1. Lactose (allolactose) binds repressor
2. Repressor releases from operator
3. RNA polymerase transcribes genes
4. Lactose metabolized

Function: Inducible system - genes turned ON when substrate present

trp Operon (Repressible)

Tryptophan synthesis genes

Without tryptophan (ON):

1. Repressor inactive (can't bind operator)
2. RNA polymerase transcribes genes
3. Tryptophan synthesized

With tryptophan (OFF):

1. Tryptophan (corepressor) binds repressor
2. Activated repressor binds operator
3. Blocks transcription

Function: Repressible system - genes turned OFF when product present

Eukaryotic Gene Regulation

More complex than prokaryotes:

• Chromatin structure
• Transcription factors
• Alternative splicing
• mRNA stability
• Translation control
• Post-translational modifications

Chromatin Structure

Histone modifications:

• Acetylation: loosens chromatin (genes accessible - ON)
• Methylation: can activate or repress (depends on location)
• Phosphorylation: various effects

DNA methylation:

• Addition of methyl groups to cytosine
• Usually silences genes
• Heritable (epigenetic)

Chromatin remodeling:

• Euchromatin: loosely packed, genes active
• Heterochromatin: tightly packed, genes inactive

Transcription Factors

Activators:

• Promote transcription
• Help RNA polymerase bind
• Bind to enhancers (DNA sequences)

Repressors:

• Inhibit transcription
• Block activators or RNA polymerase
• Bind to silencers

Enhancers and silencers:

• Can be far from gene
• DNA loops bring them near promoter

Control Elements

Proximal control elements:

• Near promoter
• TATA box, CAAT box, GC box

Distal control elements:

• Far from promoter
• Enhancers and silencers

Epigenetics

Changes in gene expression without DNA sequence changes

Mechanisms:

1. DNA methylation: adds methyl groups to DNA
2. Histone modification: acetylation, methylation, etc.
3. Chromatin remodeling: changes DNA packaging

Characteristics:

• Can be heritable (passed to daughter cells)
• Can be reversible
• Influenced by environment
  • Diet, stress, toxins, behavior

Examples:

• X-inactivation in females (Barr body)
• Genomic imprinting: parent-specific expression
• Cancer: abnormal methylation patterns

Post-Transcriptional Regulation

mRNA processing:

• Alternative splicing (one gene → multiple proteins)
• 5' cap and poly-A tail additions

mRNA stability:

• Some mRNAs degraded quickly
• Others stable for long time
• Controlled by sequences in 3' UTR

microRNA (miRNA) and siRNA:

• Small RNAs that bind mRNA
• Block translation or cause degradation
• Gene silencing

Levels of Gene Regulation

1. Chromatin structure (access to DNA)
2. Transcription (RNA synthesis)
3. RNA processing (splicing, capping, tailing)
4. mRNA stability (degradation)
5. Translation (protein synthesis)
6. Post-translational (protein modifications)

Key Concepts

1. lac operon: inducible, turned ON by lactose
2. trp operon: repressible, turned OFF by tryptophan
3. Chromatin modifications control gene accessibility
4. Transcription factors (activators/repressors) control transcription
5. Epigenetics: heritable changes without DNA sequence change
6. Multiple levels of regulation in eukaryotes
7. miRNA and siRNA silence genes post-transcriptionally

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:

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