second partial diploid analyzed
lacI+ P O+ Z− Y+ A+
F’ lacI− P O+ Z+ Y− A+
GENOTYPE
NO LACTOSE
SENZA INDUTTORE
b-galactosidase −
permease

28. lacI+ P O+ Z− Y+ A+ F’ lacI− P O+ Z+ Y− A+ GENOTYPE WITH LACTOSE
CON INDUTTORE
b-galactosidase +
permease

29. Regulatory mutants identified
GENE
MUTATION
PHENOTYPE
lacI
lacI-
synthesis constitutive of 3 enzymes
lacO
lacOc
lacIs
No synthesis even with lactose
lacP
lacP-
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

30. 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

31. The positive regulatory model
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
CAP (Catabolite Activator Protein)
cAMP (AMPcyclic)
cAMP-CAP binding increases the affinity of CAP for a site adjacent to lacP
RNA polymerase
The binding of the CAP-cAMP complex to DNA favors RNA polymerase recruitment to the promoter
CAP and cAMP are involved in operons of arabinose and galactose

32. 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

33. Corismic acid ->Tryptophan
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
P262

34. 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.

35. Palindromic seq. rich in G:C followed by A:T
Transcriptional attenuation
trpR P O
trpE
trpD
trpC
trpB
trpB
leader (14AA)
Attenuator
Palindromic seq. rich in G:C followed by A:T 1 2 3 4
codon trp
leader
162 nt
mRNA
Second level of regulation -> attenuation
The presence of the tRNA-trp loaded causes premature termination
of operon transcription -> truncated transcript (140nt)

36. 1 2 3 4
mRNA 1 2 3 4
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 1 2 3 4
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?

37. The ribosome behaviour during translation of the leader peptide dictates the activity of the RNA polymerase 1 2 3 4
mRNA
Leader peptide
AUG
UGA
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 4 1 2
AUG
UGA
WITH TRYPTOPHAN -> Termination stem-loop->OPERON TRP NOT TRANSCRIBED

38. 1 2 3 4
mRNA
Leader peptide
AUG
UGA
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 3 4 1
AUG
UGA

39. RNA Polymerase terminates transcription
WITH TRYPTOPHAN -> ATTENUATION ->OPERON trp ATTENUATED 2 3 4
RNA Polymerase terminates
transcription
3-4 STEM-LOOP TERMINATION
ABSENCE OF TRYPTOPHAN -> 2-3 LOOP ->OPERON trp NOT ATTENUATED 2 3
RNA Polymerase moves on

40. 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