CH19 - Regulating the Flow of Genetic Information

Flow of Gene-Expression Regulation – Big Picture

• All instructions for life are encoded in the genome, but only a subset is expressed at any moment.
  • Selective expression conserves energy and lowers cancer risk (mis-expression can drive tumorigenesis).
• Check-points where expression can be modulated
  • DNA access / chromatin state (not covered deeply here)
  • $\textbf{Transcription initiation}$ – ON/OFF decision for RNA polymerase (RNAP) at the promoter.
  • Post-transcriptional RNA processing – e.g. intron removal, 5′-capping, 3′ poly-A, RNA editing.
  • mRNA stability – decay vs. stabilization → copies available for translation.
  • Translation – initiation efficiency, ribosome access.
  • Post-translational protein modification (phosphorylation, methylation, etc.).
  • Protein transport/localization – cytoplasm ↔ nucleus, membranes, periplasm (in Gram-negative bacteria).
  • Protein degradation – half-life control via proteasome (eukaryotes) or bacterial proteases.

Transcription Initiation: Core Concepts

• RNAP must bind promoter, change from closed complex (dsDNA) → open complex (melted bubble) to start RNA synthesis.
• Bacterial promoter consensus sites: $-10$ (TATAAT) & $-35$ (TTGACA) recognized by σ-factor.

Constitutive vs. Regulated Genes

• Constitutive (“house-keeping”) – expressed constantly at the same level; e.g. cell-wall enzymes. (Illustrated as always ≈3 mRNA copies.)
• Regulated genes – expression changes in response to signals (nutrients, stress, etc.).
  • Control proteins = transcription factors (TFs) / regulators.

Activators & Repressors (Local Control)

• Activator binding → stabilizes RNAP → ON (positive regulation). Site often just called “activator site.”
• Repressor binding → blocks RNAP → OFF (negative regulation). Binding sequence frequently called operator.

Long-Range Control via DNA Looping

• Regulators can bind distal sites and contact RNAP by looping.
  • Activator-mediated looping: distant site ↔ promoter → ON.
  • Repressor-mediated looping between two operators → loop occludes promoter → OFF.
• Helper proteins that bend DNA = architectural regulators; bridge proteins = co-activators / co-repressors.

Insulators (Eukaryotes)

• DNA elements + insulator proteins that prevent cross-talk between adjacent genes (block an enhancer for gene A from activating gene B).

Signal Integration Example – lac Operon (E. coli)

• Three structural genes (lacZYA) needed for lactose import & catabolism.
• Cell must evaluate two signals:
  1. Lactose present/absent → binds LacI repressor.
  2. Glucose present/absent → sets cAMP level → cAMP·CRP activator status.
• Logic table (simplified):
  • Glucose present (preferred C-source) ⇒ operon OFF, regardless of lactose.
  • Glucose absent & lactose present ⇒ high cAMP, CRP binds activator site; lactose inactivates LacI → RNAP stabilized → operon strongly ON.
  • Lactose absent ⇒ LacI binds operator → OFF.

Effector Molecules & Regulatory Logic

• Small ligands (effectors) bind TFs, altering DNA affinity.
• Four canonical classes (combine Positive/Negative with Inducible/Repressible):
  • Negative–Inducible: effector (inducer) removes repressor → ON (e.g. lactose/LacI).
  • Negative–Repressible: effector (co-repressor) enables repressor DNA binding → OFF (e.g. tryptophan/TrpR).
  • Positive–Inducible: effector enables activator binding → ON (e.g. cAMP/CRP).
  • Positive–Repressible: effector causes activator release → OFF.
• Mnemonic: “Inducer present ⇒ expression ON; Co-repressor present ⇒ expression OFF.”

Operons & Combinatorial Control

• Operon / polycistronic RNA – single promoter/terminator controlling multiple ORFs (e.g. lacZYA, trpEDCBA).
• Eukaryotes: “combinatorial control” – limited set of TFs used in distinct combinations for many genes (saves energy). Example scheme:
  • Gene X: TF A + B + C + D + E
  • Gene Y: TF B + C + E
  • Gene Z: TF A + B + D + F + G

Structural Basis of Transcriptional Regulation

Why the Major Groove?

• Major groove presents more H-bond donors/acceptors & methyl groups → richer “chemical barcode” for base identification versus minor groove.

Regulatory Sites Often Inverted Repeats

• Homodimeric TFs bind palindromic DNA; each monomer contacts one half-site.

Canonical DNA-Binding Motifs

Modular Domain Architecture

• TFs often = DNA-binding domain + separate regulatory (activation/repression) domain.
• Classic experiment (Kadoch/Kadner et al.): fused LexA DNA-binding domain to Gal4 activation domain; reporter with LexA site upstream of GAL promoter → β-galactosidase expressed only when both modules present ⇨ domains are functionally separable.
• Concept borrowed by yeast two-hybrid screens.

Post-Transcriptional Regulation

Attenuation (Bacteria)

• Acts like a conditional intrinsic terminator.
• Example: trp operon
  • Leader RNA can fold into 3:4 terminator stem-loop followed by U-tract → RNAP falls off.
  • High charged tRNA
    t_Trp allows ribosome to quickly clear region 1➜2, promoting 3:4 pairing (terminator) ⇒ transcription stops when Trp abundant.
  • Low Trp ➜ ribosome stalls, 2:3 anti-terminator forms ⇒ transcription continues through trpEDCBA genes.

Transcription Elongation Control (Eukaryotes)

• RNAP II associates with multiple elongation factors; phosphorylation pattern of CTD and factor availability modulate pause/release.

Alternative Splicing

• Different splice-site use alters exon composition.
  • Splicing activators/repressors (SR proteins, hnRNPs) bind enhancer/silencer elements to select sites.
  • Example diagram: full inclusion of exons 1-4 vs. skipping exon 2 when inner splice sites blocked.

mRNA Stability & Localization

• 5′ cap + 3′ poly-A protect transcript; also bind export & translation factors.
• Small RNAs (sRNAs in bacteria; miRNA/siRNA in eukaryotes):
  • Base-pair to mRNA → inhibit ribosome binding (often Shine-Dalgarno), stimulate or prevent degradation.
  • Dicer processes dsRNA → 21-25 nt miRNA; loaded into RISC; outcomes:
  • Perfect pairing → mRNA cleavage
  • Imperfect pairing → translational repression / deadenylation.

Post-Translational Modifications (PTMs)

• Phosphorylation, acetylation, methylation, ubiquitination, glycosylation, etc.
• Example – JAK/STAT pathway (cytokine signaling):
  1. Cytokine binds receptor → dimerizes JAK kinases.
  2. JAKs auto-phosphorylate then phosphorylate STAT.
  3. Phospho-STAT dimerizes, exposes NLS → imported to nucleus.
  4. STAT acts as TF → target gene ON.
• Histone PTMs alter chromatin structure, impacting large swaths of transcription.

Protein Degradation

• Eukaryotes: proteins tagged with poly-ubiquitin (Ub) via E1/E2/E3 enzymes.
  • Ub chain recognized by $26\,\text{kDa}$ proteasome core → ATP-dependent unfolding and proteolysis.
• Bacteria: no ubiquitin; oxidized/misfolded proteins expose hydrophobic patches → recognized by bacterial proteasome-like complexes (e.g. ClpXP, Lon).

Ethical, Philosophical & Practical Notes

• Precise regulation conserves limited cellular energy; mis-regulation can waste resources or induce disease.
• Cancer prevention: avoiding inappropriate gene activation (e.g. proto-oncogenes) lowers mutational pressure.
• Biotechnology: manipulating promoters, TF motifs, or Ub-tags enables synthetic-biology circuits, therapeutic protein control, or gene-silencing therapies (siRNA/miRNA, CRISPRi).