1. Protein Complexes in S. cerevisiae and E. coli A Focus on Transcription NIH April 7, 2003

2. ATGTAA Primer 1 Primer 2 TRP1 Marker Protein A Calmodulin Binding Peptide Primer 1 Primer 2 TEV Protease Site PCR, Transform, Select for TRP + + Tandem Affinity Purification (TAP) Tagging Strategy for S. cerevisiae 1. 2. Rigaut et al. (1999) Nat. Biotech. 17, 1030 - - 1032. Targeted Gene

3. attL exo bet gam cI857 (cro-attR-bioA) P L P R Δ Carboxy-terminal Tagging in E. coli TAP or SPA KAN STOP CODON Exo/Bet – λ recombinase + E. coli RecBCD – Exonuclease V λ Gam - Temperature sensitive cI repressor – inactive at 42°C CHROMOSOMAL ORF

4. Identification of Protein Complexes in E. coli InfC TAP SPA TAP YacL RpoD SufD SufC SufB YacL RpoD SufD SufC SufB RpoB,C HepA RpoD RpoA YacL NusG SufB SufD SufC RpsA InfC RpsC,D RpsG RpsE,F,K,M,J InfC

5. PROGRESS IN PURIFYING E. coli PROTEIN COMPLEXES A. Tagging of Essential Proteins TAP tags: 91 / 96 SPA tags: 95 / 96 B. Tagging of the 192 Most Highly Conserved, Non-ribosomal, Essential Proteins 188 / 192 C. Overall Progress (March 2003) Tagging attempts for 616 genes (15% of all genes) 559 tagged genes (91%) 468 successful purifications (76%)

6. Compositions and Structures of Protein Complexes Should Also be Determined for Other Important Bacteria Streptococcus pneumoniae Staphylococcus aureus Mycobacterium tuberculosis

8. Elp3-TAPElp1 Elp2 Elp4 Elp6 Elp5 Elp3-TAPSpt6-TAPSpt6 Iws1 Chd1 Spt16-TAP Ctr9 Pob3 Cdc73 CkaI CkaII CkbII CkbI Psh1 Histones Rtf1 Paf1 Leo1Spt16-TAP TAP Purification of Various Elongation Factors Elongator Spt6/Iws1 FACT

9. “Old” and “New” Elongator Gene Deletions Have Similar Effects on Gene Expression Wild type /elp1 deletion Wild type /elp6 deletion

10. Salt Effect in the Purification of Yeast FACT no tag Spt16-TAP Pob3-TAP Pob3-TAPSpt16-TAP 150 mM NaCl 125 mM NaCl Spt16-TAP Pob3 Pob3-TAP Spt16

11. RNA Polymerase II Elongator (Elp1, 2, 3, 4, 5, 6) TFIIS TFIIF Spt5 Spt6 Iws1 Paf1 Cdc73 Rtf1 Leo1 Spt16/Pob3 (FACT) Psh1 Ctr9 Histones Chd1 (Tfg1, Tfg2, Tfg3) Protein Interactions Involved in Transcriptional Elongation (2001) Casein Kinase II Spt4 Phosphorylation? Ctk1, Ctk2,Ctk3 Fcp1 36 Polypeptides

12. A Strategy for IDs of Stable Complexes and Weak Interactions Two Affinity Purification Steps SDS-PAGE Gel Bands Trypsin Digestion MALDI-TOF Mass Spectrometry Identification of Stably Associated Proteins LCQ-Deca Ion Trap Mass Spectrometry Identification of Stably and Weakly Associated Proteins Active Protein For Assays NO GEL! Trypsin Digestion

13. Components: (Exosome) Protein Complex Clustergrams

14. DIAGONALIZED CLUSTERING DEFINES PROTEIN COMPLEXES AND THEIR INTERACTIONS

15. 35S 27S 20S U2 25S 18S U2 U1 7S 5.8S L 5.8S S WT TET- IPI1 TET- IPI2 TET- IPI3 WT Ipi1-TAP Ipi2 Ipi3 Ipi1-TAP 45 66 97 kDa No tag THE IPI COMPLEX IS REQUIRED FOR RIBOSOMAL RNA PROCESSING

16. The Method of Extract Preparation Can Make a Big Difference

17. Erb1- TAP No Tag kDa 97 66 45 31 Erb1- TAP No Tag kDa 97 66 45 31 180000 g 45 min 60000g 30 min Erb1-TAP Nop7 Ytm1 Erb1-TAP Effect of Centrifugation on the Purification of the Erb1/Nop7/Ytm1 Complex

18. Careful Biophysical Characterization of Protein Complexes is Very Important if They are to be Used for Structure Determination Purifications must be scaled up to generate enough material (cost ~$5000 per purification from 1 kg of yeast) Preparations must be homogeneous - extract preparation method must be optimal - salt concentration during preparation must be appropriate - choice of tagged subunits must be appropriate Biophysical methods should be used to determine - homogeneity - subunit stoichiometry - native molecular weight - presence of metal ions and other bound co-factors It will then be possible to mix together protein complexes in equimolar amounts and determine co-structures for interacting protein complexes

19. Purification of Tagged RNA Polymerase II Identification of Iwr1 no tag Rpb3 Rpb1 Rpb2 Rpb3-TAP Rpb4 Rpb5 Rpb6 Ydl115c (Iwr1) kDa 97 66 45 31 21

20. Iwr1 is an evolutionarily conserved, gene specific, elongation factor that interacts with RNA polymerase II.

21. Affinity Purified Protein Complexes are Usually Active

22. SET Domain SPRY Domain WD-40 Repeats Trx related PHD Finger WD-40 Repeats Implicated in regulation of X linked dosage compensated genes Set1 Compass60 Compass50 Compass40 Compass35 Compass25 Compass15 Compass30 Tandem Affinity Purification of COMPASS

23. Subunits of COMPASS are Essential for H3 Lys4 Methylation in vivo COMPASS Methylates Histone H3 Lys4 In Vitro COMPASS (purified Cps60-TAP) Anti-H3 Methyl K4 WT set1  cps60  cps50  cps40  cps30  cps25   H3 Methyl K4

24. Set2- TAP No Tag Rpb1 Rpb2 Set2- TAP 97 66 45 Rpb1 (H5) Rpb1 (H14) Set2-TAP Rpb1 (8WG16) No Tag Tandem Affinity Purification of Set2

25. CH 3 Promoter Coding Region 3’ Untranslated I II RNAPII Ser2 Ser5 Set2 P TFIIH Mediator RNAPII Ser2 Ser5 Paf1C P GTFs CH 3 444 44444444 4444444444 36 COMPASS CH 3 Paf1C CH 3 H3 K4

26. CH 3 IV CH 3 4 44444444 4 36 CH 3 Ser2 Ser5 RNAPII CH 3 III CH 3 4 44444444 4 36 CH 3 Ser2 Ser5 RNAPII P Paf1C CH 3 Ctk1C Set2 Promoter Coding Region 3’ Untranslated 44 CH 3 36 H3 K36

27. Extending the Network: Genetics of Synthetic Lethality in S. cerevisiae A B C X Y Z P X X X = synthetic growth defect OR synthetic lethality

28. SUBSET OF THE GENETIC INTERACTIONS INVOLVING SET2

29. IgG INPUT 1 TATAA PMA1 -304 -47 5 2018 2290 6 32873500 1(ATG)2757(STOP) 2 168 376 3 584 807 4 1010 1250 2823 3277 ChIP Distinguishes Localization in Various Regions of a Gene Hpr1 123456 Coding Regions Rna14 123456 3‘ Untranslated Tfg2 2345 Promoter Promoter 16 Spt16 123456 All Three All Three TFIIFTREX FACT CFIA

30. Localization of Iwr1 on Drosophila Polytene Chromosomes Strategy: make peptide antibodies against Drosophila homologues (15 aa N- and C-terminus) Iwr1 C-terminal RNAPII CTD (H5) Merge

31. Paf1CMediator Rad6C Set3C COMPASS Elongator PRELIMINARY CLUSTERING OF THE GENETIC DATA RXT

32. What About Mammalian Protein Complexes? A. Transfection the tagged protein is overproduced non-stoichiometric complexes are purified spurious protein-protein interactions are expected B. Stable cell lines production of the tagged protein can be regulated, but an appropriate level of the tagged protein is hard to achieve stoichiometric protein complexes will be obtained only if the tagged protein is underproduced cell type specificity is hard to achieve

33. “Knock-in” ES Cells and Mice The Perfect $100,000,000 Solution and Resource the C-terminally tagged protein will usually be produced at the correct level cell type specificity, developmental specificity, and intracellular localization of tagged proteins will be determined by immunofluorescence using antibody against the tag cell-type and tissue-type variation in the compositions of protein complexes will be determined by affinity purification and mass spectrometry an important genetic resource will be available in the form of frozen sperm: tagged genes can be combined with deleted genes simply by mating mice structures of mammalian protein complexes will be determined purified protein complexes will be available for activity assays purified protein complexes will be available for high throughput screens

34. Acknowledgments Nevan Krogan Joyce Li Stephan Zhang Yan Xue Guaqing Zhong Grace Guo Atanas Lalev Nira Datta Ashkan Golshani Robin Haw St. Louis Ali Shilatifard Mark Johnston Andrew Emili Charlie Boone Huiming Ding Tim Hughes Gerard Cagney Amy Tong Ainslie Parsons Mark Robinson Greenblatt Laboratory University of Toronto Affinium Pharmaceuticals Dawn Richards Veronica Canadien Bryan Beattie Harvard University Steve Buratowski Minkyu Kim