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 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

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.

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 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

+ − (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

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

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

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+ F’ lacI+ P Oc Z+ Y- A+ GENOTYPE NO LACTOSE NO INDUCER b-galactosidase + permease − (mutated)

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

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

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

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

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 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

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

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

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.

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)

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?

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

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

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

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