mcb 317 genetics and genomics mcb 317 topic 10, part 2, a story of transcription
TRANSCRIPT
MCB 317Genetics and Genomics
MCB 317 Topic 10, part 2,A Story of Transcription
Deletion andLinker Scanner Analysis
In vitro Txn Assay
Promoter not sufficient in vivo
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
Identifying Proteins that Regulate Transcription
Experimental Continuum
• Biochemistry– Chemistry– Enzymology– Crystallography– Protein purification– Molecular biology
• Genetics– Population– Mutant screens– Classical e.g. Epistasis– Molecular genetics– Molecular biology
Fields-of-study function in conjunctionfew biologists/biochemists adhere to a particular sub-field
Genomics
Proteomics
“Factors” are required for specific txn of genes in vitro
gene gene
+
RNAPI, II or III
Radioactive nts+
+ RNAP
+ radiact. nts
+ Nuclear Extract
“Random” initiation Initiation at startSites 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 includedin 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 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
What do factors do?
• 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. Footprinting2. Gel mobility shift assay = Band shift assay3. Affinity purification
Bandshift = simple assay to monitorpurification
Assay fractionsfor 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 RNAPIIPromoter function:
it binds TATAit binds TAFsit 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
TFIIH = Helicase and RNAPII CTD kinase
How would you generate the data for a step-wise assembly model?
Stable pre-initiation Complex (PIC)
TFIIH
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 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