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The GAL4 Regulon PowerPoint Presentation
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The GAL4 Regulon

The GAL4 Regulon

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The GAL4 Regulon

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  1. Simple regulatory systems: The GAL4 Regulon

  2. + Glucose (= no Galactose) no Glucose + Galactose Gal3p Gal3p

  3. Gal4 binds to UASG sites and regulates genes involved in Galactose metabolism

  4. The dissociation model

  5. The non-dissociation model

  6. TBP TBP Active Gal4 protein recruits TfIId TfIId no Galactose GAL1 UASG TATA + Galactose (no Glucose) GAL1 UASG TATA

  7. Chromatin Immunoprecipitation (“chromatin-IP” or “chIP”) Proteins cross-linked with Formaldehyde (FA) to sheared DNA Antibody binds to specific protein Enrichment of protein-DNA complexes Naked DNA, ~ 200 bp average Amplified DNA detectable by Standard gel-electrophoresis

  8. DNA ladder TBP

  9. TBP binding to the GAL1 TATA box requires an activator (an example for chIP: chromatin Immunoprecipitation)

  10. Activation Domains

  11. Gal4 protein is modular

  12. Domain swapping proves modularity

  13. Most transcriptional activators have independent domains

  14. An transcription factor may use a co-activator or co-repressor Co-regulator = common term for either co-activator or co-repressor

  15. Activation Domains • In contrast to DNA-binding domains, activation domains (ADs) often do not have a conserved three-dimensional structure and are not well defined. • Rather, activation domains are defined by amino acid • content and often contain small repeats. For instance, Gal4 AD is rich in acidic and hydrophobic amino acids. • The potency of an activation domain depends on its overall charge, resulting in “sticky” surfaces. This variation from domain to domain and allows fine tuning of interactions between ADs and Mediator and/or GTFs.

  16. The Mediator Complex

  17. TFIId requires activators for binding to DNA Step 1 Step 3 Step 2

  18. Problem: Something is missing… Problem I: Highly purified Gal4 + RNA Polymerase II + all general transcription factors (= GTF’s) failed to initiate transcription in vitro. Problem II: Overexpressing Gal4 (an activator) in yeast reduced the activating strength of other activators, a phenomenon called “squelching”. This suggested that Gal4 was recruiting a limiting factor in yeast cells and thus interfered with the other activator. Experiments in crude yeast extracts showed that adding GTF’s or RNA polymerase II did not relieve squelching. This squelching was independent of the Gal4 DNA-binding domain, but required the activating region of Gal4.

  19. The missing component turns out to be a giant multi-protein complex: the Mediator

  20. Mediator: Major Findings Mediator interacts with the CTD tail of RNA Pol II and stimulates TFIIH to initiate CTD phosphorylation. Once all yeast Mediator proteins were identified, it turned out that 13 of the corresponding genes were already known because they were identified in genetic screens that affect transcriptional activation and repression. The most dramatic mutation corresponds to a yeast gene encoding Med17 (aka SRB4). This mutation is temperature-sensitive and yeast cells raised at the permissive temperature survive. Once shifted to the restrictive temperature, transcription of ALL Pol II-dependent genes in yeast is massively affected.

  21. The activator-bypass experiment

  22. DNA-binding Domains • Structural considerations of the DNA double helix • Families of DNA-binding proteins

  23. DNA-binding domains: Let’s look at DNA first

  24. The minor groove harbors little chemical Information G:C C:G A:T T:A

  25. Helix-Turn-Helix

  26. The Helix-Turn-Helix motif was reinvented multiple times Homeodomain (yeast MatA1) TEA domain (human TEF-1) Anbanandam A et al. PNAS 2006;103:17225-17230

  27. Basic Region Leucine zipper (bZIP) Dimer of two large a-helices that form a coiled coil Examples: FOS and JUN

  28. Basic Region Helix-loop-Helix domain (bHLH) Example: MyoD

  29. The C2H2 zinc finger domain Example: TFIIIA

  30. Nuclear Receptors bind DNA via a pair of C4 zinc fingers Four cysteines are complexed with a Zn++ ion

  31. Structural properties of zinc fingers binding to DNA C2H2 type C4 type (nuclear receptors)

  32. Dimerization domains often occur in combinations