Download MCB317 Topic 10, part 4, A Story of Txn Sp14

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
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
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
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
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