mcb 317 genetics and genomics mcb 317 topic 10, part 4 a story of transcription
TRANSCRIPT
![Page 1: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/1.jpg)
MCB 317Genetics and Genomics
MCB 317 Topic 10, part 4A Story of Transcription
![Page 2: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/2.jpg)
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
![Page 3: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/3.jpg)
Co-activators and Chromatin Remodeling Complexes
![Page 4: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/4.jpg)
Co-activators & chromatin remodeling complexes not shown
![Page 5: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/5.jpg)
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?
![Page 6: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/6.jpg)
Purify Polymerases
Immuno-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators
![Page 7: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/7.jpg)
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?
![Page 8: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/8.jpg)
Concept:
Comprehensive view of a molecular process requires both Genetics and Biochemistry
![Page 9: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/9.jpg)
GeneticScreens
(Yeast mostly) Coactivators
ChromatinRemodelingComplexes
Basal Factors
Activators
Mediator
RNAP II
![Page 10: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/10.jpg)
Purify Polymerases
Immuno-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators
![Page 11: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/11.jpg)
![Page 12: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/12.jpg)
![Page 13: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/13.jpg)
![Page 14: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/14.jpg)
Coding RegionPrUAS
Enzyme involved in sucrose metabolism
![Page 15: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/15.jpg)
Coding RegionPrUAS
Enzyme involved in sucrose metabolism
![Page 16: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/16.jpg)
![Page 17: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/17.jpg)
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
![Page 18: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/18.jpg)
Our snf- screen = many complementation groups = many genes
snf1snf2snf3snf4snf5snf6snf7snf8…..
Which, if any encode txn factors?Secondary screen to identify possible txn factors
![Page 19: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/19.jpg)
Genetic Screens:
Primary screen
Secondary screen(s)
![Page 20: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/20.jpg)
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
![Page 21: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/21.jpg)
![Page 22: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/22.jpg)
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
![Page 23: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/23.jpg)
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
![Page 24: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/24.jpg)
![Page 25: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/25.jpg)
![Page 26: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/26.jpg)
![Page 27: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/27.jpg)
Purify Polymerases
Immuno-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators
![Page 28: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/28.jpg)
Back to GCN5
![Page 29: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/29.jpg)
Continuing Concept:
Comprehensive view of a molecular process requires both Genetics and Biochemistry
![Page 30: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/30.jpg)
Histone Modification
Histone Code
![Page 31: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/31.jpg)
![Page 32: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/32.jpg)
![Page 33: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/33.jpg)
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
![Page 34: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/34.jpg)
Activators
One function of activators is to act as a “platform” that recruits (binds) Co-activators
![Page 35: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/35.jpg)
Purify Polymerases
Immuno-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators
![Page 36: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/36.jpg)
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?
![Page 37: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/37.jpg)
![Page 38: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/38.jpg)
![Page 39: MCB 317 Genetics and Genomics MCB 317 Topic 10, part 4 A Story of Transcription](https://reader035.vdocument.in/reader035/viewer/2022062407/56649dc75503460f94abb758/html5/thumbnails/39.jpg)
Purify Polymerases
Immuno-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators