Chapter 7 - Gene Regulation

• All cells have the same DNA - Express different proteins, 30,000 gene in human cell, 10-20,000 genes expressed in any cell
• Proteins common to all cells - polymerases, repair enzymes, ribosomal proteins
• Proteins found only in certain cells - Insulin by beta cells in Islets of Langerhans of the pancreas
• Stages in Gene to Functional Protein Regulation:

  1. Transcriptional control
  2. RNA processing control
  3. RNA transport and localization control
  4. Translational control
  5. mRNA degradation control
  6. Protein activity control
• Stages in Gene to Function Protein Regulation - DNA/Chromatin, Transcription, RNA processing, Nuclear export, mRNA degradation, Protein synthesis, Protein folding/modification, Protein degradation
• DNA/Chromatin:

  • DNA methylation - decreases transcription
  • Histone deacetylation - decreases transcription
  • Placement of nucleosomes - beads less active than string / 30nm less active than beads
  • Heterochromatin - transcriptionally inactive genes
• Transcription:

  • Most economical way to regulate
  • Promoters - general TF, proximal regions, 100-200 base pairs upstream of start site
  • Enhancers - activators and repressors, can be 1000s of base pairs away from promoter, upstream or downstream
• Promoter and General TF:

  • TATAA box
  • All general transcriptional factors needed
  • RNA polymerase - Carboxyl terminal domain
• TATAA box - change in one nucleotide decreases transcription
• Carboxyl terminal domain (CTD):

  • Hepta-nucleotide sequence - several repeats
  • Ser/Thr must be phosphorylated by TFIIH to function properly
• Hepta-nucleotide sequence repeats: Tyr - Ser - Pro - Thr - Ser - Pro - Ser
• Enhancers - Activators and Repressors

  • Enhancer DNA - up to 50,000 nucleotide in length
  • Transcription factor - 5-10% of 30,000 genes code for regulatory proteins
  • Each gene uses different DNA sequences and TF
• Transcription Factors:

  • Made by RNA polymerase II
  • Specific domains: DNA binding domain, NLS, Activation domain, Dimerization domain
  • May need to be phosphorylated or modified to be functional
• NLS domain: Allows proteins in cytoplasm to move to nucleus and bind with DNA
• Dimerization domain: 2 proteins must come together, quaternary structure
• DNA/Protein Interactions:

  • relies on exact fit between DNA and proteins - complimentary between helix and protein
  • Hydrogen bonds, Ionic bonds, hydrophobic interactions - ~20 formed
  • Some of the tightest and most specific interactions in biology
• How Enhancers Work - Proteins bind to DNA Sequences:

  • Loop out DNA between enhancer and promoter
  • Binds to (or promotes) assembly of general TFs
• How Enhancers Work - Structural proteins

  • Binds DNA and creates bend allowing other TF to then interact properly
• Other Enhancer functions:

  • Recruit HATs
  • Have histone acetylase activity
  • Recruit chromatin remodeling complexes
• DNA Binding Domains: Helix turn helix / Helix loop helix, Zinc Fingers, Leucine Zippers
• Helix-Turn-Helix and Helix-Loop-Helix: 2 alpha helices connected by short chain of amino acids (turn) or slightly longer chain (loop)
• Helix-Turn/Loop-Helix binds as hetero- or homo dimers to DNA, Ex. E-Box site
• E-box site - CACGTG
• E-box site (CACGTG) examples: c-myc, max, USF-1, USF-2
• USF: Upstream stimulatory factor
• Zinc Fingers

  • Uses amino acid side chains and Zn++ ions
  • Binds to G rich areas of DNA
  • Type I - C2H2
  • Type II - C4
• Zinc Fingers Type I - C2H2

  • Alpha helix and Beta pleated sheet
  • Zinc held in place by 2-cys and 2-his side chains
  • Bind as monomers
• Zing Finger Type II - C4

  • Steroid receptor superfamily
  • 2 alpha helices
  • Zinc held in place by 4-cys side chains
  • Bind as hetero- or homodimers to major groove
• Leucine Zipper - Dimers containing an alpha helix

  • C-terminus of DNA binding site has Leucine every 7 amino acids which forms a coiled coil (dimerization domain)
• Leucine Zipper - just beyond dimerization interface the 2 alpha helices form a Y shaped structure which binds to the DNA
• Leucine Zipper Ex.: AP-1 site - TGACTCA
• AP-1 site - TGACTCA: c-jun, Jun B, Jun D, c-fos, Fos B, Fra-1, Fra-2
• AP-1 site types can bind in different combinations to regulate gene expression
• Protein DNA Interaction Protocols:

  • DNase I Footprinting
  • Electrophoretic Mobility Shift Assay (EMSA) - Supershift and Depletion
  • Chromatin Immunoprecipitation (ChIP) Assay
• Dimerization Domains: allows mix and match proteins that activate/repress transcription, Combinational control
• Combinatorial control: combinations of proteins regulate cellular processes instead of an individual protein
• NLS Domains - transcription factors typically made/held in cytoplasm

  • Wait for signal to go to the nucleus and activate genes
  • Often involves phosphorylation
• Activation Domains

  • High acidic amino acid concentrations (glutamic acid or aspartic acid), Proline rich, Ser/Thr rich (may have more than 1)
  • May act to recruit other TFs and general TFs
• Transcription Factor Repressors

  • Form heterodimers with transcriptional activators (lack activation domains)
  • Compete with activators for DNA binding (lack activation domains)
  • Interact with general TFs (block binding)
  • Recruit repressive chromatin remodeling complexes and HDACs
• Protein/Promoter Function Protocols

  • Proteins Function Analysis - Transfection (Stable vs Transient), Over Expression, Antisense Vectors
  • Promoter Function Analysis - Reporter Genes