Transcript Reading assignment - life.illinois.edu

MCB 317
Genetics and Genomics
MCB 317 Topic 10, part 2,
A Story of Transcription
Deletion and
Linker Scanner Analysis
In vitro
Txn Assay
Promoter not
sufficient in vivo
Identify and define TBP
and basal factors
Extract +
Prom.-Enh.
Basal Facts. +
Prom.-Enh.
Activated Txn
(Enhanced) &
Regulated Txn
Extract +
Prom.-Enh.
Identification of
Enhancers
Activators
Co-activators + Enhancer &
TBP & TAFs
Promoter
“Activated” txn & Regulated txn
Identifying Proteins that
Regulate Transcription
Experimental Continuum
• Biochemistry
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Chemistry
Enzymology
Crystallography
Protein purification
Molecular biology
Proteomics
• Genetics
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Population
Mutant screens
Classical e.g. Epistasis
Molecular genetics
Molecular biology
Genomics
Fields-of-study function in conjunction
few biologists/biochemists adhere to a particular sub-field
“Factors” are required for specific txn of genes in vitro
gene
gene
+
RNAPI, II or III
+
Radioactive nts
“Random” initiation
+ RNAP
+ radiact. nts
+ Nuclear Extract
Initiation at start
Sites and txn of
specific genes
Conclusions
1. Cellular (nuclear) molecules other than the
RNAPs mediate specificity of txn. Different than
prokaryotes.
2. “Factors” recruit RNAPs to their Promoters!
How to Identify Txn “Factors”?
Controls allow interpretation
of results.
One way to look at controls:
Positive control insures that
Assay or experiment
is working properly.
Negative control sets the
“baseline” or defines the
“signal-to-noise” ratio
Controls need to be included
in virtually all experiments
RNAPII Txn
• General transcription factors: TFIIA, TFIIB, TFIID, TFIIE,
TFIIF, TFIIH
• General factors also called Basal Factors
• Basal factors plus RNAPII sufficient for txn in vitro from a
gene and promoter
• Required for txn of all RNAPII genes
• Factors are (protein) complexes
• Subunits: TFIIA = 2, TFIID ~ 15, TFIIE = 2, TFIIF = 3, TFIIH =
10 subunits
• “Core” RNAPII = 12 subunits in yeast
• Factors v. Fractions (Fxn)
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
What do factors do?
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•
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•
•
Do they bind DNA?
Are they enzymes that modify the polymerase?
Do they bind RNAPII (ala Sigma Factor)?
Do they do any of a hundred other things?
Are they artifacts? [TFIIC, TFIIG]
Order of Addition Assay
How to identify where factors bind to genes?
1. Footprinting
2. Gel mobility shift assay (band shift assay)
Nucleases
•
•
•
•
Site specific (restriction enzymes)
Exonucleases
DNAases
RNAases
“Footprinting”
DNAase I
Footprinting
DNAase “nicks”
one strand
Gel Mobility Shift = Band Shift
= EMSA
(Electrophoretic Mobility Shift Assay)
“Native gels =
Gels in which
proteins remain
functional
How to purify factors that bind DNA?
How to assay for DNA binding proteins?
1. Footprinting
2. Gel mobility shift assay = Band shift assay
3. Affinity purification
Bandshift = simple
assay to monitor
purification
Assay fractions
for factors by band
shift gel
Gel Mobility Shift Assay = Band Shift Assay
“Free” DNA = unbound DNA; only small % needs to be bound
Why is small amount of DNA shifted
experimentally significant?
How does this compare to footprinting?
Combine gel shift and Footprinting
DNAase -> Run Gel -> cut out shifted bands
-> denature -> sequencing gel
Lodish 11-13
Affinity purification of
DNA binding proteins
Which txn proteins bind DNA?
• General txn factors?
• Activators?
“Real” Data: actual basal factors, not hypothetical fxns
Therefore, TFIID must contain additional
components that are required for activated
levels of txn
TAFs = TBP Associated Factors
Results without Experiment
TFIID is a complex of ~15 proteins:
TBP plus 14 TAFs
TBP is at the heart of RNAPII
Promoter function:
it binds TATA
it binds TAFs
it binds basal factors
A current model for assembly of the
general txn factors at promoters
Does this process actually occur
in vivo?
Remember, we’ll talk about it
later
Stable pre-initiation Complex
(PIC)
TFIIH
TFIIH = Helicase and RNAPII CTD kinase
How would you generate the data for a step-wise assembly
model?
Title
“Complex” binds multiple sites
Cooperativity: strong sites help binding at weak sites
Order of binding- is TBP/TFIID always first?
How are txn by RNAP I, RNAP II and
RNAP III similar and dissimilar?
RNAP I Txn
RNAP III Txn
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