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MCB 317 Genetics and Genomics

MCB 317 Genetics and Genomics. MCB 317 Topic 10, part 4 A Story of Transcription. Deletion and Linker Scanner Analysis. Identify and define TBP and basal factors. In vitro Txn Assay. Extract + Prom.- Enh. Basal Facts. + Prom.- Enh. Promoter sufficient in vitro. Activated Txn

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MCB 317 Genetics and Genomics

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  1. MCB 317Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription

  2. Deletion and Linker Scanner Analysis Identify and define TBP and basal factors In vitro Txn Assay Extract + Prom.-Enh. Basal Facts. + Prom.-Enh. Promoter sufficient in vitro Activated Txn (Enhanced) & Regulated Txn Extract + Prom.-Enh. In vivo Txn Assay Promoter not Sufficient Activators Co-activators + Enhancer & TBP & TAFs Promoter “Activated”txn & Regulated txn Identification of Enhancers

  3. Co-activators and Chromatin Remodeling Complexes

  4. Co-activators & chromatin remodeling complexes not shown

  5. How could we have missed Co-activators and Chromatin Remodelling Complexes for 20+ years?How to study what’s going on and what’s important in vivo?

  6. In vitro“chromatin” Assembly Purify Polymerases Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitrotxn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes

  7. Strength of Genetics as a Tool Strength of Genetics: Is a Gene Important in vivo? Limitation of biochemistry: Does an in vitro assay recapitulate the entire in vivo process?

  8. Concept: Comprehensive view of a molecular process requires both Genetics and Biochemistry

  9. RNAP II Basal Factors Activators Genetic Screens (Yeast mostly) Coactivators Chromatin Remodeling Complexes Mediator

  10. In vitro“chromatin” Assembly Purify Polymerases Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitrotxn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes

  11. UAS Pr Coding Region Enzyme involved in sucrose metabolism

  12. UAS Pr Coding Region Enzyme involved in sucrose metabolism

  13. Genetic Screens: Primary screen and initial characterization of mutants Screen for mutants Phenotype due to a single mutation? Dominant or Recessive? Complementation tests

  14. Our snf- screen = many complementation groups = many genes snf1 snf2 snf3 snf4 snf5 snf6 snf7 snf8….. Which, if any encode txn factors? Secondary screen to identify possible txn factors

  15. Genetic Screens: Primary screen Secondary screen(s)

  16. SUC2 encodes an enzyme that metabolizes sucrose. SUC2 txn is induced in response to sucrose Transform Reporter into each of our mutant strains: snf1-, snf2-, snf3-, snf4-, snf5-, snf6-, etc. Three key complementation groups identified: SNF2, SNF5 and GCN5

  17. Raise antibodies to Snf2 and Snf5 proteins and use them to purify the native proteins from wild-type yeast cells Snf2 and Snf5 are part of the same large protein complex

  18. Nuclease Protection Assay = variation on footprinting that provides information on where histones bind and on which bases and strands of the DNA faces outward on the nucleosome surface and which face inward

  19. In vitro“chromatin” Assembly Purify Polymerases Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitrotxn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes

  20. Back to GCN5

  21. Continuing Concept: Comprehensive view of a molecular process requires both Genetics and Biochemistry

  22. Histone Modification Histone Code

  23. Lodish 11-32 UAS = Upstream Activation Site = Yeast Enhancer Gcn4 is an Activator Gcn5 is a subunit of a co-activator (SAGA) that has histone acetylase activity

  24. Activators One function of activators is to act as a “platform” that recruits (binds) Co-activators

  25. In vitro“chromatin” Assembly Purify Polymerases Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitrotxn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes

  26. How could we have missed Co-activators and Chromatin Remodelling Complexes for 20+ years?How to study what’s going on and what’s important in vivo?

  27. In vitro“chromatin” Assembly Purify Polymerases Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitrotxn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes

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