Regulation of Gene Expression

Constitutive gene expression:

• Expression that occurs at a relatively constant level regardless of cellular conditions
• Associated with housekeeping genes (e.g., enzymes of central metabolic pathways)
• Not responsive to external signals or molecular cues

Regulated gene expression:

• Expression levels rise and fall in response to molecular signals
• Two main types:
  • Inducible: expression increases under specific conditions (e.g., DNA repair enzymes induced by DNA damage)
  • Repressible: expression decreases in response to a signal (e.g., tryptophan biosynthesis enzymes repressed when tryptophan is abundant)

This distinction allows cells to optimize energy expenditure by producing proteins only when needed.

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.

Despite specific interactions like glutamine-adenine and arginine-guanine binding, no simple recognition code exists because:

1. Multiple recognition strategies:

   • The same base pair can be recognized in different ways by different proteins
   • For example, A-T pairs can be recognized via hydrogen bonds (glutamine with adenine) or through van der Waals interactions with thymine's methyl group

2. Context dependence:

   • The three-dimensional structure of the protein affects how amino acids interact with DNA
   • Neighboring amino acids influence binding specificity
   • Water molecules can mediate interactions between proteins and DNA

3. Conformational flexibility:

   • Both DNA and protein structures can adapt during binding
   • Subtle conformational changes can significantly alter binding specificity

4. Cooperative effects:

   • Multiple relatively weak interactions work together for specificity
   • The pattern of interactions across several base pairs determines binding strength and specificity

This complexity means researchers cannot simply examine a DNA-binding protein's sequence and predict its DNA target sequence.

The dual regulation of the lac operon demonstrates metabolic efficiency through:

1. Resource conservation:

   • Bacteria avoid synthesizing unnecessary enzymes when preferred energy sources are available
   • Lac enzymes are only fully expressed when:
     • Lactose is present (to be utilized)
     • Glucose is absent (preferred carbon source)

2. Hierarchical energy utilization:

   • Glucose is metabolized first (requires fewer steps)
   • Secondary sugars like lactose are only utilized when glucose is depleted
   • This prioritization ensures optimal energy yield per metabolic effort

3. Regulatory economy:

   • Negative regulation (Lac repressor) prevents wasteful expression in lactose absence
   • Positive regulation (CRP-cAMP) links expression to cellular energy status
   • Integration of these signals creates a logical AND gate for gene expression

This system allows bacteria to rapidly adapt to changing nutrient conditions while minimizing unnecessary protein synthesis.

ppGpp signaling mechanism:

• Synthesis:

  1. RelA protein (stringent factor) binds to ribosomes with uncharged tRNAs
  2. Catalyzes: GTP + ATP → pppGpp + AMP
  3. Phosphohydrolase converts pppGpp to ppGpp

• Regulatory effects:

  • Binds β subunit of RNA polymerase
  • Alters promoter specificity
  • Decreases rRNA and tRNA transcription
  • Reduces translation machinery production
  • Reprograms transcription for stress adaptation

Comparison with cAMP:

• Similarities:

  • Both act as starvation signals in bacteria
  • Both modify RNA polymerase activity
  • Both regulate large sets of genes (regulons)
  • Both use nucleotide second messengers

• Differences:

  • ppGpp responds to amino acid starvation; cAMP to carbon source limitation
  • ppGpp generally represses growth-related genes; cAMP activates alternative carbon utilization
  • Different synthesis mechanisms (RelA vs. adenylyl cyclase)
  • Different interaction sites on RNA polymerase

Together, these systems allow bacteria to adapt metabolism to different resource limitations.

RecA functions as a molecular switch through:

• Sensor role: Detects DNA damage by binding to single-stranded DNA gaps • Coprotease activity: Not a traditional protease but facilitates self-cleavage of target proteins • Dual functionality: Serves both as a DNA repair protein and a regulatory molecule

Distinguished from typical repressors by: • Signal-dependent activation rather than inactivation • Catalyzes destruction of other regulatory proteins rather than being the regulatory protein itself • Functions through proteolysis rather than conformational change or dissociation • Creates an irreversible regulatory event (cleavage) rather than a reversible binding/unbinding • Amplifies its own expression through a positive feedback loop (RecA levels increase 50-100 fold)

This represents a sophisticated regulatory paradigm where protein destruction rather than allosteric change mediates the cellular stress response.

The GAL gene system in yeast:

1. Components:

   • GAL genes: Encode enzymes for galactose metabolism (GAL1, GAL2, GAL7, GAL10, etc.)
   • Scattered across chromosomes (no operons like in bacteria)
   • Regulated by three key proteins: Gal4p, Gal80p, Gal3p

2. Promoter structure:

   • Contains TATA box and Inr sequences
   • Includes Upstream Activator Sequence (UASG) recognized by Gal4p

3. Regulatory mechanism:

   • Negative regulation: Gal80p forms complex with Gal4p, preventing activation
   • Signal detection: Galactose binds to Gal3p
   • Positive regulation: Galactose-bound Gal3p interacts with Gal80p, freeing Gal4p to function as an activator
   • Gal4p then recruits coactivator complexes to GAL promoters

4. Multilevel control:

   • Additional factors: SAGA complex (histone acetylation), SWI/SNF (nucleosome remodeling), mediator complex
   • Glucose override: Catabolite repression system overrides GAL regulation when glucose is present

This system demonstrates:

• Coordinated regulation of scattered genes via common promoter elements
• Integration of positive and negative regulatory mechanisms
• Signal-responsive transcriptional control
• Interplay between specific DNA-binding factors and general transcription machinery

Early Drosophila embryo development:

1. Pre-fertilization preparation:

   • Egg cell forms surrounded by 15 nurse cells and layer of follicle cells
   • Nurse and follicle cells deposit critical mRNAs and proteins into egg cell
   • These maternal factors will direct early development

2. Post-fertilization nuclear divisions:

   • Synchronized nuclear divisions every 6-10 minutes
   • No plasma membranes form around nuclei (syncytium)
   • Between 8th-11th divisions, nuclei migrate to egg periphery
   • Forms syncytial blastoderm (monolayer of nuclei surrounding common yolk-rich cytoplasm)

3. Cellular blastoderm formation:

   • Membrane invaginations surround nuclei
   • Creates layer of defined cells
   • Mitotic cycles lose synchrony
   • Cell fates determined by maternal mRNAs/proteins

4. Role of maternal mRNAs:

   • Remain dormant until fertilization
   • Provide proteins needed before zygotic transcription begins
   • Establish anterior-posterior and dorsal-ventral polarity
   • Create morphogen gradients that direct spatial organization

DNA-binding transactivators facilitate transcription through multiple pathways:

1. They bind to specific DNA sequences (like UAS - Upstream Activator Sequences) through DNA-binding domains
2. Their activation domains (often acidic, glutamine-rich, or proline-rich) interact with:
   • TFIID complex (containing TBP and TAFs)
   • Mediator complex (which interacts with RNA polymerase II's CTD)
3. These interactions stabilize the binding of RNA polymerase II and its associated factors
4. They recruit chromatin remodeling complexes (such as SWI/SNF) and histone acetyltransferases
5. The combined effect facilitates formation of the preinitiation transcription complex

Example: In yeast, the Gal4p transactivator binds to UASG sequences and recruits the transcriptional machinery needed to express galactose metabolism genes when galactose is present.

1. Egg chamber formation: Oocyte surrounded by nurse cells and follicle cells
2. Maternal mRNA localization: bicoid mRNA deposited anteriorly, nanos mRNA posteriorly
3. Maturation into unfertilized egg
4. Fertilization: Two nuclei form within the egg
5. Nuclear divisions: Multiple synchronous nuclear divisions create a syncytium
6. Nuclear migration: Nuclei migrate to the periphery of the embryo
7. Syncytial blastoderm formation: Nuclei form a layer at the periphery
8. Pole cell formation: Special cells form at posterior end (future germ cells)
9. Membrane invagination: Cell membranes grow inward between nuclei
10. Cellular blastoderm formation: Complete monolayer of cells forms at periphery

Example: This process creates the cellular foundation for body axis formation and segmentation, with anterior cells receiving more Bicoid protein while posterior cells have more Nanos protein.