-
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
-
4 types of DNA elements that TFs bind to
- Promoter core element
- Upstream promoter sites
- Enhancer elements
- Response (regulatory) elements
-
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
-
Upstream promotor element
- TATA box not generally sufficient due to repression by histones
- ~10bp sequences within -50 to -150 site
- eg CCAAT and GC
-
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
-
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
-
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
-
Histone Acetylases
- Activate gene expression, by acetylating lysine and reducing its positive charge
- eg CBP
-
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)
-
Response elements
- GRE: cortisol -> GR -> hsp90 dissociation -> GR dimerisation and movement into nucleus
- CRE: cAMP -> PKA -> CREB phosphorylation -> CBP binds -> CBP binds CRE
-
Tissue specific gene expression
- MyoD: TF in myoblasts
- Oct2: light and heavy immunoglobulin genes in B cell lymphocytes
|
|
