Regulation of Gene Expression

Secondary Operator Sites - Structure and Location:

• O₁: Main operator, adjacent to promoter
• O₂: Located ~410 bp downstream within Z gene
• O₃: Located ~90 bp upstream within I gene
• All sites bind Lac repressor, but with different affinities

Functional Significance:

• DNA looping: Repressor binds O₁ and either O₂ or O₃ simultaneously
• Tetrameric repressor structure enables binding to distant sites
• Creates stable repression complex with DNA looped out between sites

Contribution to Repression:

• Increases effective repressor concentration near O₁
• With all operators intact: 1000-fold repression
• With only O₁ present: 100-fold repression
• Provides redundancy in repression mechanism
• Ensures tight regulation despite low repressor numbers (~20 tetramers/cell)

Significance: The cooperative binding to multiple sites demonstrates a sophisticated mechanism for amplifying regulatory control beyond what a single binding site could achieve, allowing effective regulation with minimal protein investment.

A prokaryotic operon is a functional genetic unit in bacteria where multiple genes are transcribed together as a single polycistronic mRNA.

Key structural components:

1. Regulatory sequences:

   • Activator binding site: Where activator proteins bind to enhance transcription
   • Promoter: Where RNA polymerase binds to initiate transcription
   • Operator: Where repressor proteins bind to inhibit transcription

2. Coding sequences:

   • Multiple genes (e.g., genes A, B, C) transcribed as a unit
   • All genes under control of a single promoter
   • All genes regulated coordinately

This organization allows bacteria to efficiently coordinate the expression of functionally related genes (such as those in a metabolic pathway) in response to environmental conditions.

Advantages to prokaryotes:

• Allows coordinated expression of genes involved in related functions
• Provides efficient resource utilization by expressing all proteins needed for a pathway simultaneously
• Enables rapid response to environmental changes
• Reduces genetic "overhead" by using a single promoter and regulatory system
• Simplifies regulation of metabolic pathways and stress responses

Rare in eukaryotes because:

• Eukaryotic mRNAs generally undergo 5' capping which functions best with one coding sequence
• Eukaryotic translation requires ribosome attachment at the 5' end and scanning to start codons
• Eukaryotes have complex post-transcriptional processing including splicing
• Eukaryotic gene expression relies more on combinatorial control with multiple transcription factors
• Eukaryotes often need independent regulation of related genes in different tissues or developmental stages

This difference reflects the more complex regulatory needs of multicellular organisms versus the efficiency requirements of unicellular prokaryotes.

The major groove of DNA presents more distinctive chemical groups that differ between base pairs:

• Major groove contains hydrogen bond donors and acceptors that uniquely identify each base pair • The methyl group of thymine protrudes into the major groove, allowing T/A discrimination • Functional groups in the major groove are positioned to form specific hydrogen bonds with amino acid side chains (Arg, Gln, Asn, Lys)

The minor groove has fewer distinguishing features between base pairs, making it less useful for sequence-specific recognition.

Example: Transcription factors like the Lac repressor use helix-turn-helix motifs to interact with major groove elements to achieve highly specific DNA binding.

The trp operon employs two distinct regulatory mechanisms:

1. Repressor-operator interaction:

   • When tryptophan levels are high, tryptophan binds to the Trp repressor protein
   • This binding causes a conformational change in the repressor
   • The activated repressor binds to the operator, blocking RNA polymerase
   • Result: Transcription initiation is prevented

2. Transcriptional attenuation:

   • Occurs after transcription has begun
   • When tryptophan levels are high, transcription terminates prematurely at the attenuator
   • This produces a truncated mRNA that doesn't include the structural genes
   • Result: Full operon transcription is halted even if some transcription began

The dual regulation ensures tight control over tryptophan biosynthesis enzyme production exactly when needed.

Anterior vs. Posterior Regulation:

Anterior Region:

• Dominated by Bicoid gradient (highest at anterior)
• Bicoid acts as transcriptional activator of hunchback
• Bicoid functions as translational repressor of caudal
• High concentration of Hunchback protein
• Low concentration of Caudal protein
• Transcriptional activation is primary mechanism
• Head and thoracic structures develop

Posterior Region:

• Dominated by Nanos gradient (highest at posterior)
• Nanos acts as translational repressor (via Pumilio) of hunchback
• Nanos cannot directly activate transcription
• Absence of Bicoid allows caudal mRNA translation
• High concentration of Caudal protein
• Low concentration of Hunchback protein
• Translational repression is primary mechanism
• Abdominal structures develop

Both regions use combination of protein gradients and regulatory mechanisms, but with inverse distributions and different emphases on transcriptional versus translational control.

Translational feedback regulation of r-proteins:

• Mechanism:

  1. One r-protein in each operon functions as translational repressor
  2. This protein binds to mRNA near translation start site of (usually) first gene
  3. Binding blocks translation of all genes in operon through translational coupling
  4. Repressor r-protein binds with higher affinity to rRNA than to its mRNA

• Regulatory logic:

  • When r-protein synthesis exceeds available rRNA, excess r-proteins bind mRNA
  • When rRNA is abundant, r-proteins preferentially bind rRNA, allowing translation

• Balancing effect:

  • Links r-protein synthesis to rRNA availability
  • Prevents wasteful production of excess r-proteins
  • Coordinates assembly of functional ribosomes

This elegant system ensures stoichiometric production of r-proteins relative to rRNA, optimizing resource allocation for this energy-intensive process.

The GAL regulatory system involves three key proteins that control galactose metabolism genes:

1. Gal4p: DNA-binding transactivator that binds to UASG (upstream activator sequence) elements
2. Gal80p: Repressor that forms a complex with Gal4p, preventing activation
3. Gal3p: Sensor protein that binds galactose

Activation mechanism:

• In absence of galactose: Gal80p binds to Gal4p, blocking its activation function
• When galactose is present: Galactose binds to Gal3p
• Gal3p-galactose complex interacts with Gal80p
• This interaction causes a conformational change in Gal80p
• Gal4p is released from inhibition and can activate transcription
• Transcription proceeds through intermediary complexes (TFIID or mediator) and RNA polymerase II

This system ensures galactose-metabolizing enzymes are only produced when galactose is available as a carbon source.

The Drosophila melanogaster life cycle includes:

1. Oocyte → fertilization → egg (Day 0)
2. Early embryo (no segmentation)
3. Late embryo (segmented with defined head, thorax, abdomen)
4. Hatching (Day 1)
5. Larval stages (three instars separated by molts)
6. Pupation (Day 5)
7. Complete metamorphosis within pupal case
8. Adult emergence (Day 9)

The entire cycle from egg to adult takes approximately 9 days under standard laboratory conditions.

Note: This process exemplifies complete metamorphosis, where the adult form differs radically from immature stages.

The trp operon employs two complementary regulatory mechanisms that provide sophisticated control over tryptophan biosynthesis:

1. Repressor-Operator Control (Primary Gate)

• Mechanism: Binary on/off switch at transcription initiation
• Key components: Trp repressor protein, operator sequence
• Function: When tryptophan (co-repressor) binds to repressor, the complex blocks RNA polymerase attachment
• Response threshold: Activated at higher tryptophan concentrations

2. Transcriptional Attenuation (Fine-Tuning Mechanism)

• Mechanism: Controls transcription after initiation through premature termination

• Key component: 162-nucleotide leader region (trpL) with four critical sequences:

  1. Sequence 1: Encodes 14-amino acid leader peptide containing two consecutive Trp codons

     • Functions solely as regulatory element with no other cellular purpose
     • Translation rate directly senses tryptophan availability

  2. Sequence 2: Complementary to sequence 3

  3. Sequence 3: Pivotal sequence that can pair with either sequence 2 OR sequence 4

  4. Sequence 4: Forms terminator-like attenuator structure with sequence 3

• In high tryptophan conditions:

  • Ribosome translates sequence 1 rapidly through Trp codons
  • Ribosome physically blocks sequence 2
  • Sequences 3 and 4 pair to form terminator hairpin
  • RNA polymerase dissociates, prematurely terminating transcription

• In low tryptophan conditions:

  • Ribosome stalls at Trp codons due to limited charged tRNA^Trp
  • Sequence 2 remains available to pair with sequence 3
  • The 2:3 pairing prevents attenuator (3:4) formation
  • Transcription continues into structural genes

Integration of Both Systems

• Together provide ~700-fold regulation range
• Repressor acts as coarse control; attenuation provides fine-tuning
• Demonstrates how protein-DNA, RNA structure, and translation can collectively regulate gene expression
• Economical solution that conserves cellular resources by responding to subtle changes in tryptophan levels