protein complexes in s. cerevisiae and e. coli a focus on transcription
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
Protein Complexes in S. cerevisiae and E. coliA Focus on Transcription
NIH April 7, 2003
ATG TAA
Primer 1Primer 1
Primer 2Primer 2
TRP1TRP1 MarkerMarkerProtein AProtein A
CalmodulinCalmodulinBinding Binding PeptidePeptide
Primer 1Primer 1
Primer 2Primer 2
TEV TEV Protease SiteProtease Site
PCR, Transform, Select PCR, Transform, Select for TRPfor TRP++
Tandem Affinity Purification (TAP) Tagging Strategy for S. cerevisiae
1.
2.
RigautRigaut et al. (1999) et al. (1999) Nat. Nat. Biotech.Biotech. 17, 103017, 1030--1032.1032.
Targeted GeneTargeted Gene
attL exo bet gam cI857 (cro-attR-bioA)
PL PR 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
Identification of Protein Complexes in E. coli
InfC
TAP SPA
SPA TAPY
acL
Rpo
DS
ufD
Suf
CS
ufB
Yac
LR
poD
Suf
D
Suf
CS
ufB
RpoB,CHepA
RpoD
RpoA
YacLNusG
SufBSufD
SufC
RpsA
InfCRpsC,D
RpsG
RpsE,F,K,M,J
InfC
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%)
Compositions and Structures of Protein Complexes Should Also be Determined for Other Important Bacteria
• Streptococcus pneumoniae
• Staphylococcus aureus
• Mycobacterium tuberculosis
•
•
•
Transcription cycle for RNA Polymerase II
P
PP
PPP
PP
P
PP
PPP
PP
AAAAAA
CTD-Kinases
Promoter Escape TerminationElongationInitiation
m7Gp
m7Gp
AAAAAA
CTD-Phosphatase
RNAPII
Holo-RNAPII
Elongation factors
Accessory factors
GTFs and Mediator
SRB 10/11
CTD kinase
m7Gpm7Gp
Elp3-TAPElp3-TAP
Elp1Elp1
Elp2Elp2
Elp4Elp4
Elp6Elp6
Elp5Elp5
Elp3-TAPElp3-TAP
Spt6-TAPSpt6-TAP
Spt6Spt6
Iws1Iws1
Chd1Chd1Spt16-TAPSpt16-TAP
Ctr9Ctr9
Pob3Pob3
Cdc73Cdc73CkaICkaI
CkaIICkaII
CkbIICkbII
CkbICkbI
Psh1Psh1
Histones Histones
Rtf1Rtf1
Paf1Paf1
Leo1Leo1
Spt16-TAPSpt16-TAP
TAP Purification of Various Elongation Factors
Elongator Spt6/Iws1FACT
-4
-3
-2
-1
0
1
2
3
-4 -3 -2 -1 0 1 2 3 4
“Old” and “New” Elongator Gene Deletions Have Similar Effects on Gene Expression
Wild type /elp1 deletion
Wil
d ty
pe /e
lp6
dele
tion
Salt Effect in the Purification of Yeast FACTSalt Effect in the Purification of Yeast FACT
no tagno tagno tagno tag no tagno tagSpt16-TAPSpt16-TAPPob3-TAPPob3-TAPPob3-TAPPob3-TAP Spt16-TAPSpt16-TAP
150 mM NaCl150 mM NaCl 125 mM NaCl125 mM NaCl
Spt16-TAPSpt16-TAP
Pob3Pob3Pob3-TAPPob3-TAP
Spt16Spt16
RNA Polymerase IIRNA Polymerase II Elongator (Elp1, 2, 3, 4, 5, 6)
TFIIS
TFIIF
Spt5
Spt6
Iws1
Paf1
Cdc73
Rtf1
Leo1
Spt16/Pob3 Spt16/Pob3 (FACT)(FACT)
Psh1Ctr9
Histones
Chd1
(Tfg1, Tfg2, Tfg3)
Protein Interactions Involved in Transcriptional Elongation (2001)
Casein Kinase II
Spt4
Phosphorylation?Ctk1, Ctk2,Ctk3
Fcp1
36 Polypeptides36 Polypeptides
A Strategy for IDs of Stable Complexes and Weak Interactions
Two Affinity Purification Steps
SDS-PAGE
Gel Bands
Trypsin Digestion
MALDI-TOFMass Spectrometry
Identification of Stably Associated Proteins
LCQ-Deca Ion TrapMass Spectrometry
Identification of Stably and Weakly Associated Proteins
ActiveProtein
ForAssays
NO GEL!
Trypsin Digestion
MIPS Functional Classification Catalogue (259 categories)
Category p-value In Category from Cluster k frRNA processing 6.64E-11 RRP43 RRP45 RRP46 SKI6 RRP4 DIS3 6 63rRNA transcription 1.94E-09 RRP43 RRP45 RRP46 SKI6 RRP4 DIS3 6 109
MIPS Complexes Catalogue (315 categories)
Category p-value In Category from Cluster k fExosome complex -1.33E-15 RRP43 RRP45 RRP46 SKI6 RRP4 DIS3 6 7rRNA processing complexes 1.75E-14 RRP43 RRP45 RRP46 SKI6 RRP4 DIS3 6 18RNA processing complexes 2.42E-09 RRP43 RRP45 RRP46 SKI6 RRP4 DIS3 6 113
MIPS Protein Classes Catalogue (190 categories)
Category p-value In Category from Cluster k fExonucleases 8.19E-09 SKI6 RRP4 DIS3 3 43'->5' exoribonucleases 8.19E-09 SKI6 RRP4 DIS3 3 4Nucleases 2.05E-08 SKI6 RRP4 DIS3 3 5
GO Cellular Component (240 categories)
Category p-value In Category from Cluster k fexosome (RNase complex) -2.66E-15 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 8 10cytoplasmic exosome (RNase complex) -2.66E-15 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 8 10nuclear exosome (RNase complex) -2.22E-15 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 11nucleus 6.99E-11 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 470intracellular 7.18E-08 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 1011cell 1.65E-06 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 1431
GO Biological Process (461 categories)
Category p-value In Category from Cluster k fmacromolecule catabolism -1.55E-15 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 8 21RNA metabolism -1.33E-15 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 8 19mRNA catabolism -1.33E-15 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 8 19ribosome biogenesis -6.66E-16 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 164transcription, from Pol I promoter -4.44E-16 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 6635S primary transcript processing 1.44E-15 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 41cytoplasm organization and biogenesis 4.17E-11 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 444cell organization and biogenesis 8.30E-11 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 479catabolism 4.80E-10 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 8 331metabolism 7.73E-07 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 8 836cell growth and/or maintenance 6.05E-06 RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6 9 1652
YCR035C RRP43YGR095C RRP46YGR195W SKI6YOL142W RRP40YNL232W CSL4YOL021C DIS3YOR001W RRP6YHR069C RRP4YDR280W RRP45
Components:(Exosome)
Protein Complex Clustergrams
DIAGONALIZED CLUSTERING DEFINES PROTEIN COMPLEXES AND THEIR INTERACTIONS
35S
27S
20S
U2
25S
18S
U2
U1
7S
5.8SL
5.8SS
WT
TET-
IPI1
TET-
IPI2
TET-
IPI3
WT
Ipi1
-TA
PIpi2
Ipi3
Ipi1-TAP 45
66
97
kDa
No
tag
THE IPI COMPLEX IS REQUIRED FOR RIBOSOMAL RNA PROCESSING
The Method of Extract Preparation Can Make a Big Difference
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
Nop7Nop7
Ytm1
Ytm1
Erb1-TAP
Effect of Centrifugation on the Purification of the Erb1/Nop7/Ytm1 Complex
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
Purification of Tagged RNA Polymerase IIIdentification of Iwr1
no tag Rpb3
Rpb1Rpb2
Rpb3-TAP
Rpb4
Rpb5
Rpb6
Ydl115c (Iwr1)
kDa
97
66
45
31
21
Iwr1 is an evolutionarily conserved, gene specific, elongation factor thatinteracts with RNA polymerase II.
Affinity Purified Protein Complexes are Usually Active
SET Domain
SPRY Domain
WD-40 Repeats
Trx related
PHD Finger
WD-40 Repeats
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
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
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
CH3
CH3 CH3
CH3
CH3 CH3
CH3 CH3
Promoter Coding Region 3’ Untranslated
I
IIRNAPII
Ser2
Ser5
Set2
P
TFIIH Mediator
RNAPII
Ser2
Ser5
Paf1C
P
GTFsCH3 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
4 4 4 4 4 4 4 4 4 4 4
4 4 4 4 4 4 4 4 4 4
36 36 36 36 36 36 36 36 36 36 36
36 36 36 36 36 36 36 36 36 36
COMPASS
CH3
CH3 CH3
Paf1C
CH3 CH3
CH3
CH3 CH3
H3 K4
CH3
CH3 CH3 CH3 CH3CH3 CH3
IV
CH3
CH3 CH3
CH3
CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3
4 4 4 4 4 4 4 4 4 4
36 36 36 36 36 36 36 36 36
CH3 CH3 CH3 CH3
Ser2
Ser5
RNAPII
CH3
CH3 CH3
CH3
CH3 CH3 CH3 CH3CH3 CH3
III
CH3
CH3 CH3
CH3
CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3
4 4 4 4 4 4 4 4 4 4
36 36 36 36 36 36 36 36
CH3 CH3 CH3
CH3
CH3 CH3
Ser2
Ser5
RNAPII
PPaf1C
CH3 CH3
Ctk1C
Set2
Promoter Coding Region 3’ Untranslated
4 4
CH3
CH3 CH3
CH3
CH3 CH3
36 36 36 36
H3 K36
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
SUBSET OF THE GENETIC INTERACTIONS INVOLVING SET2
IgG
INPUT
1
TATAA
PMA1
-304 -47
5
2018 2290
6
3287 3500
1(ATG) 2757(STOP)
2
168 376
3
584 807
4
1010 1250
2823 3277
ChIP Distinguishes Localization in Various Regions of a Gene ChIP Distinguishes Localization in Various Regions of a Gene
Hpr1Hpr1
1 2 3 4 5 6
Coding RegionsCoding Regions
Rna14Rna14
1 2 3 4 5 6
3‘ Untranslated 3‘ Untranslated
Tfg2Tfg2
2 3 4 5
PromoterPromoter
1 6
Spt16
1 2 3 4 5 6
All ThreeAll Three
TFIIF TREX FACTCFIA
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
Paf1CMediator
Rad6C
Set3CCOMPASS
Elongator
PRELIMINARY CLUSTERING OF THE GENETIC DATA
RXT
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
• cell type specificity is hard to achieve
“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
Acknowledgments
Nevan Krogan Joyce LiStephan Zhang Yan Xue Guaqing Zhong Grace GuoAtanas Lalev Nira DattaAshkan Golshani Robin Haw
St. LouisAli ShilatifardMark Johnston
Andrew Emili Charlie Boone Huiming Ding Tim HughesGerard Cagney Amy Tong Ainslie Parsons Mark Robinson
Greenblatt Laboratory
University of Toronto
Affinium PharmaceuticalsDawn RichardsVeronica CanadienBryan Beattie
Harvard UniversitySteve BuratowskiMinkyu Kim