1 / 31

Protein Complexes, Networks, and Pathways

Protein Complexes, Networks, and Pathways. components of protein complexes. interactions between protein complexes. functional pathways. Accessory Proteins. Pol III Subunit. α. TopB. τ. RecQ. γ. δ. δ `. ε. Ssb. χ. ψ. DNA Polymerase III Core Enzyme and Clamp Loader Complexes.

haru
Télécharger la présentation

Protein Complexes, Networks, and Pathways

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Protein Complexes, Networks, and Pathways • components of protein complexes • interactions between protein complexes • functional pathways

  2. Accessory Proteins Pol III Subunit α TopB τ RecQ γ δ δ` ε Ssb χ ψ DNA Polymerase III Core Enzyme and Clamp Loader Complexes -AP ψ-AP θ-AP α-AP ε-AP χ * • purification of a core enzyme • subunit (αθε) yields sub- • stoichiometric amounts of the • clamp-loader complex (γδδ’χψ) • purification of a clamp- • loader subunit (γδδ’χψ) • yields sub-stoichiometric • amounts of core enzyme * * * • the clamp loader is also • associated with Ssb, TopB, • and RecQ * • TopB and RecQ are also • associated with RNaseH1, • which degrades RNA primers

  3. REPLICATION FORK IN E. coli Ssb Primase RNA primer DnaB helicase RNase HI RecQ Topoisomerase III ψ χ Clamp loader complex α ε θ βSliding clamp Core Polymerase

  4. Interaction Network for DNA Polymerase III DNA pol III

  5. THE MOST HIGHLY CONSERVED ESSENTIAL E. coli PROTEINS TARGETS FOR NEW BROAD-SPECTRUM ANTIBIOTICS • The 189 most highly conserved non-ribosomal E. coli proteins known to be essential in at least one bacterial species were chosen for study • 176 of these genes were successfully tagged with SPA or TAP tags. • 168 (89 %) of the proteins were successfully purified. • 188 distinct interacting proteins were identified, 46 of which represent hypothetical proteins or proteins of unknown function. CURRENT STATUS: More than 1000 proteins tagged and purified

  6. Predicting protein functions using microarray expression data (Wu et al. (2002), Nature Genetics) Protein translocation (2) Cell Wall (9) DNA Repair (11) Vesicular transport (13) Recombination (15) Cell cycle control (17) Phosphate metabolism (19) Protein folding (29) Nucleotide metabolism (38) Cell Stress (39) Protein degradation (63) Pol II transcription (65) Protein modification (66) Mating response (79) Meiosis (97) Chromatin chromosome structure (100) Lipid fatty-acid and sterol metabolism (139) Carbohydrate metabolism (179) Small molecule transport (192) Amino-acid metabolism (249) RNA processing and modification (285) Energy generation (292) Protein synthesis (370) 80% 70% 60% 50% 40% 30% Anticipated accuracy (based on characterized genes) Essential Conserved 456 microarray experiments 1,655 uncharacterized yeast ORFs RNA processing and modification (285 genes)

  7. RNA Processing and Modification Genes • 91 representative genes from literature and databases • 88 from transcriptional co-regulation • 28 homologous to human nucleolar proteins

  8. The Program for RNA Processing and Modifying Proteins Mutants (deletions, TET promoters) TAP tagged strains Cell pellets Purification Phenol extraction DNase I SDS PAGE Trypsin digestion Labeling of RNA MALDI-TOF MS Gel Densitometry LC-MS/MS Hybridization to Microarray

  9. Non-coding RNA Microarray

  10. <-0.3 -0.2 -0.1 0 0.1 0.2 >0.3 Missing data = grey Log(ratio) MICROARRAY ANALYSIS OF RNA PROCESSING DEFECTS A0 A1 D A2 A3 B1 E C2 C1 B2 Mutated geneGO biological process 3' UTR processing defective (rnt1) RNT1 35S primary transcript processing YGR251W*** biological_process unknown ECM16 processing of 20S pre-rRNA HCA4 35S primary transcript processing UTP20 biological_process unknown UTP22 biological_process unknown UTP19 biological_process unknown SAS10 establishment and/or maintenance of chromatin YOR287C biological_process unknown UTP11 processing of 20S pre-rRNA YGR272C*** biological_process unknown UTP6 processing of 20S pre-rRNA FYV7*** biological_process unknown RRP9 35S primary transcript processing RPS4A protein biosynthesis UTP4 processing of 20S pre-rRNA NOP7 ribosomal large subunit biogenesis UTP9 processing of 20S pre-rRNA YHR040W biological_process unknown YOR004W biological_process unknown NAN1 processing of 20S pre-rRNA CBF5 35S primary transcript processing MRD1 35S primary transcript processing BUD22 bud site selection NSR1*** biological_process unknown BUD21 processing of 20S pre-rRNA RPS1B protein biosynthesis POL5 DNA dependent DNA replication DBP6 35S primary transcript processing NOP4 rRNA processing PXR1 biological_process unknown YLR022C biological_process unknown MAK16 ribosomal large subunit biogenesis RNA1 rRNA-nucleus export RRP43 35S primary transcript processing NSR1 rRNA processing DBP8 35S primary transcript processing FYV5 biological_process unknown BRX1 ribosomal large subunit assembly and maintenance TIF6 processing of 27S pre-rRNA RPL17A protein biosynthesis NOP15 ribosomal large subunit biogenesis NOC3 DNA replication initiation NUP82 NLS-bearing substrate-nucleus import YOL022C biological_process unknown PRP42 mRNA splicing YNL313C biological_process unknown LSM5 mRNA splicing SSU72 transcription initiation from Pol II promoter GLC7 glycogen metabolism YDR365C biological_process unknown CDC33 regulation of cell cycle PRP5 lariat formation, 5'-splice site cleavage PRP39 mRNA splicing RTS2 biological_process unknown KRE28 biological_process unknown YMR269W biological_process unknown EPL1 histone acetylation RRP12 processing of 20S pre-rRNA RPS18B protein biosynthesis LOC1 ribosomal large subunit biogenesis YOR006C biological_process unknown RIO1 S phase of mitotic cell cycle YLR435W*** biological_process unknown FAP7*** response to oxidative stress TSR1 ribosome biogenesis and assembly DRE3 cell growth and/or maintenance LSM6 mRNA splicing RPS0A protein biosynthesis NOC2 biological_process unknown PTA1 mRNA cleavage RPP1 rRNA processing POP1 rRNA processing SNM1 rRNA processing LSM1 deadenylation-dependent decapping GRC3*** cell growth and/or maintenance MRT4 ribosomal large subunit biogenesis IPI3 biological_process unknown YDR412W*** biological_process unknown NOG2 mRNA splicing MAK5 rRNA processing SDA1 actin cytoskeleton organization and biogenesis IPI1 biological_process unknown MDN1 protein complex assembly IPI2 biological_process unknown NUG1 biological_process unknown YDL062W biological_process unknown YDL063C biological_process unknown RAI1 RNA catabolism RRP6 35S primary transcript processing LRP1*** biological_process unknown TIF4631 translational initiation YNL227C endocytosis MPE1 mRNA cleavage RRP46 35S primary transcript processing 5' ETS and A2 cleavage (U3 processome- like) A2 cleavage (snoRNP-like) D-A2 processing (RIO1-like) A2-B1 processing (RNAseP-like) E-C2 (exosome/ pre-60S-like) A0 A1 D A2 A3 B1 E C2 C1 B2 5’ETS 18S ITS1 5.8S ITS2 25S 3’ETS

  11. Ipi1-TAP No tag kDa 97 Ipi2 66 Ipi3 5’ETS18SITS1 5.8S ITS2 25S 3’ETS5S 45 Ipi1-TAP IPI (Involved in Processing ITS2) Complex WT TET-IPI1 TET-IPI2 TET-IPI3 WT C A B 35S 27S 20S TET-IPI1 TET-IPI2 TET-IPI3 U2 25S 18S rRNA U2 U1 7S 5.8SL 5.8SS

  12. Protein Complexes in RNA Processing • 317 proteins “clustered” into complexes (~stoichiometric) • 56 complexes (2-17 subunits) • 36 singleton proteins • 172 additional sub-stoichiometric proteins (possible • interactions between complexes)

  13. TAP Tagged Proteins Met-Glut tRNA Synthetase Dbp3 C Gar1/Cbf5 Nop58/Sik1 Dbp7/Rrp5 Mtr4 Complex Mdm20/Nat3 Noc Complex Sit4 Complex U4/U6.U5 tri-snRNP U6 Specific snRNP Ydr117c/Rps4b Complex mRNA Cap-Binding/eIF4F Exosome CLUSTERING TO ORGANIZE PROTEIN COMPLEXES RNA Polymerase III RNA Polymerase II RNA Polymerase I Rrb1/Rpl3 Purified Proteins Cbc2/Sto1 Nop7 Complex TREX RSC Mediator Diagonal-gram goes here TFIIIC U1 snRNP Arx1 Pwp1/Nop12/Brx1 Nap1 UTP “C” Spt16 Casein Kinase II Ykl088w 19S Proteosome eIF3 tIF2 Kap95/Srp1 Rli1 Prp19 mRNA Cleavge/Polyadenylation Imd Gir 20S Proteosome Bcp1 Ccr4 Ipi Kap104/Nab2 Rcl1/Bms1 RNase P SRP Translation Release Factor Trm1/Nsa1 Trm7 UTP “B” Eft2/Hgh1

  14. A Network Diagram Using Protein Complexes

  15. Association of RNA-Processing Complexes with Specific RNAs RRP9 - Rrp9 (1) UTP13 - UTP B (6) UTP18 - UTP B (6) UTP9 - UTP A (8) UTP10 - UTP A (8) UTP8 - UTP A (8) RRP5 - Dbp7/Rrp5 (4) PRT1 - eIF3 (7) TIF35 - eIF3 (7) NIP1 - eIF3 (7) DBP3 - Dbp3/Bmh1/Nsr1 (3) HCA4 - Hca4 (1) NOP58 - Nop58/Sik1 (3) NPL3 - Npl3 (1) GAR1 - Gar1/Cbf5 (6) YGR283C - Gar1/Cbf5 (6) RRP8 - Rrp8 (1) YDR365C - Ydr365c (1) NOC2 - Noc (3) ERB1 - Nop7/Erb1/Ytm1 (3) NOP7 - Nop7/Erb1/Ytm1 (3) NOP12 - Pwp1/Brx1/Nop12 (3) PWP1 - Pwp1/Brx1/Nop12 (3) RRP45 - Exosome (12) RRP46 - Exosome (12) RRP6 - Exosome (12) IPI1 - Ipi (3) PRP19 - Prp19 (4) PUF6 - Puf6 (1) RNT1 - Rnt1 (1) YBR025C - Ybr025c (1) RPN6 - 19S Proteosome (17) PRE8 - 20S Proteosome (13) PUS1 - Pus1 (1) TRM1 - Trm1/Nsa1 (2) CBC2 - Cbc2/Sto1 (2) STO1 - Cbc2/Sto1 (2) LHP1 - SRP (3) THS1 - Ths1 (1) TSR2 - Tsr2 (1) BRR2 - U4/U6.U5 tri-snRNP (2) PRP3 - U6-specific snRNP core (14) LSM3 - U6-specific snRNP core (14) LSM5 - U6-specific snRNP core (14) KEM1 - U6-specific snRNP core (14) UTP22 - UTP22/RRP7 (2) UTP “B” B A UTP “A” C D eIF3 Nop58/Sik1 F E G H Nop7 Complex I Exosome J L tagged proteins K 19S Proteasome M N 20S Proteasome O P SRP Q U6-Specific snRNP R Tyr2 Ile2 Trp2 Thr3 M-Ile purified proteins snR78 snR77 snR76 snR75 snR74 snR73 snR72 snR190 U14 U3 snR36 snR43 snR38 RPR1 MRP TLC1 ScR1 YDR064w EF-1g RPL30 18S 5.8S 25S 5S U1 U2 U4 U5 U6 5’ ETS ITS1 ITS2 3’ ETS rRNA snoRNA tRNA snRNA misc. mRNA

  16. Steps in the RNA Polymerase II Transcription Cycle Initiation Elongation Termination General transcription factors Mediator CTD kinases Elongation factors Cleavage, polyadenylation, and termination factors chromatin-modifying enzymes

  17. 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

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

  19. Protein Interactions Involved in Transcriptional Elongation (2002) Spt5 Spt4 TFIIS Ctk1 Iwr1 Spt6 Phosphorylation? Elongator (Elp1, 2, 3, 4, 5, 6) Iws1 RNA Polymerase II Casein Kinase II Fcp1 Set2 Chd1 TFIIF Paf1 (Tfg1, Tfg2, Tfg3) Cdc73 Paf1-C Ctr9 Spt16/Pob3 (FACT) Psh1 COMPASS (8 polypeptides) Rtf1 Leo1 Histones 55 Polypeptides

  20. X Y Z Theoretical Pathways in S. cerevisiae A X B = synthetic lethality X X C P

  21. MATa MATa X set2 xxx Mating D a/a wild-type Sporulation MATa Haploid Selection (MFA1pr-HIS3) Double Mutant Selection

  22. Hits Two Dimensional Hierachical Clustering Identifies Similar Patterns of Genetic Interactions: Clusters of Query Genes Y axis Clusters of Interacting Genes (Hits) X axis MMS4 MUS81 RTT107 YBR094W HST3 TOP1 CDC8 RAD50 RAD52 CDC45 CDC7 DBF4 CSM3 MRC1 TOF1 ELG1 POL32 RAD27 ORC2 ORC5 RAD9 RAD53 ESC2 SGS1 RAD24 ARP1 DYN1 PAC1 NBP2 NUM1 BIK1 TUB3 CHL1 MAD2 CHL4 YDR332W CTF18 CTF8 CTF4 DCC1 BIM1 KAR3 GIM3 YKE2 GIM4 PAC10 TUB2 CLB4 KAR9 KIP3 ARP6 GOD1 HTZ1 SET2 ARC40 ARP2 BNI1 CLA4 KRE1 SLT2 SHS1 SMY1 BNI4 PHO85 BBC1 CDC42-118 YDR437W CNB1 CNB1/1 ERG11 RIC1 YPT6 HST1 YJR070C APP1 RAS2 Query Genes

  23. 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

  24. Bdf1 Yaf9 Purification of the SWR1 Complex Swr1-TAP Vps72-TAP Swc1-TAP No Tag Aor1-TAP Vps71-TAP MR Swr1-TAP Swr1 kDa Vps72-TAP Vps72 Swc1-TAP 97 Swc1 66 Arp4/God1 Rvb1/Rvb2/Arp6 Aor1-TAP 45 Act1 Aor1 Vps71-TAP Vps71 31 Translocated to MLL in Acute Myeloid Leukemia

  25. Swr1-TAP Swc1-TAP Vps71-TAP Aor1-TAP Vps72-TAP Swr1 Swc1 Vps72 Aor1 Vps71 God1 Arp6 Act1 Arp4 Yaf9 Rvb1 Rvb2 Bdf1

  26. ORGANIZING PROTEINS INTO PATHWAYS • Microarray analysis • Synthetic genetic array analysis Concept: proteins with the same function should have similar effects on gene expression and similar genetic interactions

  27. Clustering of Microarray Data Groups Proteins into Complexes and Pathways SIF2 RAD6 BRE1 SDC1 RTF1 PAF1 BRE5 SWC4 SWC2 SET3 RXT2 DEP1 SWR1 LEO1 HOS2 SAP30 RXTC RAD6C PAF1C SWR1C SET3C Histone Trans-Modification Pathway

  28. SWR1C Bdf1 Htz1 Step 2 Step 3 Step 1 H4-Ac Silent Heterochromatin

  29. Extending Pathway Analysis to Multiple Systems The Case of ER to Golgi Transport • ~400 non-essential proteins localize to ER and Golgi • purify these proteins and organize into protein complexes • use SGA to organize the proteins and protein complexes • into functional pathways

  30. Structural Genomics of Protein Complexes • Production of protein complexes • - co-overexpression • - purification from a natural source • - synthesis in vitro • Structure determination • - high resolution by x-ray crystallography • - low resolution by electron microscopy

  31. Acknowledgments Greenblatt Laboratory Affinium Pharmaceuticals Dawn Richards Veronica Canadien Bryan Beattie Kamal Boura Nevan Krogan Joyce Li Guaqing Zhong Grace Guo Atanas Lalev Nira Datta Robin Haw University of Toronto Andrew Emili Charlie Boone Tim Hughes Gerard Cagney Amy Tong Mark Robinson Gareth Butland Huiming Ding WenTao Peng Owen Ryan UCSF Erin O’Shea Jonathan Weissman Harvard University Steve Buratowski Minkyu Kim Michael Keough

More Related