MCB 317
Genetics and Genomics
MCB 317 Topic 10, part 5
A Story of Transcription
How was mediator identified?
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
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
Affinity purification of
RNAPII identified
mediator (as did a
genetic screen)
Lodish 11-35
Mass Spectrometer
Mass Spectrometer
Two spectrometers working in tandem
1. Separate “large” fragments of proteins
2. Those fragments analyzed by a second spectrometer
-> masses of peptides
3. Masses of peptides = sequence of peptide fragments
4. 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
Protein
Subunit
1
Protein
Complex
2
Genes
(encoding)
other
subunits
1. Immuno-affinity purification
2. Mass-spec and genomic database search
Biochemistry
Subunits of Protein
Complex
1
Protein
2
6
4
Ab
9
5
Orthologs and
Paralogs
Gene
7
3
8
Expression
Pattern
10
Mutant Gene
Mutant Organism
11
Genetics
Biochemistry
1
Subunits of Protein
Complex
12
Protein
2
6
4
Ab
13
9
5
Orthologs and
Paralogs
Gene
7
3
8
Expression
Pattern
10
Mutant Gene
Mutant Organism
11
Genetics
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
Marker
Gene
Gene Disruption in Yeast
Yeast Gene Disruption
YFG in yeast chromosome:
UAS
Pr
YFG
Pr
URA3
URA3 on plasmid:
UAS
PCR
UAS
Pr
URA3
Yeast Gene Disruption
Transform PCR Product into yeast
Select for URA3
UASYFG
PrY
UASURA3
PrU
URA3
UASYFG
PrY
YFG
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
Biochemistry
1
Subunits of Protein
Complex
12
Protein
2
6
4
Ab
13
9
5
Orthologs and
Paralogs
Gene
7
3
8
Expression
Pattern
10
Mutant Gene
Mutant Organism
11
Genetics
Gene Replacement
Replace 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 Replacement
Replace Chromosomal at Native Locus
YFG
Replace YFG with URA3
URA3
Transform with mutant allele
URA3
yfgSelect on media containing FOA
yfg-
Biochemistry
1
Subunits of Protein
Complex
12
Protein
2
6
4
Ab
13
9
5
Orthologs and
Paralogs
Gene
7
3
8
Expression
Pattern
10
Mutant Gene
Mutant Organism
11
Genetics
Epitope Tagging
Peptide
(epitope)
YFG
YFG
YFP
YFP
A commercially available
Antibody will now recognize YFP
Epitope Tagging genes in the yeast genome
YFG (coding region)
Stop codon
Tag
Stop codon
Marker
Epitope Tagging genes in the yeast genome
YFG (coding region)
Tag
Marker
PCR
Transform
Select for Marker
YFG (coding region)
Tag
Marker
Is YFP part of a complex?
If so, what other proteins are in the complex?
1. Identify YFG (genetic screen for instance)
2. Epitope tag
3. Immuno-affinity purification
4. Mass spec
Green Fluorescent Protein (GFP)
Hartwell 19-18
GFP fusion
YFG (coding region)
GFP
Marker
PCR
Transform
Select for Marker
YFG (coding region)
GFP
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
Biochemistry
1
Subunits of Protein
Complex
12
Protein
2
6
4
Ab
13 14
5
9
Orthologs and
Paralogs
Gene
7
3
8
Expression
Pattern
10
Mutant Gene
Mutant Organism
11
Genetics
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
F
A
YFP
D
B
E
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
On plasmid (gene encoding):
Gal4 BD
Gal4 AD
In chromosome:
UASGAL4
Pr
HIS3
Yeast strain with plasmid: His prototroph
Yeast strain without plasmid: His auxotroph
Yeast two-hybrid assay
Txn
UASGAL4
Pr
HIS3
Growth on minimal media lacking histidine
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
On plasmid 1 (gene encoding):
Gal4 BD
On plasmid 2 (gene encoding):
Gal4 AD
In chromosome:
UASGAL4
Pr
HIS3
Yeast strain with plasmid: His auxotroph
Yeast strain without plasmid: His auxotroph
Yeast two-hybrid assay
Gal4 AD
NO Txn
UASGAL4
Pr
HIS3
NO Growth on minimal media lacking histidine
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
F
A
YFP
On plasmid 1 (gene encoding):
On plasmid 2 (gene encoding):
Gal4 BD
A
YFP
Gal4 AD
D
B
E
Yeast two-hybrid assay
A
Gal4 AD
YFP
Tx
n
UASGAL4
Pr
HIS3
Growth on minimal media lacking histidine
Two-hybrid
Assay
Growth on
minimal media
lacking histidine
No growth on
minimal media
lacking histidine
Two fusion genes and a reporter gene
Yeast two-hybrid assay
An assay for mapping protein interaction domains
On plasmid 1
(gene encoding):
On plasmid 2
(gene encoding):
Growth on Minimal
Media lacking Histidine
Gal4 BD
Y
A
Gal4 AD
-
Gal4 BD
F
A
Gal4 AD
+
Gal4 BD
P
A
Gal4 AD
-
Domain F = region of YFP that binds to subunit A
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
F
A
YFP
Reporter
Strain
WT
bg-
A
Gal4 AD
+
+
+
E
Gal4 AD
+
+
D
B
G
E
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
F
D
F
A
YFP
D
A
E
B
G
Delete
Gene B
YFP
G
F
A
YFP
D
B
G
F
A
E
Delete
Gene G
YFP
D
B
E
E
Biochemistry
1
Subunits of Protein
Complex
12
Protein
2
6
4
Ab
13 14
5
9
Orthologs and
Paralogs
Gene
7
3
8
Expression
Pattern
10
Mutant Gene
Mutant Organism
11
Genetics
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
On plasmid 1 (gene encoding):
Gal4 BD
YFP
Start with a yeast strain containing reporter gene and plasmid 1,
tranform with cDNA fusion library in plasmid 2:
On plasmid 2 (gene encoding):
Subunit clone
Gal4 AD
GROWTH on Minimal
Media lacking Histidine
cDNA library
Gal4 AD
Random clone
Gal4 AD
NO Growth on Minimal
Media lacking Histidine
Yeast two-hybrid assay
An assay in yeast for protein-protein interactions
FOR ANY ORGANISM: H = HUMAN
Gal4 BD
On plasmid 1 (gene encoding):
YFHP
Start with a strain containing reporter gene and plasmid 1, tranform with
cDNA fusion library in plasmid 2:
On plasmid 2 (gene encoding):
HSubunit clone
HcDNA library
Gal4 AD
GROWTH on Minimal
Media lacking Histidine
Gal4 AD
Random Hclone
Gal4 AD
NO Growth on Minimal
Media 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)
Biochemistry
1
Subunits of Protein
Complex
12
Protein
2
6
4
Ab
15
Orthologs and
Paralogs
16
9
5
Gene
13
7
3
8
Expression
Pattern
17
10
Mutant Gene
14
Mutant Organism
11
Genetics
Molecular Genetics Summary
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Column Chromatograpy (ion exch, gel filtr) + in vitro assay
A. Make Polyclonal Ab; B. Make Monoclonal Ab
Western blot, in situ immuno-fluorescence (subcellular, tissue)
Screen expression library (with an Ab)
Screen library with degenerate probe
Protein expression (E. coli)
A. Differential hybridization
A. Northern blot (RNA), in situ hybridization (RNA or Protein), GFP tag (Protein
pattern and sub-cellular localization)
A. low stringency hybridization; B. computer search/clone by phone; C.
computer search PCR
Clone by complementation (yeast, E. coli)
A. Genetic screen; B. genetic selection
Immuno-affinity purification + Mass spec + Computer search
In vitro mutagenesis (site directed, deletion, etc)
Gene replacement (Yeast, Homologous Recombination)
Epitope tag + immuno-affinity chromatography
Yeast two-hybrid analysis and screens
RNAi
How do all the txn complexes and components
interact in vivo to bring about transcription?
Some data for
looping =
two-hybrid
interactions
Taf-activator
Basal-activator