Download Jack Greenblatt

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
1.
ATG
TAA
Targeted Gene
Primer 2
2.
Primer 1
Protein A
Calmodulin
Binding
Peptide
Rigaut et al. (1999) Nat.
Biotech. 17, 1030-1032.
TRP1 Marker
Primer 2
TEV
Protease Site
PCR, Transform, Select
for TRP+
Carboxy-terminal Tagging in E. coli
Temperature sensitive cI repressor
– inactive at 42°C
attL
exo bet gam
cI857 (cro-attR-bioA)
PL
PR
Exo/Bet – λ recombinase
+
CHROMOSOMAL ORF
TAP or SPA KAN
Δ
λ Gam
-
STOP CODON
E. coli RecBCD – Exonuclease V
Identification of Protein Complexes in E. coli
TAP
RpoB,C
HepA
RpoD
TAP
SPA
RpsA
SufB
SufD
RpoA
InfC
SufC
YacL
NusG
InfC
SufB
SufC
SufD
RpoD
YacL
YacL
RpoD
SufD
SufC
SufB
SPA
InfC
RpsC,D
RpsG
RpsE,F,K,M,J
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
GTFs an d
Mediator
Elongation factors
RNAPII
S RB 10/11
Accessory factors
AAA
AA A
CTD kinase
CTD-Phosphatase
Initiation
HoloRNAPII
m7Gp
CTD-Kinases
m7Gp
Termination
Elongation
Promoter Escape
P
P P
P P P
P
m7Gp
P
P
P P
P P P
P
m7Gp
P
AAA
A
AA
TAP Purification of Various Elongation Factors
Elp3-TAP
Spt6-TAP
Chd1
Spt16-TAP
Spt6
Elp1
Ctr9
Rtf1
Pob3
Paf1
Leo1
Psh1
Cdc73
CkaI
CkaII
CkbI
Elp2
Elp3-TAP
Elp4
Spt16-TAP
Iws1
CkbII
Elp5
Elp6
Elongator
Spt6/Iws1
Histones
FACT
“Old” and “New” Elongator Gene Deletions Have Similar
Effects on Gene Expression
3
2
1
0
-4
-3
-2
-1
0
1
-1
-2
-3
-4
Wild type /elp1 deletion
2
3
4
Salt Effect in the Purification of Yeast FACT
150 mM NaCl
Pob3-TAP no tag
125 mM NaCl
no tag Pob3-TAP Spt16-TAP
Spt16-TAP no tag
Spt16
Spt16-TAP
Pob3-TAP
Pob3
Protein Interactions Involved in Transcriptional Elongation (2001)
Spt5
TFIIS
Spt6
Ctk1, Ctk2,Ctk3
Elongator
(Elp1, 2, 3,
4, 5, 6)
Phosphorylation?
Iws1
RNA Polymerase II
Casein Kinase II
Fcp1
TFIIF
(Tfg1, Tfg2, Tfg3)
Spt4
Paf1
Chd1
Cdc73
Ctr9
Rtf1
Spt16/Pob3
(FACT)
Leo1
Histones
36 Polypeptides
Psh1
A Strategy for IDs of Stable Complexes and Weak Interactions
Two Affinity Purification Steps
NO GEL!
SDS-PAGE
Gel Bands
Trypsin Digestion
MALDI-TOF
Mass Spectrometry
Identification of
Stably Associated Proteins
Trypsin Digestion
Active
Protein
For
Assays
LCQ-Deca Ion Trap
Mass Spectrometry
Identification of
Stably and Weakly
Associated Proteins
Protein Complex Clustergrams
MIPS Functional Classification Catalogue (259 categories)
Category
rRNA processing
rRNA transcription
p-value In Category from Cluster
6.64E-11 RRP43 RRP45 RRP46 SKI6 RRP4 DIS3
1.94E-09 RRP43 RRP45 RRP46 SKI6 RRP4 DIS3
k f
6
63
6 109
MIPS Complexes Catalogue (315 categories)
Category
Exosome complex
rRNA processing complexes
RNA processing complexes
p-value
-1.33E-15
1.75E-14
2.42E-09
In Category from Cluster
RRP43 RRP45 RRP46 SKI6 RRP4 DIS3
RRP43 RRP45 RRP46 SKI6 RRP4 DIS3
RRP43 RRP45 RRP46 SKI6 RRP4 DIS3
k f
6
7
6
18
6 113
MIPS Protein Classes Catalogue (190 categories)
Category
Exonucleases
3'->5' exoribonucleases
Nucleases
p-value
8.19E-09
8.19E-09
2.05E-08
In Category from Cluster
SKI6 RRP4 DIS3
SKI6 RRP4 DIS3
SKI6 RRP4 DIS3
k f
3
3
3
4
4
5
GO Cellular Component (240 categories)
Category
exosome (RNase complex)
cytoplasmic exosome (RNase complex)
nuclear exosome (RNase complex)
nucleus
intracellular
cell
p-value
-2.66E-15
-2.66E-15
-2.22E-15
6.99E-11
7.18E-08
1.65E-06
In Category from Cluster
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
k
8
8
9
9
9
9
f
k
8
8
8
9
9
9
9
9
8
8
9
f
10
10
11
470
1011
1431
GO Biological Process (461 categories)
Category
macromolecule catabolism
RNA metabolism
mRNA catabolism
ribosome biogenesis
transcription, from Pol I promoter
35S primary transcript processing
cytoplasm organization and biogenesis
cell organization and biogenesis
catabolism
metabolism
cell growth and/or maintenance
p-value
-1.55E-15
-1.33E-15
-1.33E-15
-6.66E-16
-4.44E-16
1.44E-15
4.17E-11
8.30E-11
4.80E-10
7.73E-07
6.05E-06
In Category from Cluster
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40
RRP43 RRP45 RRP46 SKI6 RRP4 CSL4 DIS3 RRP40 RRP6
Components:
(Exosome)
YCR035C RRP43
YGR095C RRP46
YGR195W SKI6
YOL142W RRP40
YNL232W CSL4
YOL021C DIS3
YOR001W RRP6
YHR069C RRP4
YDR280W RRP45
21
19
19
164
66
41
444
479
331
836
1652
DIAGONALIZED CLUSTERING DEFINES PROTEIN COMPLEXES AND THEIR INTERACTIONS
No tag
Ipi1-TAP
WT
TET-IPI1
TET-IPI2
TET-IPI3
WT
THE IPI COMPLEX IS REQUIRED FOR RIBOSOMAL RNA PROCESSING
35S
kDa
97
27S
20S
Ipi2
66
Ipi3
U2
Ipi1-TAP
45
25S
18S
U2
U1
7S
5.8SL
5.8SS
The Method of Extract Preparation Can Make a Big Difference
Effect of Centrifugation on the Purification of the Erb1/Nop7/Ytm1 Complex
180000 g 45 min
60000g 30 min
Erb1- No
TAP Tag
Erb1-TAP
Erb1- No
Tag
TAP
kDa
97
Erb1-TAP
kDa
97
Nop7
66
Nop7
Ytm1
66
Ytm1
45
31
45
31
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 II
Identification of Iwr1
no
tag
kDa
Rpb3
Rpb1
Rpb2
97
66
Rpb3-TAP
45
Rpb4
31
21
Rpb5
Ydl115c (Iwr1)
Rpb6
Iwr1 is an evolutionarily conserved,
gene specific, elongation factor that
interacts with RNA polymerase II.
Affinity Purified Protein Complexes are Usually Active
Tandem Affinity Purification of COMPASS
SET Domain
Set1
SPRY Domain
Compass60
Compass50
Trx related
WD-40 Repeats
Compass40
PHD Finger
Compass35
WD-40 Repeats
Compass30
WD-40 Repeats
Compass25
Compass15
Implicated in regulation of
X linked dosage
compensated genes
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
WT
set1
cps60 cps50 cps40
cps30 cps25
 H3 Methyl K4
Tandem Affinity Purification of Set2
Set2- No Tag
TAP
Rpb1
Set2-TAP
No Tag
Rpb2
Set2TAP
97
66
45
Rpb1
(H5)
Rpb1
(H14)
Rpb1
(8WG16)
I
GTFs
TFIIH
3’ Untranslated
Coding Region
Promoter
MediatorCH3
RNAPII
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
4
4
4
4
4
36
36
36
36
36
CH3 CH3
4
36
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3
4
4
4
4
4
36
36
36
36
36
Paf1C
P
Ser2
Ser5
CH3
II
CH3
CH3
CH3 CH3
CH3
4
4
36
36
CH3
CH3
CH3
CH3 CH3
CH3 CH3
RNAPII
COMPASS
Set2
CH3 CH3
Ser5
CH3
CH3
CH3 CH3
CH3 CH3
CH3 CH3
4
4
4
4
4
4
36
36
36
36
36
36
Paf1C
P
Ser2
CH3 CH3
H3 K4
CH3 CH3
4
36
CH3
4
36
CH3
III
3’ Untranslated
Coding Region
Promoter
CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3 CH3
CH3 CH3
4
4
4
36
36
36
CH3
4
CH3 CH3
4
Ctk1C
36
36
4
4
RNAPII
36
36
Set2
CH3
P
CH3 CH3
CH3 CH3
CH3 CH3
Paf1C
CH3
CH3
4
CH3 CH3
4
4
36
CH3 CH3
36
36
CH3 CH3
CH3
4
4
36
36
CH3
H3 K36
Ser2
Ser5
CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3
4
IV
36
4
4
36
36
4
36
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3
4
CH3 CH3
4
4
4
4
4
36
36
36
36
36
CH3
CH3
CH3
CH3
CH3
Ser2
Ser5
CH3
CH3
CH3
RNAPII
CH3
CH3
Extending the Network: Genetics of Synthetic Lethality in S. cerevisiae
A
X
X
B
X
Y
X
C
Z
P
= synthetic growth
defect
OR
synthetic lethality
SUBSET OF THE GENETIC INTERACTIONS INVOLVING SET2
ChIP Distinguishes Localization in Various Regions of a Gene
Promoter
Coding Regions
3‘ Untranslated
All Three
Tfg2
Hpr1
Rna14
Spt16
12 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
TREX
CFIA
FACT
IgG
INPUT
TFIIF
1
-304
-47
TATAA
2
3
4
168 376 584 807 1010 1250
1(ATG)
5
2018
6
2290
3287
2757(STOP)
2823 3277
PMA1
3500
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
PRELIMINARY CLUSTERING OF THE GENETIC DATA
COMPASS
Rad6C
Set3C
Mediator
RXT Elongator
Paf1C
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
“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
Acknowledgments
Greenblatt Laboratory
Nevan Krogan
Joyce Li
Stephan Zhang
Yan Xue
Guaqing Zhong
Grace Guo
Atanas Lalev
Nira Datta
Ashkan Golshani Robin Haw
Affinium Pharmaceuticals
Dawn Richards
Veronica Canadien
Bryan Beattie
University of Toronto
Andrew Emili
Gerard Cagney
Charlie Boone
Amy Tong
Harvard University
Steve Buratowski
Minkyu Kim
Huiming Ding
Ainslie Parsons
St. Louis
Ali Shilatifard
Mark Johnston
Tim Hughes
Mark Robinson