Lac Operon - Classic Gene Regulation Model:

(a) Components:

Structural genes (transcribed together):

• lacZ: Codes for β-galactosidase (cleaves lactose → glucose + galactose)
• lacY: Codes for permease (transports lactose into cell)
• lacA: Codes for transacetylase (modifies lactose metabolites)

Regulatory sequences:

• Promoter (P): RNA polymerase binding site
• Operator (O): Repressor binding site (overlaps promoter)
• CAP-cAMP binding site: Positive control element

Regulatory gene:

• lacI: Codes for lac repressor protein (located upstream, has own promoter)

Structure:

    lacI gene         CAP site  P    O      lacZ    lacY    lacA
5'—————[——]—————————[——][——][——]———[——]———[——]———[——]———3'
         ↓                                   ↓       ↓       ↓
    Repressor                          β-gal   Permease  Acetylase

(b) Absence of Lactose (Operon OFF):

Situation: No lactose available, don't need lac enzymes

Step 1: lacI gene constitutively expressed

• lac repressor protein continuously made
• Repressor is active (no lactose to inactivate it)

Step 2: Repressor binds to operator

• Blocks RNA polymerase from transcribing
• Steric hindrance - polymerase can't proceed
• Negative control (repressor blocks transcription)

Step 3: No transcription of structural genes

• lacZ, lacY, lacA not transcribed
• No β-galactosidase, permease, or transacetylase made
• Cell doesn't waste energy making unneeded enzymes

State: Repressed (OFF)

         Repressor (active)
              ↓ binds
    P    [O]  lacZ  lacY  lacA
————[——][🛑]————————————————
         ↑
    RNA pol blocked
    
Result: NO TRANSCRIPTION

(c) Presence of Lactose (Operon ON):

Situation: Lactose available, need enzymes to metabolize it

Step 1: Lactose enters cell (basal permease)

• Small amount of permease always present
• Lactose converted to allolactose (by basal β-gal)

Step 2: Allolactose binds repressor

• Acts as inducer
• Causes conformational change in repressor
• Repressor can no longer bind operator
• Inactivates repressor

Step 3: Operator is free

• RNA polymerase can now bind promoter
• Transcription proceeds

Step 4: Structural genes transcribed

• Single polycistronic mRNA produced
• Contains all three genes (lacZ, lacY, lacA)

Step 5: Translation

• β-galactosidase: breaks down lactose
• Permease: imports more lactose
• Transacetylase: detoxifies metabolites

State: Induced (ON)

Lactose → Allolactose
                ↓ binds
         Repressor (inactive)
                      
    P    O   lacZ  lacY  lacA
————[——][——]———————————————
    ↓
RNA pol transcribes
    ↓
mRNA → Proteins

Result: ACTIVE TRANSCRIPTION

(d) CAP-cAMP and Glucose Repression:

Concept: Even with lactose, operon works poorly if glucose present

Why? Glucose is preferred carbon source

• Catabolite repression (glucose effect)
• Cell prefers glucose over lactose (more efficient)

Mechanism - Positive Control:

When glucose is LOW:

Step 1: cAMP levels increase

• Glucose inhibits adenylyl cyclase
• No glucose → enzyme active → more cAMP

Step 2: cAMP binds CAP (Catabolite Activator Protein)

• CAP = CRP (cAMP Receptor Protein)
• CAP-cAMP complex forms

Step 3: CAP-cAMP binds near promoter

• Enhances RNA polymerase binding
• Bends DNA, helps position RNA pol correctly
• Positive regulation (stimulates transcription)

Step 4: Strong transcription

• With lactose (repressor off) AND CAP-cAMP (enhancer on)
• Maximum expression of lac operon

When glucose is HIGH:

Step 1: cAMP levels decrease

• Glucose present → adenylyl cyclase inhibited

Step 2: Little CAP-cAMP complex

• CAP without cAMP doesn't bind DNA well

Step 3: Weak transcription

• Even if lactose present (repressor off)
• RNA polymerase binds poorly without CAP-cAMP
• Low expression of lac operon

Four States:

| Glucose | Lactose | CAP-cAMP | Repressor | Transcription | |---------|---------|----------|-----------|---------------| | High | Absent | No | Bound | None (OFF) | | High | Present | No | Off | Low (weak ON) | | Low | Absent | Yes | Bound | None (OFF) | | Low | Present | Yes | Off | High (strong ON) |

Logical operation: $\text{Transcription} = \text{(NOT repressor)} \land \text{CAP-cAMP}$

Complete regulation diagram:

LOW GLUCOSE + LACTOSE PRESENT = MAXIMUM TRANSCRIPTION

         cAMP (high)
            ↓
         CAP-cAMP (forms)
            ↓ binds
    [CAP site]  P    O   lacZ
————[——✓——]—[——][——]———————
                 ↑
        Repressor OFF (lactose bound it)
        RNA pol binds strongly
        ↓
    Strong transcription +++

HIGH GLUCOSE (even with lactose) = LOW TRANSCRIPTION

         cAMP (low)
            ↓
    [CAP site]  P    O   lacZ  
————[——✗——]—[——][——]———————
                 ↑
        Repressor OFF (lactose bound it)
        RNA pol binds weakly
        ↓
    Weak transcription +

Key Concepts:

Negative control: Repressor blocks (default OFF) Positive control: CAP-cAMP enhances (booster)

Inducible operon: Turned ON by substrate (lactose)

• Makes sense: only make enzymes when substrate available

Contrast with repressible operon (trp):

• Turned OFF by end product (tryptophan)
• Makes sense: don't make enzymes when product abundant

$\boxed{\text{lac operon: Induced by lactose, enhanced by low glucose (CAP-cAMP)}}$

Evolutionary advantage:

• Saves energy (only make what's needed)
• Adapts to environment quickly
• Coordinate regulation of related genes