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

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

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

Co-activators and Chromatin Remodeling Complexes

Co-activators & chromatin remodeling complexes not shown

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?

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

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?

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

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

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

Coding RegionPrUAS Enzyme involved in sucrose metabolism

Coding RegionPrUAS Enzyme involved in sucrose metabolism

Genetic Screens: Primary screen and initial characterization of mutants 1. Screen for mutants 2. Phenotype due to a single mutation? 3. Dominant or Recessive? 4. Complementation tests

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

Genetic Screens: Primary screen Secondary screen(s)

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

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

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

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

Back to GCN5

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

Histone Modification Histone Code

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

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

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

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?

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