RNA transcription in eukaryotes

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RNA transcription in eukaryotes
2012-05-12 08:21:32
RNA transcription eukaryotes eukaryotic eukaryote

RNA transcription in eukaryotes and experimental
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  1. 3 types of RNA pol: what? where?
    • RNA Pol I: pre-rRNA, nucleolus
    • RNA Pol II: pre-mRNA, snRNA, miRNA, nucleoplasm
    • RNA Pol III: tRNA, 5S rRNA, snRNA, nucleoplasm
    • all have ~ 12 subunits, and require additional TFs
  2. 4 types of DNA elements that TFs bind to
    • Promoter core element
    • Upstream promoter sites
    • Enhancer elements
    • Response (regulatory) elements
  3. Promoter 'core element'
    • TATA box: -25, 5'TATAYYAY3'
    • TBP: binds to TATA and bends the DNA to create a stage for the assembly of other proteins
    • TBP with TAFs form TFIID
    • Pol II and TFIIA, B, E, F, H bind to form PIC
  4. Upstream promotor element
    • TATA box not generally sufficient due to repression by histones
    • ~10bp sequences within -50 to -150 site
    • eg CCAAT and GC
  5. Transcription factor structure
    • Activation domain: rich in acidic/glu/pro residues
    • DNA binding
    • Can splice two parts and use them experimentally, eg by putting regulatory gene fragments upstream of a assayable gene (eg firefly luciferase) and measuring outcome
  6. DNA Footprinting
    • Label dsDNA with 32P
    • Add binding protein - prevents endonuclease digestion
    • Analyse sizes of cleaved ssDNA by autoradiography
    • Compare on denaturing gel to find protected binding site
  7. Chromatin Immunoprecipitation (ChIp)
    • Provides evidence for binding sites of TFs in cell
    • Crosslink chromatin and DNA binding proteins to DNA in live cells
    • Cut DNA into small fragments (sonication with ultrasonic devices)
    • Purify TF and associated DNA with a specific anti-TF antibody
    • Remove TF and other proteins using proteases
    • Amplify pure DNA left
  8. Histone Acetylases
    • Activate gene expression, by acetylating lysine and reducing its positive charge
    • eg CBP
  9. Enhancer elements
    • Upstream/downstream/introns
    • eg AP1, AP2 recognised by c-Fos and c-Jun
    • tend to be long (~100bp) and far away (many kbp)
  10. Response elements
    • GRE: cortisol -> GR -> hsp90 dissociation -> GR dimerisation and movement into nucleus
    • CRE: cAMP -> PKA -> CREB phosphorylation -> CBP binds -> CBP binds CRE
  11. Tissue specific gene expression
    • MyoD: TF in myoblasts
    • Oct2: light and heavy immunoglobulin genes in B cell lymphocytes