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