Post on 24-Dec-2015
MCB 317Genetics and Genomics
MCB 317 Topic 10, part 4A Story of Transcription
Deletion andLinker Scanner Analysis
In vitro Txn Assay
Promotersufficient in vitro
Identification of Enhancers
Identify and define TBPand basal factors
Extract +Prom.-Enh.
Basal Facts. +Prom.-Enh.
Activated Txn(Enhanced) &Regulated Txn
Extract +Prom.-Enh.
ActivatorsCo-activators + Enhancer &TBP & TAFs Promoter
“Activated” txn & Regulated txn
In vivo Txn AssayPromoter 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-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“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
GeneticScreens
(Yeast mostly) Coactivators
ChromatinRemodelingComplexes
Basal Factors
Activators
Mediator
RNAP II
Purify Polymerases
Immuno-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“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 mutants2. Phenotype due to a single mutation? 3. Dominant or Recessive?4. Complementation tests
Our snf- screen = many complementation groups = many genes
snf1snf2snf3snf4snf5snf6snf7snf8…..
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-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators
Back to GCN5
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
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-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“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-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators