Regulation of Gene Expression

Molecular Mechanisms for DNA-Binding Specificity:

1. Multiple Specific Contacts:

   • Direct readout: Specific hydrogen bonds between protein side chains and DNA bases
   • Each individual contact contributes to overall specificity
   • Cumulative effect of 5-10 specific contacts creates high selectivity

2. Cooperative Binding:

   • Binding of one protein subunit facilitates binding of others
   • Seen in dimeric and tetrameric repressors (e.g., Lac repressor)
   • Dramatically increases overall affinity for specific sequences

3. Conformational Complementarity:

   • Protein structure precisely matches DNA shape at target sites
   • Indirect readout: recognition of sequence-dependent DNA deformability

4. Palindromic Recognition Sites:

   • Match the inherent symmetry of dimeric binding proteins
   • Double the specificity by requiring matching at two half-sites

5. Kinetic Discrimination:

   • Higher dissociation rates from non-specific sites
   • Longer residence times at specific sequences

Example: Lac repressor discriminates between operator and non-specific DNA by ~10⁶-fold and binds to operator sites with Kd ≈ 10⁻¹⁰ M, enabling precise regulation despite the presence of millions of competing DNA sequences.

Inducer Molecules:

• Bind to repressor proteins, causing dissociation from DNA
• Enable transcription by removing the repression
• Function in inducible systems (normally off, turned on by signal)
• Example: Allolactose in lac operon
  • Binds to Lac repressor, causing conformational change
  • Repressor dissociates from operator
  • Allows RNA polymerase to transcribe lactose utilization genes

Co-repressor Molecules:

• Bind to inactive repressor proteins, enabling DNA binding
• Prevent transcription by facilitating repression
• Function in repressible systems (normally on, turned off by signal)
• Example: Tryptophan in trp operon
  • Binds to Trp repressor, causing conformational change
  • Repressor-tryptophan complex binds to operator
  • Blocks RNA polymerase from transcribing tryptophan biosynthesis genes

Both mechanisms modify protein-DNA interactions through allosteric changes to regulatory proteins, but they operate in opposite directions and typically regulate different classes of genes (catabolic vs. biosynthetic).

Lactose in E. coli follows two distinct metabolic fates:

1. Hydrolysis Pathway (Primary):

   • Catalyzed by β-galactosidase
   • Cleaves lactose into galactose and glucose
   • Products enter glycolysis and the Leloir pathway
   • Purpose: Energy production and carbon utilization
   • Represents ~95-99% of lactose metabolism

2. Transglycosylation Pathway (Secondary):

   • Also catalyzed by β-galactosidase
   • Converts lactose to allolactose (β-1,6 linkage instead of β-1,4)
   • Represents only ~1-5% of lactose metabolism
   • Purpose: Regulatory signaling, not energy production
   • Critical for lac operon induction

Significance:

• Dual pathways create an efficient regulatory circuit
• Minor transglycosylation product (allolactose) serves as the actual inducer
• This arrangement ensures that β-galactosidase production is directly tied to substrate availability
• Demonstrates how metabolic and genetic regulatory networks are integrated in prokaryotes

The tetrameric structure of the Lac repressor enables its function through:

1. Multi-point DNA binding:

   • The repressor functions as a tetramer composed of four identical subunits
   • The tetramer operates as two dimers tethered together
   • Each dimer has its own DNA-binding domain
   • This allows simultaneous binding to two operator sites (O₁ and O₂, or O₁ and O₃)

2. DNA looping mechanism:

   • When bound to two operators, the repressor creates a loop in the DNA
   • This loop wraps around the repressor
   • The looping increases binding stability by 10-100 fold compared to binding at a single site
   • This provides extremely tight repression (Kd ≈ 10⁻¹⁰ M)

3. Allosteric regulation:

   • The tetrameric structure contains inducer binding sites distant from DNA-binding domains
   • When inducer molecules bind, they cause a conformational change
   • This change propagates through the protein structure
   • The DNA-binding domains become disordered, preventing operator binding

Example: This tetrameric arrangement allows for extraordinary specificity, enabling the ~20 repressor tetramers per cell to find and bind their target sequences among 4.6 million base pairs with a discrimination factor of 10⁶.

Hydrogen bonding patterns provide specificity through:

• Each base pair presents a unique "signature" of hydrogen bond donors and acceptors in the major groove • Adenine-Thymine pairs display:

• The N6 and N7 positions of adenine form hydrogen bonds with Gln or Asn
• Thymine's methyl group creates a hydrophobic recognition point

• Guanine-Cytosine pairs display:

• N7 and O6 of guanine form hydrogen bonds with Arg residues
• Cytosine presents different acceptor/donor patterns than thymine

These patterns allow regulatory proteins to "read" the DNA sequence without unwinding the double helix, achieving binding specificity factors of 10⁴ to 10⁶ over non-specific sequences.

• Catabolite repression: Mechanism that restricts expression of genes for secondary sugar metabolism when glucose is present

• CRP-cAMP complex role:

• CRP (cAMP Receptor Protein) binds cAMP when glucose is low
• CRP-cAMP binds near lac promoter and stimulates transcription 50-fold
• Acts as positive regulator responding to glucose levels
• Interacts directly with RNA polymerase α subunit
• Stabilizes open complex formation with promoter

• Combined regulation:

• Lac operon requires BOTH absence of repressor (high lactose) AND presence of CRP-cAMP (low glucose)
• Without CRP-cAMP, the lac promoter is inherently weak
• Full expression occurs only when glucose is low and lactose is high
• This prevents wasteful production of lactose-metabolizing enzymes when glucose is available

• Lac operon (inducible system):

• Negative regulation: Repressor active by default, blocks transcription
• Induced by presence of substrate (lactose/allolactose)
• Positive regulation via CRP-cAMP responds to glucose levels
• Regulated by substrate availability (lactose) and preferred energy source (glucose)
• Two independent regulatory mechanisms working in concert

• Trp operon (repressible system):

• Negative regulation: Repressor activated by excess product (tryptophan)
• Repressed by high levels of pathway end product
• Additional attenuation mechanism provides fine-tuning
• 700-fold range of expression based on tryptophan levels
• Regulated at both transcription initiation and elongation stages

• Key conceptual difference:

• Lac operon: "Turn on when substrate is present" (synthesis when needed)
• Trp operon: "Turn off when product is abundant" (prevent overproduction)

Distinguishing features: • Contains both a helix-loop-helix domain and elements of a leucine zipper in a single structure • The second helix of the HLH directly continues into a dimerization domain with leucine residues • Has fewer leucine repeats than a pure leucine zipper (may have as few as one pair) • Maintains the loop structure absent in pure leucine zippers • Combines DNA binding and dimerization functions in a more integrated manner

Functional advantages: • Enhanced structural stability through multiple interaction points • Greater versatility in partner selection for dimerization • More precise alignment of DNA-binding regions • Ability to form both homodimers and heterodimers with different regulatory outcomes • Can respond to multiple signaling pathways simultaneously

This hybrid structure, exemplified in transcription factors like Max, allows for more sophisticated regulation and greater combinatorial control over gene expression than either motif alone would provide.

Eukaryotic DNA-binding transactivators contain three major types of activation domains:

1. Acidic domains: Rich in negatively charged amino acids (aspartic acid, glutamic acid)

   • Example: Gal4p contains an acidic activation domain
   • Critical for function regardless of exact sequence

2. Glutamine-rich domains: Contain ~25% glutamine residues

   • Example: Sp1 transcription factor uses glutamine-rich domains
   • Typically found near GC box recognition sites

3. Proline-rich domains: Contain >20% proline residues

   • Example: CTF1 (CCAAT-binding transcription factor 1)
   • Found in factors that bind CCAAT elements

Each domain type interacts with the transcriptional machinery through different mechanisms while maintaining their specialized DNA-binding functions.

Bicoid Gradient and Function

• Morphogen gradient: Bicoid (Bcd) protein forms a concentration gradient that decreases from anterior to posterior
• Primary functions:
  • Defines the anterior-posterior (A-P) axis
  • Acts as a transcription factor for segmentation genes
  • Functions as a translational repressor for specific mRNAs

Threshold-Dependent Mechanism

• Spatial gene regulation:

  • Different target genes require specific Bicoid concentration thresholds for activation
  • Only activated where Bicoid concentration exceeds gene-specific thresholds
  • Creates precise boundaries between developmental domains

• Concentration-dependent outcomes:

  • High (anterior): Activates anterior-specific genes
  • Intermediate (middle): Activates different gene sets
  • Low/absent (posterior): Permits posterior-specific development

• Example: Hunchback protein expression occurs at high levels in anterior regions due to Bicoid activation

Mutant Phenotype (bcd⁻/bcd⁻)

• "Double-posterior" phenotype:

  • Complete absence of head and thorax structures
  • Two posterior ends develop instead
  • Embryo is non-viable

• Rescue: Normal development can be restored by injecting bicoid mRNA into the anterior region of mutant eggs

Significance

• This system elegantly converts a simple gradient into complex spatial patterning with discrete boundaries
• Demonstrates how a single morphogen can generate multiple distinct developmental domains
• Represents a fundamental mechanism for translating positional information into cell fate decisions