MCB 317 Genetics and Genomics MCB 317 Topic
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 Identify and define TBP and basal factors Extract + Prom. -Enh. Basal Facts. + Prom. -Enh. Activated Txn (Enhanced) & Regulated Txn Extract + Prom. -Enh. In vivo Txn Assay Promoter not Sufficient Identification of Enhancers Activators Co-activators + Enhancer & TBP & TAFs Promoter “Activated” txn & Regulated txn
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 Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitro “chromatin” Assembly In vitro txn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes
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
RNAP II Basal Factors Genetic Screens (Yeast mostly) Activators Coactivators Chromatin Remodeling Complexes Mediator
Purify Polymerases Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitro “chromatin” Assembly In vitro txn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes
UAS Pr Coding Region Enzyme involved in sucrose metabolism
UAS Pr Coding Region Enzyme involved in sucrose metabolism
Genetic Screens: Primary screen and initial characterization of mutants 1. 2. 3. 4. Screen for mutants Phenotype due to a single mutation? Dominant or Recessive? Complementation tests
Our snf- screen = many complementation groups = many genes snf 1 snf 2 snf 3 snf 4 snf 5 snf 6 snf 7 snf 8…. . Which, if any encode txn factors? Secondary screen to identify possible txn factors
Genetic Screens: Primary screen Secondary screen(s)
SUC 2 encodes an enzyme that metabolizes sucrose. SUC 2 txn is induced in response to sucrose Transform Reporter into each of our mutant strains: snf 1 -, snf 2 -, snf 3 -, snf 4 -, snf 5 -, snf 6 -, etc. Three key complementation groups identified: SNF 2, SNF 5 and GCN 5
Raise antibodies to Snf 2 and Snf 5 proteins and use them to purify the native proteins from wild-type yeast cells Snf 2 and Snf 5 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 Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitro “chromatin” Assembly In vitro txn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes
Back to GCN 5
Continuing Concept: Comprehensive view of a molecular process requires both Genetics and Biochemistry
Histone Modification Histone Code
Lodish 11 -32 UAS = Upstream Activation Site = Yeast Enhancer Gcn 4 is an Activator Gcn 5 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 Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitro “chromatin” Assembly In vitro txn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes
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 Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitro “chromatin” Assembly In vitro txn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes
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