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

2. How was mediator identified?

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

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

9. Mass Spectrometer

10. 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

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

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

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

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

16. 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

17. Gene Deletion in Yeast by Homologous Recombination Marker Gene

18. Gene Disruption in Yeast

19. Yeast Gene Disruption UASYFGPr YFG in yeast chromosome: URA3 on plasmid: UASURA3Pr PCR UASURA3Pr

20. Yeast Gene Disruption UAS YFG YFGPr Y UAS URA3 URA3Pr U Transform PCR Product into yeast Select for URA3 UAS YFG Pr Y UAS URA3 Pr U URA3

21. 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

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

23. Gene Replacement Replace Chromosomal at Native Locus YFG yfg

24. Gene Replacement by Counterselection URA3 Ura3 XY Uracil

25. 5 FOA is an analog of the Substrate of the Ura3 Enzyme URA3 Ura3 5 FOAToxic Product URA3 cells dead on media containing 5-FOA ura3 cells alive on media containing 5-FOA

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

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

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

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

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

31. 1.Identify YFG (genetic screen for instance) 2.Epitope tag 3.Immuno-affinity purification 4.Mass spec Is YFP part of a complex? If so, what other proteins are in the complex?

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

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

34. 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

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

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

37. Yeast two-hybrid assay An assay in yeast for protein-protein interactions Gal4 BDGal4 AD UAS GAL4 PrHIS3 On plasmid (gene encoding): In chromosome: Yeast strain with plasmid: His prototroph Yeast strain without plasmid: His auxotroph

38. Yeast two-hybrid assay Gal4 BDGal4 AD UAS GAL4 PrHIS3 Txn Growth on minimal media lacking histidine

39. Yeast two-hybrid assay An assay in yeast for protein-protein interactions Gal4 BD Gal4 AD UAS GAL4 PrHIS3 In chromosome: Yeast strain with plasmid: His auxotroph Yeast strain without plasmid: His auxotroph On plasmid 1 (gene encoding): On plasmid 2 (gene encoding):

40. Yeast two-hybrid assay Gal4 BD UAS GAL4 PrHIS3 NO Txn Gal4 AD NO Growth on minimal media lacking histidine

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

42. Gal4 AD Yeast two-hybrid assay UAS GAL4 PrHIS3 Gal4 BD Tx n A YFP Growth on minimal media lacking histidine

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

44. Yeast two-hybrid assay An assay for mapping protein interaction domains On plasmid 1 (gene encoding): On plasmid 2 (gene encoding): Gal4 BD Gal4 AD A Y Gal4 BD F P Gal4 AD A A Growth on Minimal Media lacking Histidine - - + Domain F = region of YFP that binds to subunit A

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

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

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

48. Yeast two-hybrid assay An 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 AD Random clone Gal4 AD Subunit clone GROWTH on Minimal Media lacking Histidine NO Growth on Minimal Media lacking Histidine

49. Yeast two-hybrid assay An 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 AD Random Hclone Gal4 AD HSubunit clone GROWTH on Minimal Media lacking Histidine NO Growth on Minimal Media lacking Histidine

50. Note: different reporters- screens and selections

51. 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)

52. Ab Protein Expression Pattern Gene Orthologs and Paralogs Mutant Gene Biochemistry Genetics Mutant Organism 1 2 3 4 7 8 5 6 9 10 11 Subunits of Protein Complex 12 14 13 15 16 17

53. Molecular Genetics Summary 1.Column Chromatograpy (ion exch, gel filtr) + in vitro assay 2.A. Make Polyclonal Ab; B. Make Monoclonal Ab 3.Western blot, in situ immuno-fluorescence (subcellular, tissue) 4.Screen expression library (with an Ab) 5.Screen library with degenerate probe 6.Protein expression (E. coli) 7.A. Differential hybridization 8.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 PCR 10.Clone by complementation (yeast, E. coli) 11.A. Genetic screen; B. genetic selection 12.Immuno-affinity purification + Mass spec + Computer search 13.In vitro mutagenesis (site directed, deletion, etc) 14.Gene replacement (Yeast, Homologous Recombination) 15.Epitope tag + immuno-affinity chromatography 16.Yeast two-hybrid analysis and screens 17.RNAi

54. 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