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