MCB 317 Genetics and Genomics

1. MCB 317Genetics and Genomics MCB 317 Topic 10, part 5 A Story of Transcription

2. How was mediator identified?

3. In vitro“chromatin” Assembly Purify Polymerases Genetic Screens “Histone” Biochemistry Immuno-affinity Purification, Mass Spec In vitrotxn of in vitro “chromatin” Coactivators Mediator Activators Chromatin Remodeling Complexes

4. 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?

5. 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

7. Immuno-affinity purification

8. Affinity purification of RNAPII identified mediator (as did a genetic screen) Lodish 11-35

9. Mass Spectrometer

10. Mass Spectrometer Two spectrometers working in tandem • Separate “large” fragments of proteins • Those fragments analyzed by a second spectrometer -> masses of peptides • Masses of peptides = sequence of peptide fragments • Computer compares sequence of peptide fragments with predicted products of genes in genome to identify the gene that encodes the protein

11. One Subunit -> Complex Protein -> Immuno-affinity purification -> mass spec -> genome database -> genes that encode subunits of a complex

13. 1 Protein Complex Protein Subunit 2 Genes (encoding) other subunits Immuno-affinity purification Mass-spec and genomic database search

14. Biochemistry Subunits of Protein Complex 1 Protein Orthologs and Paralogs 2 6 9 5 4 Gene Ab 7 3 8 Expression Pattern Mutant Gene 10 Mutant Organism 11 Genetics

15. Biochemistry Subunits of Protein Complex 1 12 Protein Orthologs and Paralogs 13 2 6 9 5 4 Gene Ab 7 3 8 Expression Pattern Mutant Gene 10 Mutant Organism 11 Genetics

16. Yeast Genome Manipulation via Homologous Recombination • Gene disruption • Determine null phenotype of a gene • Gene replacement • Create mutant alleles of a gene [ptmut, deletion series, etc] • Epitope TAG • GFP fusions and protein localization

17. Gene Deletion in Yeast by Homologous Recombination Marker Gene

18. Gene Disruption in Yeast

19. Yeast Gene Disruption YFG in yeast chromosome: UAS Pr YFG URA3 on plasmid: UAS Pr URA3 PCR UAS Pr URA3

20. UASYFG PrY YFG UASYFG PrY UASURA3 PrU URA3 Yeast Gene Disruption Transform PCR Product into yeast Select for URA3 UASURA3 PrU URA3

21. Delete one copy of YFG in a diploid strain Sporulate, dissect tetrads. If your gene is essential, only two spores will form colonies; if it is not essential all four will form colonies Compare null phenotype to phenotype of your alleles Gene Disruption in Yeast

22. Biochemistry Subunits of Protein Complex 1 12 Protein Orthologs and Paralogs 13 2 6 9 5 4 Gene Ab 7 3 8 Expression Pattern Mutant Gene 10 Mutant Organism 11 Genetics

23. Gene ReplacementReplace Chromosomal at Native Locus YFG yfg

24. Gene Replacement by Counterselection URA3 Ura3 X Y Uracil

25. 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

26. YFG URA3 Gene ReplacementReplace Chromosomal at Native Locus Replace YFG with URA3 Transform with mutant allele URA3 yfg- Select on media containing FOA yfg-

27. Biochemistry Subunits of Protein Complex 1 12 Protein Orthologs and Paralogs 13 2 6 9 5 4 Gene Ab 7 3 8 Expression Pattern Mutant Gene 10 Mutant Organism 11 Genetics

28. YFP Epitope Tagging Peptide (epitope) YFG YFG YFP A commercially available Antibody will now recognize YFP

29. Epitope Tagging genes in the yeast genome YFG (coding region) Stop codon Tag Marker Stop codon

30. Epitope Tagging genes in the yeast genome YFG (coding region) Tag Marker PCR Transform Select for Marker YFG (coding region) Tag Marker

31. Is YFP part of a complex? If so, what other proteins are in the complex? Identify YFG (genetic screen for instance) Epitope tag Immuno-affinity purification Mass spec

32. Green Fluorescent Protein (GFP) Hartwell 19-18

33. GFP fusion YFG (coding region) GFP Marker PCR Transform Select for Marker YFG (coding region) GFP Marker

34. Yeast Genome Manipulation via Homologous Recombination • Gene disruption • Determine null phenotype of a gene • Gene replacement • Create mutant alleles of a gene [ptmut, deletion series, etc] • Epitope TAG • GFP fusions and protein localization

35. Biochemistry Subunits of Protein Complex 1 12 Protein Orthologs and Paralogs 13 14 2 6 9 5 4 Gene Ab 7 3 8 Expression Pattern Mutant Gene 10 Mutant Organism 11 Genetics

36. Yeast two-hybrid assayAn assay in yeast for protein-protein interactions F A E D YFP B

37. Gal4 BD Gal4 AD Yeast two-hybrid assayAn assay in yeast for protein-protein interactions On plasmid (gene encoding): In chromosome: UASGAL4 Pr HIS3 Yeast strain with plasmid: His prototroph Yeast strain without plasmid: His auxotroph

38. Gal4 BD Gal4 AD Yeast two-hybrid assay Txn UASGAL4 Pr HIS3 Growth on minimal media lacking histidine

39. Yeast two-hybrid assayAn assay in yeast for protein-protein interactions Gal4 BD On plasmid 1 (gene encoding): On plasmid 2 (gene encoding): Gal4 AD In chromosome: UASGAL4 Pr HIS3 Yeast strain with plasmid: His auxotroph Yeast strain without plasmid: His auxotroph

40. Yeast two-hybrid assay Gal4 AD NO Txn Gal4 BD UASGAL4 Pr HIS3 NO Growth on minimal media lacking histidine

41. A Gal4 AD Yeast two-hybrid assayAn assay in yeast for protein-protein interactions F A E D YFP B Gal4 BD YFP On plasmid 1 (gene encoding): On plasmid 2 (gene encoding):

42. Gal4 AD Yeast two-hybrid assay A YFP Txn Gal4 BD UASGAL4 Pr HIS3 Growth on minimal media lacking histidine

43. Two-hybrid Assay Growth on minimal media lacking histidine No growth on minimal media lacking histidine Two fusion genes and a reporter gene

44. A A A Gal4 AD Gal4 AD Gal4 AD Yeast two-hybrid assayAn 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 + Gal4 BD F - Gal4 BD P Domain F = region of YFP that binds to subunit A

45. A Gal4 AD Yeast two-hybrid assayAn assay in yeast for protein-protein interactions F A E D YFP B G Reporter Strain E Gal4 AD + + WT b- g- + - + +

46. Yeast two-hybrid assayAn assay in yeast for protein-protein interactions F E D F A E A D YFP B YFP Delete Gene B G G F F A E A E D D YFP B YFP B Delete Gene G G

47. Biochemistry Subunits of Protein Complex 1 12 Protein Orthologs and Paralogs 13 14 2 6 9 5 4 Gene Ab 7 3 8 Expression Pattern Mutant Gene 10 Mutant Organism 11 Genetics

48. Yeast two-hybrid assayAn assay in yeast for protein-protein interactions Gal4 BD YFP On plasmid 1 (gene encoding): Start with a yeast strain containing reporter gene and plasmid 1, tranform with cDNA fusion library in plasmid 2: On plasmid 2 (gene encoding): cDNA library Gal4 AD Subunit clone Gal4 AD Random clone Gal4 AD GROWTH on Minimal Media lacking Histidine NO Growth on Minimal Media lacking Histidine

49. Yeast two-hybrid assayAn assay in yeast for protein-protein interactionsFOR ANY ORGANISM: H = HUMAN Gal4 BD YFHP On plasmid 1 (gene encoding): Start with a strain containing reporter gene and plasmid 1, tranform with cDNA fusion library in plasmid 2: On plasmid 2 (gene encoding): HcDNA library Gal4 AD HSubunit clone Gal4 AD Random Hclone Gal4 AD GROWTH on Minimal Media lacking Histidine NO Growth on Minimal Media lacking Histidine

50. Note: different reporters- screens and selections