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Protein Complexes in S. cerevisiae and E. coli A Focus on Transcription

Protein Complexes in S. cerevisiae and E. coli A Focus on Transcription. NIH April 7, 2003. Tandem Affinity Purification (TAP) Tagging Strategy for S. cerevisiae. Primer 1. Primer 1. 1. ATG. TAA. Targeted Gene. Targeted Gene. Primer 2. Primer 2. 2. Primer 1. Primer 1. Marker.

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Protein Complexes in S. cerevisiae and E. coli A Focus on Transcription

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

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

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

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

  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

  7. Elp3-TAP Spt6-TAP Spt16-TAP Chd1 Spt6 Spt16-TAP Elp1 Ctr9 Rtf1 Elp2 Pob3 Paf1 Elp3-TAP Leo1 Psh1 Cdc73 Elp4 Iws1 CkaI CkaII CkbI CkbII Elp5 Elp6 Histones TAP Purification of Various Elongation Factors Spt6/Iws1 Elongator FACT

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

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

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

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

  12. Protein Complex Clustergrams Components: (Exosome)

  13. DIAGONALIZED CLUSTERING DEFINES PROTEIN COMPLEXES AND THEIR INTERACTIONS

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

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

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

  17. 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

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

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

  20. Affinity Purified Protein Complexes are Usually Active

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

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

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

  24. CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 COMPASS Paf1C CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 Paf1C 3’ Untranslated Coding Region Promoter I GTFs Mediator TFIIH 4 4 4 4 4 4 4 4 4 4 4 RNAPII 36 36 36 36 36 36 36 36 36 36 36 P Ser2 Ser5 4 4 4 4 4 4 4 4 4 4 RNAPII II 36 36 36 36 36 36 36 36 36 36 Set2 P H3 K4 Ser2 Ser5

  25. CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 Ctk1C Paf1C Set2 RNAPII CH3 P Ser2 Ser5 3’ Untranslated Coding Region Promoter 4 4 4 4 4 4 4 4 4 4 4 4 III 36 36 36 36 36 36 36 36 36 36 36 36 CH3 CH3 CH3 H3 K36 4 4 4 4 4 4 4 4 4 4 RNAPII IV 36 36 36 36 36 36 36 36 36 CH3 CH3 CH3 CH3 CH3 Ser2 Ser5

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

  27. SUBSET OF THE GENETIC INTERACTIONS INVOLVING SET2

  28. Promoter Coding Regions 3‘ Untranslated All Three Tfg2 Hpr1 Rna14 Spt16 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 -304 584 2018 3287 3500 168 1010 -47 376 807 1250 2290 1(ATG) 2757(STOP) TATAA 2823 3277 PMA1 ChIP Distinguishes Localization in Various Regions of a Gene IgG INPUT FACT TFIIF TREX CFIA

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

  30. PRELIMINARY CLUSTERING OF THE GENETIC DATA Set3C COMPASS Mediator Paf1C Rad6C RXT Elongator

  31. What About Mammalian Protein Complexes? A. Transfection • the tagged protein is overproduced • non-stoichiometric complexes are purified • spurious protein-protein interactions are expected • cell type specificity is hard to achieve 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

  32. “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 • 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 • 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

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

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