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