Post on 23-Dec-2015
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MCB 317Genetics and Genomics
MCB 317 Topic 10, part 5A Story of Transcription
How was mediator identified?
Purify Polymerases
Immuno-affinityPurification, Mass Spec
Mediator
In vitro “chromatin”Assembly
GeneticScreens
In vitro txnof in vitro“chromatin”
Coactivators
ChromatinRemodelingComplexes
“Histone”Biochemistry
Activators
RNAPs Purified Based on in vivo txn of naked genomic DNA- nonpecific synthesis of RNA,
but…
… is the “structure” (subunit composition) of RNAPII the same in vivo as defined in vitro?
Hypothesis: Steps involved in purification of RNAPs may have dissociated some subunits.
Test: “Purify” RNAPII by the most gentle method possible
Method: Immunoaffinity purification and Immunoprecipitation from crude extracts
Immuno-affinity purification
Lodish 11-35
Affinity purification of RNAPII identified mediator (as did a genetic screen)
Mass Spectrometer
Mass Spectrometer
Two spectrometers working in tandem1. Separate “large” fragments of proteins2. Those fragments analyzed by a second spectrometer
-> masses of peptides3. Masses of peptides = sequence of peptide fragments4. Computer compares sequence of peptide fragments
with predicted products of genes in genome to identify the gene that encodes the protein
One Subunit -> Complex
Protein -> Immuno-affinity purification -> mass spec -> genome database -> genes that encode subunits of a complex
Genes (encoding)
other subunits
ProteinSubunit
Protein Complex
1
2
1. Immuno-affinity purification2. Mass-spec and genomic database search
Ab
Protein
ExpressionPattern
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
Subunits of Protein Complex
Ab
Protein
ExpressionPattern
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
Subunits of Protein Complex12
13
Yeast Genome Manipulation via Homologous Recombination
• Gene disruption– Determine null phenotype of a gene
• Gene replacement– Create mutant alleles of a gene [pt mut, deletion series,
etc]• Epitope TAG• GFP fusions and protein localization
Gene Deletion in Yeast by Homologous Recombination
MarkerGene
Gene Disruption in Yeast
Yeast Gene Disruption
UAS YFGPr
YFG in yeast chromosome:
URA3 on plasmid:
UAS URA3Pr
PCR
UAS URA3Pr
Yeast Gene Disruption
UASYFG YFGPrY
UASURA3 URA3PrU
Transform PCR Product into yeastSelect for URA3
UASYFG PrY UASURA3 PrU URA3
Gene Disruption in Yeast
1. Delete one copy of YFG in a diploid strain
2. Sporulate, dissect tetrads.
3. If your gene is essential, only two spores will form colonies; if it is not essential all four will form colonies
4. Compare null phenotype to phenotype of your alleles
Ab
Protein
ExpressionPattern
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
Subunits of Protein Complex12
13
Gene ReplacementReplace Chromosomal at Native Locus
YFG
yfg
Gene Replacement by Counterselection
URA3
Ura3
X Y Uracil
5 FOA is an analog of the Substrate of the Ura3 Enzyme
URA3
Ura3
5 FOA Toxic Product
URA3 cells dead on media containing 5-FOA
ura3 cells alive on media containing 5-FOA
Gene ReplacementReplace Chromosomal at Native Locus
YFG
yfg-
URA3
Replace YFG with URA3
URA3
Transform with mutant allele
Select on media containing FOA
yfg-
Ab
Protein
ExpressionPattern
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
Subunits of Protein Complex12
13
Epitope Tagging
Peptide(epitope)
YFG
YFP
YFG
YFP
A commercially availableAntibody will now recognize YFP
Epitope Tagging genes in the yeast genome
Tag Marker
Stop codon
YFG (coding region)
Stop codon
Epitope Tagging genes in the yeast genome
Tag Marker
YFG (coding region)
YFG (coding region) Tag Marker
PCRTransformSelect for Marker
1. Identify YFG (genetic screen for instance)2. Epitope tag3. Immuno-affinity purification4. Mass spec
Is YFP part of a complex? If so, what other proteins are in the complex?
Green Fluorescent Protein (GFP)
Hartwell 19-18
GFP fusion
GFP Marker
YFG (coding region)
YFG (coding region) GFP Marker
PCRTransformSelect for Marker
Yeast Genome Manipulation via Homologous Recombination
• Gene disruption– Determine null phenotype of a gene
• Gene replacement– Create mutant alleles of a gene [pt mut, deletion series,
etc]• Epitope TAG• GFP fusions and protein localization
Ab
Protein
ExpressionPattern
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
Subunits of Protein Complex12
1413
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
EB
A DF
YFP
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
Gal4 BD Gal4 AD
UASGAL4 Pr HIS3
On plasmid (gene encoding):
In chromosome:
Yeast strain with plasmid: His prototrophYeast strain without plasmid: His auxotroph
Yeast two-hybrid assay
Gal4 BD
Gal4 AD
UASGAL4 Pr HIS3
Txn
Growth on minimal media lacking histidine
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
Gal4 BD
Gal4 AD
UASGAL4 Pr HIS3
In chromosome:
Yeast strain with plasmid: His auxotrophYeast strain without plasmid: His auxotroph
On plasmid 1 (gene encoding):
On plasmid 2 (gene encoding):
Yeast two-hybrid assay
Gal4 BD
UASGAL4 Pr HIS3
NO Txn
Gal4 AD
NO Growth on minimal media lacking histidine
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
On plasmid 1 (gene encoding):
On plasmid 2 (gene encoding):
EB
A DF
Gal4 BD
Gal4 ADA
YFP
YFP
Gal4 AD
Yeast two-hybrid assay
UASGAL4 Pr HIS3Gal4 BD Tx
n
A
YFP
Growth on minimal media lacking histidine
Two fusion genes and a reporter gene
Two-hybridAssay
Growth on minimal media lacking histidine
No growth on minimal media lacking histidine
Yeast two-hybrid assayAn assay for mapping protein interaction domains
On plasmid 1 (gene encoding):
On plasmid 2(gene encoding):
Gal4 BD Gal4 ADAY
Gal4 BD F
Gal4 BD P
Gal4 ADA
Gal4 ADA
Growth on MinimalMedia lacking Histidine
-
-
+
Domain F = region of YFP that binds to subunit A
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
EB
A DF
Gal4 AD
YFP
EGal4 ADAReporter
Strain
WTb-g-
G
+ +
+++
-
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
EB
A DF
YFPG
EA
DF
YFP
GDeleteGene B
EB
A DF
YFPG Delete
Gene G
EB
A DYFP
F
Ab
Protein
ExpressionPattern
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
Subunits of Protein Complex12
1413
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
On plasmid 1 (gene encoding):
On plasmid 2 (gene encoding):
Gal4 BD
Gal4 AD
YFP
cDNA library
Start with a yeast strain containing reporter gene and plasmid 1, tranform with cDNA fusion library in plasmid 2:
Gal4 ADRandom cloneGal4 ADSubunit clone
GROWTH on MinimalMedia lacking Histidine
NO Growth on MinimalMedia lacking Histidine
Yeast two-hybrid assayAn assay in yeast for protein-protein interactions
FOR ANY ORGANISM: H = HUMAN
On plasmid 1 (gene encoding):
On plasmid 2 (gene encoding):
Gal4 BD
Gal4 AD
YFHP
HcDNA library
Start with a strain containing reporter gene and plasmid 1, tranform with cDNA fusion library in plasmid 2:
Gal4 ADRandom HcloneGal4 ADHSubunit clone
GROWTH on MinimalMedia lacking Histidine
NO Growth on MinimalMedia lacking Histidine
Note: different reporters- screens and selections
Some uses of two-hybrid assay
• Screen genomic library for additional subunits of a protein complex NOTE: Genes/Libraries can be from any organism
• Infer some aspects of the architecture of a complex (combine with mutant)
• Mapping interaction regions/domains
• Test candidate interactions (genes identified in a screen, for instance)
Ab
Protein
ExpressionPattern
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56
9
10
11
Subunits of Protein Complex12
14
13
15 16
17
Molecular Genetics Summary
1. Column Chromatograpy (ion exch, gel filtr) + in vitro assay2. A. Make Polyclonal Ab; B. Make Monoclonal Ab3. Western blot, in situ immuno-fluorescence (subcellular, tissue)4. Screen expression library (with an Ab)5. Screen library with degenerate probe6. Protein expression (E. coli)7. A. Differential hybridization8. A. Northern blot (RNA), in situ hybridization (RNA or Protein), GFP tag (Protein
pattern and sub-cellular localization)9. A. low stringency hybridization; B. computer search/clone by phone; C.
computer search PCR10. Clone by complementation (yeast, E. coli)11. A. Genetic screen; B. genetic selection12. Immuno-affinity purification + Mass spec + Computer search13. In vitro mutagenesis (site directed, deletion, etc) 14. Gene replacement (Yeast, Homologous Recombination)15. Epitope tag + immuno-affinity chromatography16. Yeast two-hybrid analysis and screens17. RNAi
How do all the txn complexes and components interact in vivo to bring about transcription?
Some data for looping = two-hybrid interactions
Taf-activatorBasal-activator