© 2014 Nature America, Inc. All rights reserved. Rockefeller Rockefeller University, New York, New York, USA. should Correspondence be addressed to R.G.R. ( 1 and MED7 example, (for subunits conserved most the of handful a for 50% about from ranges homology of extent the yeast, in those to relative programs transcriptional metazoan of complexity increased to yeast from eukaryotes in conserved evolutionarily are which of many subunits, 30 of sists con metazoan 2-MDa The architecture. subunit complex disease to subunits various its in mutations tie that reports by underscored also is cell the in role Mediator’s critical RNA ing noncod long with interactions through loops chromatin of lization stabi by communication enhancer- of facilitation as such processes relevant transcriptionally other in involvement Mediator phases elongation the phase PIC-establishment the to opening chromatin the from transitions mechanistic dinating in coor implicated further been has Mediator process, the of nature multistep the given recently, More conditions. certain independent) metazoan Mediator has also been shown to machinery stimulate basal (activator-II Pol the and activators bridges that cofactor a as function and ment PIC establish facilitates that coactivator critical most the as perhaps emerged has complex Mediator multisubunit the described, tivators coac of types diverse the Among recruitment. coactivator through low-level activity exhibits (basal) that elements promoter core on (PIC) complex coactivators of array an and activators type–specific cell and - (GTFs), factors transcription general between interplay a functional complex entails Activation of transcribed by eukaryotic RNA polymerase II (Pol II) approach requirement identify This associate, assays Here The Murat A Cevher reveals a critical role of the MED14 subunit Reconstitution of active human core Mediator complex nature structural & molecular biology molecular & structural nature Received 24 July; accepted 9 October; published online 10 November 2014; Laboratory Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA. These diverse Mediator-associated functions are reflected in its its in reflected are functions Mediator-associated diverse These

evolutionarily results

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. Although initially identified and characterized characterized and identified initially Although . conserved dramatically 1 both

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an 2 for . Additionally, evidence exists to suggest suggest to exists evidence Additionally, . 9 and, potentially, from the initiation to to initiation the from potentially, and,

architectural

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that

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only

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Whereas 2

, the the , coactivator after human do 1 5 i:10.1038/nsmb.291 ------.

layers in incorporation subunits and its limited yields upon purification from cell extracts. plex. These relate to its large size and heterogeneity, its many essential in manipulating com hampered,this bydifficulties in part, technical been also has complex metazoan the of understanding Furthermore, tity and mechanism of action of the active core Mediator components. iden the of understanding an to led not have studies these subunits, identification and pinpointing of the critical roles the or Mediatorof active individual transcriptionally of assembly essential the for required subunits of set minimal the of demonstration anyHowever, without complex the within organized are subunits individual how for model a suggested have Mediator yeast the of analyses recently,EM modules middle and head the within networks interaction the for predictions to led have also Yeastscreens two-hybrid on cross-linking, for protein interactions within the middle module for both the head and middle partial modules Thus, previous studies of yeast Mediator have yeast. in Mediator, provided the forrelationships especially structure-function crystal structures to tends broadly Mediator. the to and properties repressive confer complex core the with associates reversibly subunits tail individual target exclusively, not but mainly, activators enhancer-bound or promoter- specific other; each with associated are loosely of relatively module subunits tail the individual the By contrast, Pol the machinery. II with interactions in with each other and constitute a stable core; they have been implicated associated are tightly modules and middle head the composing units organized into subunits tail head, middle, and subcomplexes kinase the with modular, is humans, and yeast in both complex, of the structure overall The MED30). and MED26 example, (for nits subu metazoan-specific additional contains complex metazoan the remainder for the relationships weaker much to MED31)

of the the core usata pors hs en ae n h udrtnig of understanding the in made been has progress Substantial

the

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& G Robert Roeder RNA

[email protected]

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Laboratory Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, of

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27

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© 2014 Nature America, Inc. All rights reserved. images are shown in to contaminating polypeptides. Uncropped gel complex purified as in (Coomassie blue staining) of the H + M + 14 + 26 MED14 was Flag tagged. ( + 14) complex purified as in of a MED14-containing head + middle (H + M MED7). ( agarose (via f-MED17) and HA agarose (via HA- (H + on M) M2 after purification complex of blue staining) head the + bimodular middle ( subunits. ( head of module. blue staining) the reconstituted ( subunits. ( module. middle and reconstituted expressed of blue staining) baculovirus- (Coomassie ( subcomplexes. 1 Figure hoaorpy ( chromatography analysis.initialSequentialouraffinity in unit (ref. MED1 for requirement conditional the of Because cells. insect in (His-MED10) MED10 tagged histidine- and MED9 MED31, myc-MED21, through coexpression of Flag-tagged MED7 (f-MED7), MED19, MED4, Wereconstitutionmoduleinitiatedthe middlegenerating firstthe by Reconstitution RESULTS the architecture of yeast and human Mediator resultsarecontextthediscussedin recent ofa study solely focusedon MED14 in bridging all the main modules of the Mediator complex. Our revealing other interactions, further highlights the key structural role of MS (CX-MS) analysis of the reconstituted core complex that, while also present in the extract. We also report an in-depth cross-linking–coupledsubunit allows the Mediator to operate in the thissuggesting complementedthatthusunlesssystemscontext MED26, with of additional factors However, this complex is unable to support additionactivityto thecomplex markedly enhances inits interaction extract-basedwith Pol II. assay MED14 that show Mechanistically,weincorporated. also is MED14 resulting bimodular complex is inactive in transcriptional assays unless other,eachthe withassociatestably can modules these althoughthat We first separately reconstituted the head andMediator middle subunitsmodules. that Weare foundfound in the active PC2 form theof efficientthe Mediator.MultiBac baculovirus expression system usedisolate homogeneouswepreparationsto desirableyields,and in lish structure-function relationships for the Mediator. makes it feasible to undertake a reconstitution-based approach to estab (refs.MED26 metazoan-specific the to respect withenriched is PC2 complex) that lacks the kinase module and several tail subunits, modularity but of the Mediator and the ability to isolate an active form inherent(the The processes. transcriptional the in roles their with lations corre make to and assemblies, multimodule and modules subunits, Mediator complex, it is necessary to dissect it at the level of individual to obtain a detailed structure-function understanding of the metazoan However,subunits.order in selected ofmutant versions or wild-type express stably that lines cell HeLa of extracts nuclear from obtained characterized mainly in s e l c i t r a  d b ) ) Western of blot analysis head-module ) Western of blot analysis middle-module Here, to generate a minimal active human core Mediatorcomplexcore human active minimal a generate to Here, functionally been has complex Mediator metazoan the far, Thus

f Reconstitution of human Mediator Reconstitution ) SDS-PAGE analysis (silver staining) e c ) ) SDS-PAGE (Coomassie analysis ) SDS-PAGE (Coomassie analysis 3 1 ), we did not include this sub this includenot did we ), a

) ) SDS-PAGE analysis of upeetr Fg 1a Fig. Supplementary

Supplementary Data Set 1 the f . Asterisks in

head–middle g in vitro ) SDS-PAGE analysis e , except that the biochemical assays using preparations f and

bimodular g point

, .

b ­ ) 2 7 .

complex e a 3 Myc-MED2 0 His-MED1 to jointly express MED18, MED30 f-MED7 MED31 MED4 His-MED10 HA-MED7 advance online publication online advance f-MED17 0 1 MED31 MED22 MED21 MED20 MED MED8 MED6 MED4 Middle 4 11 , 2 9 ), - - H + and MED20, which in yeast are nonessential are yeast in which MED20, and MED18 except subunits the of all amounts stoichiometric contained Fig. 1c ( modules middle and head the between interaction a stable revealed 6 gel filtration Superose and subsequent hemagglutinin-tagged MED7 (HA-MED7) (middle module) subunits ( modules two the of subunits the coexpressed we M), + (H modules specific subunits not included in our reconstitutions.metazoan- more or one on dependentprobably is complex the with associationinputwhose subunits MED19,exceptthe of containedall MED11–MED17–MED18–MED20–MED22–MED30) ( (MED6–MED8– complex head-module a obtained we purification, specific MED30, which previously was not assigned to anyMED8, module.MED11, MED18, AfterMED19, MED20, MED22 and the metazoan- head module of the human Mediator by coexpressing f-MED17, MED6, no association with the middle module. We similarly reconstituted the omitted in further reconstitutions. MED19, although expressed, showed for Mediator function in our transcription assays (below) and thus was subunit (nonessential in yeast ingall essential subunits of the middle module ( yieldedMED4–MED7–MED10–MED21–MED31a complex contain (i) purified general transcriptionstandard two our in factors (TFIIA, TFIIB,transcriptionpreparationstimulatedbasal M + TFIID,H the whether tested TFIIE, TFIIF extract nuclearactivator-dependent intranscription NaturalMediator purified from human cells stimulates both basal and MED modules. the constituting subunits of coexpression for requirement strict a of indicative potentially results shown), not (data together mixed when complex bimodular a form to associate we that observed separately head purified and middle modules do not ( heterodimer M Supplementary Fig. 1e Supplementary Fig. Fig. b To reconstitute a complex containing both the head and middle middle and head the both containing complex a reconstitute To 2 1 1 1 f 4 , Reconstituted middle and module) (head f-MED17 through selection Sequential ). d

). ). The resulting H + M preparation ( is

HA-MED7, MED4 critical MED18, MED30 3

Natural Mediator 2 3 and thus tend to dissociate upon gel filtration filtration gel upon dissociate to tend thus and MED10 MED31 MED21 MED4 MED7 His-MED10 f-MED14 MED31 MED22 MED21 MED20 MED17

MED

for MED8 MED6 nature structural & molecular biology molecular & structural nature in vitro in 11

basal ; also described further below). Interestingly,further ; also described H +M14 c 32

transcription assays transcription and MED18, 30 , 3 f-MED17 3 ) failed to express and was not required MED22 MED20 MED * *

MED8 MED6 activated 11 g Head Fig. 1 Fig. MED18, MED30

transcription Supplementary Fig. Supplementary 1d 32 e His-MED10 Fig.1a HA-MED7 and and , 3 f-MED14 d 3 3 MED31 MED22 MED21 MED20 MED26 MED17 MED1 4 and form a labile labile a form and MED MED MED containing either containing 3– 2 1

Input Supplementary 5 , 1 8 6 4 b . Wetherefore. Fig. 1c H +M1426 Flowthrough ).The MED9

3 Elution , MED22 MED30 MED20 MED8 MED6 MED17 MED19 d ) that * - )

© 2014 Nature America, Inc. All rights reserved. An irrelevant activator (AML1-ETO) was included as a control. a control. as included was (AML1-ETO) activator irrelevant An 5×TREML. template TR-responsive the with together activator the in as performed ( indicated. as added were H + M 26) + 14 + and M + (H 14 subcomplexes Mediator and (p53) activator The (ML). template a control and 5×p53REML template in as ( function. 3 Figure ( complex ( complex 26 + M Tsai from ( ule mod head the not but module middle the with associates MED26 We MED26. that found containing complexes variant fore generated can recruit the super elongation complex to promoters content II Pol higher a have preparations Mediator support to fail depleted is subpopulation this which from extracts that shown has study a previous in cells, HeLa population Mediator cellular total the of fraction small a is PC2 MED26-containing Although cofactor(s). negative a by constraint apparent an overcome to subunit Mediator another for requirement a indicated vari result of this factors, complement nuclear ous natural more a contains extract the Because ( back extract nuclear added Mediator-depleted to when transcription basal restore to unable was ration prepa 14 + M + H the system, assay defined the in active Although MED26 human core active the Mediator preparation define complex. We H in the therefore + contained conclude that subunits M the + 14 units (lane 6 versus lane 8) and by its PC2 form (lane 6 versus lane 9). sub of set complete a containing preparation Mediator natural a by both elicited that to equivalent was stimulation Importantly,fold the ( transcription basal of strong stimulation a effected complex 14-subunit this PC4), and II Pol (GTFs, the in tested When homogeneous preparation contained stoichiometric MED14 ( and performed affinity selection via MED14 to ensure that the resulting tions that otherwise tend to be deficient in tail components tail module Westarted with MED14, which, despite its previous assignment to the lane 2), we sought to include additional subunits in our reconstitution. ( assay either in activity any show to failed modules, middle and head independent the as well as complex ( atedHeLa cell nuclear extract (NE) immunodepleted for the Mediator ( II Pol and PC4 coactivator TFIIH), and in shown are images gel Uncropped indicated. as reactions transcription the to added were subcomplexes Mediator NE. or (mock-depleted) control with template ML the from reactions ( antibody anti-MED30 by Mediator of immunodepleted (NE) extract nuclear HeLa of ( MED26). 26, MED14; 14, middle; M, head; (H, subcomplexes Mediator indicated the and PC4 and II Pol IIH), and IIF IIE, IID, IIB, (IIA, GTFs purified with performed were Reactions promoter. core late major adenovirus the containing (ML) a template from ( transcription. basal 2 Figure nature structural & molecular biology molecular & structural nature in shown are images gel Uncropped We reconstituted an H + M complex containing MED14 (H + M + 14) Supplementary Fig. 2a Fig. Supplementary Figure 2 Figure

requirement Critical roles of MED14 and MED26 in Mediator coactivator coactivator Mediator in MED26 and MED14 of roles Critical Mediator-stimulated in MED26 and MED14 of roles Critical ∆ Fig. 2 a et al. et Fig. 1 Fig. Mediator NE). ( NE). Mediator ) Autoradiogram of of ) Autoradiogram 3 5 c , is present in stoichiometric amounts in our PC2 prepara . Extract-based reactions contained the p53-responsive p53-responsive the contained reactions . Extract-based a b 2 in vitro in , except that the TR–RXR heterodimer was used as used was heterodimer TR–RXR the that , except 7 , lane 1 versus lane 2). Because the H + M preparation, g , and that it can be stably incorporated into an H + H an into incorporated stably be can it that and , and and Supplementary Fig. 2c Fig. Supplementary a b in vitro in ) Autoradiogram of of ) Autoradiogram ) Autoradiogram of of ) Autoradiogram

for transcription Supplementary Fig. 3 Fig. Supplementary c

) Autoradiogram of of ) Autoradiogram Mediator transcription assay with purified factors factors purified with assay transcription Fig. 2 Fig. in vitro in , b ), in agreement with the recent report report recent the with agreement in ), Supplementary Data Set 2 Set Data Supplementary a transcription reactions performed performed reactions transcription , lanes 1–4; lanes ,

function i. 2 Fig. 4 in vitro in . Further, MED26-containing MED26-containing Further, . in vitro in Fig. 2 Fig. Fig. 2 Fig. ) and an H + M + 14 + 26 26 + 14 + M + H an and ) Supplementary Data Set 1 Set Data Supplementary a in vitro in transcription reactions reactions transcription , lane 6 versus lane 1). 1). lane versus 6 lane , transcription reactions reactions transcription c b

, lane 7 versus lane 1). 1). lane versus 7 lane , in ). Importantly, in the the in Importantly, ). ) Western blot analysis analysis blot ) Western Fig. 2 Fig.

a

) or (ii) unfraction (ii) or ) nuclear advance online publication online advance transcription transcription 4 , 3 c 12 6 , lane 5 versus 5 lane , , and MED26 MED26 and , , 3 7 4

. extract . . We there .

∆ Mediator Mediator Fig. 1

f ). ). . ------­

plexes are rendered active, we used coimmunoprecipitationTo to understandinvestigate the mechanism wherebyMED MED14-containing Mediator com subunit MED1 missing the targets and (RXR) receptor X retinoid the with heterodimer a as functions which 6), lane versus ( (TR) receptor for thyroid-hormone the tor function coactiva no saw we experiments, control In 4). and 6 lanes versus 8 p53, activator subunit MED17 the with interact to transcriptional known is which the for function coactivator clear a assay, the H + M + 14 + 26 (but not the H + M + 14) complex exhibited Importantly, in on extract-based the MED14. dependency structural for MED26 in this context is upon superimposed a more fundamental ( assay extract-based the in transcription restore to failed complexes of into partial Inclusion other MED26 Mediator preparation. natural 14 + 26 complex restored to transcription basal the same level as did a ( system pure ( extract Mediator-depleted Fig. Fig. 2 c a ML- b a 1 Mock-depleted NE c 4 Mock-depleted NE , lanes 8–10), thus suggesting that the additional requirement that , the thus additional lanes 8–10), suggesting Natural Mediator

Control Mediator 2 1 – Control activator H +M1426 is

∆ Mock-depleted NE

∆ Mediator NE crucial Mediator NE Natural Mediator H +M1426 5 H × ∆ TR–RXR 5 H+M14 Fig. 2 Fig. TREML- Mediator NE × 9 8 7 6 5 4 3 p53REML- M ML-

for H + M a ML p53 ln 7 ess ae 8 n 9, h H M + M + H the 9), and 8 lanes versus 7 lane ,

+ + + 8 7 6 5 4 3 2 1 – – – – – – – + – – – + – 9 8 7 6 5 – 4 – 3 – 2 – 1 – – – – – – – Mediator–Pol

- M + 14 + + 9 8 7 6 5 4 3 2 1 + – + – – – – – + – – – – – – – – – + – – – – – – + – – + + + + + + + H + M + 14 Fig. 2 Fig. + – – – –

– – – – – – – – – – H H + M + 14 + 26 + – – – – – – – – – – – – – + –

c Natural Mediator + + – – –

+ + + + + + + M – – – – – – – + – , lane 11 versus lane 12), as in the the in as 12), lane versus 11 lane ,

+ + – – – PC2 Mediator

II H + M

interaction + + – – – – + H + 14 + – + + – + + – – – + + H + M + 14 b + + + – – 1 9 – – – + – M + 26 – – 2 1 1 0 + Mock-depleted NE s e l c i t r a M + 14 + 26 10 + + + + – – 1 1 3 10 Fig. 3 Fig. + 8 ∆ Mediator NE ( H + M + 26 3 + + + – – 2 9 Fig. 3 Fig. 3 11 . + H + M + 14 + 26 Natural Mediator b TFIIH MED22 MED8 MED6 MED17 MED1 MED13 12 , lane , 12 lane + + a , lane lane ,  - -

© 2014 Nature America, Inc. All rights reserved. Supplementary Tables 1 Supplementary MED14. purple, subunits; head-module gold, subunits; middle-module blue, Light depicted. are cross-links intersubunit only shown, are apart) residues (>200 cross-links intrasubunit which for MED14, for Except CX-MS. by obtained complex Mediator the of map cross-linking Residue-specific (CX-MS). MS and cross-linking chemical by revealed complex Mediator reconstituted 5 Figure ( extracts Mediator-depleted in ished previous results Mediator preparations. In agreement with our various our with reactions the supplemented Mediator-depleted further we case, latter the In extract. nuclear or control with beads DNA-bound incubated we purpose, this For ( promoter a to recruitment II Pol monitored we which in assay template lized immobi an performed we extract, nuclear in function Mediator for MED26 of requirement conditional the for basis the Tounderstand MED26 of efficiently interacting with Pol II, thereby stimulating transcription. lane 6). ( Hence,II Pola inputcritical of function75% ofto MED14up isboundcomplex to render14 H + + M capableM + H the ( ( thehead module (whether in the presence ( TFIID ( resultsfromprevious studies H + M and H + M + 14 complexes with Pol II ( theinteraction of thehead with TFIID and theinteraction of thehead, s e l c i t r a  set. data cross-linking complete subcomplexes. Recruitment of Pol II (RPB1) and TFIID (TBP) was monitored. Uncropped gel images are shown in in shown are images gel Uncropped monitored. was (TBP) TFIID and (RPB1) II Pol of Recruitment subcomplexes. ( NE HeLa Mediator-depleted or control with done were Reactions recruitment. II Pol assess to assay recruitment template immobilized an of analysis ( included. also are exposures blot western longer (inputs), 1–3 lanes For subunits. MED30) and MED14 (MED7, Mediator and RBP6) and (RPB1 II Pol for probed were immunoprecipitates Anti-MED30 M + 14). H + or H + M, (H, subcomplexes Mediator recombinant indicated the and II Pol included reactions Binding assays. interaction ( indicated. as TFIIF included also they in as were reactions Binding TFIIF.of presence the in assays interaction head ( subunits. Mediator selected and (RBP1) II Pol (TBP), TFIID for probed were (IP) immunoprecipitates Anti-MED30 indicated. as TFIID, or II Pol and module head Mediator the included reactions Binding assays. interaction Mediator of ( assays. binding the in used Mediator natural and (IID) TFIID II, Pol of preparations purified of staining) (silver analysis ( extract. nuclear in recruitment II Pol MED26-dependent and interaction 4 Figure d Fig. 4 Fig.4 ) Western blot analysis of Mediator–Pol II Mediator–Pol of analysis blot ) Western d b Fig. 4

, lane 5) was able to bind to Pol II. By contrast, and importantly,

, lane 5 andlane5 ,

overcomes Molecular architecture of the the of architecture Molecular MED14-dependent Mediator–Pol II Mediator–Pol MED14-dependent c ) Western blot analysis of Mediator- of analysis blot ) Western b , lane 7). However, in contrast to the case in yeast 4 2 , Pol II recruitment was abol Fig.4

a b

and and ) Western blot analysis analysis blot ) Western Pol c

, lane 8) of TFIIF) nor the H + M complexM + TFIIF)Hof northelane8) , II–recruitment b 2 e , except that that , except 4 show the the show ) Western blot blot ) Western 0 a , theheadmodule, alone interacted with ) SDS-PAGE ) SDS-PAGE

Fig. 4 Fig.

Fig. 4 Fig.

e

Fig.4

restriction e

). ).

- Fig.

,

c 4 ,lane 10) or absence a c ). In agreement with Pol II Head 1 MED8 0 MED1 Control Mediator IID Head module 1 RNA PolII 1 1 1 1 200 advance online publication online advance MED17 4 MED6 1 RPB1 ∆

TFIIF Mediator Fig. 4 Fig. , neither MED6 Mediator NE) and were supplemented with various recombinant Mediator Mediator recombinant various with supplemented were and NE) Mediator 268 11 7 400 d 253 + + + + + + + + 8 7 6 5 4 3 2 1 – – – – – – b - ,

+ Head module Input 10% by amine-specific, isotopically labeled disuccinimidyl suberate (DSS) disuccinimidyl labeled isotopically by amine-specific, complex 26 + 14 + M + H reconstituted the conjugated chemically we complex, Mediator the of architecture molecular the Todissect Molecular Mediator the allows to that overcome.MED26 a at reflects restriction, the level of Pol II recruitment to the promoter, conclude that the conditional requirement of MED26 in nuclear extract induce Pol II recruitment ( ( experiment the of results the paralleling and contrast, By ( II Pol purified with nor the H + M + 14 complex (lane lane 2 versus 4), lane 1). Interestingly,the neither the H latter + M complex (lane 3) of which interacted strongly RNA PolII + – MED17 + + – 600 MED8 MED6 RPB1 TFIID TBP + – – – – 1 1 + + Input 10% – + – – – + + + 1 – – – –

architecture IP anti-MED30 14 800 651 3 2 Figs. Figs. 2 – + + – – – 17 0 MED22 MED1 8 + + + + + 1 9 – MED30

5 4 + 1 0 7

c 1,000 anti-MED30 + + + – – – and 1 1 – + + + – nature structural & molecular biology molecular & structural nature Fig. 4 Fig. + + – + – + – – 7 6 2 IP

of 3 Fig. Fig. 4 ), ), the H + M + 14 + 26 complex was able to 8 d +

1 1 the 1,200 ), was able to induce Pol II recruitment. recruitment. II Pol induce to able was ), MED1 MED21

core e 13 14 0 , , lane 5 versus lane 1). Therefore, we Supplementary Data Set 2 Set Data Supplementary 5 4 d e 1,400

H +M14 RNA PolII Mediator Mock-depleted NE MED30 MED14 MED30 MED14 MED7 MED7 H + 1 1 RPB6 RPB1 ∆ Mediator NE M H MED4 1,600 Middle 1 454 Input in vitro in RPB1

+ + + + + + + 6 5 4 3 2 1 – – 2% complex TBP MED1 233 – + + – – 248 MED7 + + – – – 4 1,800 + 4 3 2 1 – MED30 IP anti- transcription transcription + – – (amino acid) 1 MED31 + + + – – – Overexposed – . – 13 Input H + M 1 2,000 H + M + 14 + 5 – H + M + 14 + 26

© 2014 Nature America, Inc. All rights reserved. and MED17 ( MED17 and MED22 and MED17 ( ( MED22 5d Fig. 5a Fig. additional interaction between MED21 and MED31 ( ( subunits middle various confirmed CX-MS–identified interactions of MED7 and MED21 with tions with selected subunit combinations ( immunoprecipita performed and subunits of omission selective by modules middle and head the of derivatives partial of series a erated dimers. (transient) of from to a arise might MED14 form pattern tendency cross-linking Polwith also perhaps II ( and modules middle and head the with interaction itself its upon facilitate and back fold potentially may subunit (170-kDa) large this that indicating thus complex, Mediator core active the in MED14 of residues N- and C-terminal between cross-links intrasubunit several identified Wealso modules. two the bridge further to serving thus MED17) and middle (MED7) components (MED6, ( head both to cross-linked subunit this function, Mediator in MED14 of role critical the to relevant mid Furthermore, the module. in dle MED21 and MED10 and module head the in MED17 not were by CX-MS. scored substoichiometric, being MED26, and MED20 MED18, subunits. constituent other the of each contact MED21 and MED7 module middle the within function hinge proposed previously ( MED30 and hub structural withina the module,also cross-linking is with MED6,MED17 MED8, human MED11, MED22of ~150–300) N-terminus the acids toward (amino region a module, head the for Especially agreement with the published studies of yeast Mediator modules two the of subunits ( modules between as well as modules middle and head an each network subunits of of extensive within between the contacts H + M + 14 + 26 complex ( reconstituted the of lysines total the of 60% represent lysines linked ( complex the of map connectivity ( to identify cross-linked peptides. We identified 277 unique cross-links ( nature structural & molecular biology molecular & structural nature ( MED17 and middle modules. middle and head between interactions intermodule blue, dark interactions; MED14 purple, head; the in interactions intramodule brown, module; middle the in interactions intramodule blue, Light approaches. biochemical and CX-MS from data composite on based complex, Mediator core human 6 Figure Supplementary Fig. Supplementary 4a Supplementary Tables 1 Tables Supplementary Importantly, the CX-MS data reveal intermodular contacts between In complementary experiments to validate the CX-MS data, we gen ln 8. iial, o te ed oue ( module head the for Similarly, 8). lane ,

), we identified complex formation between MED11 and and MED11 between formation complex identified we ), Schematic representation of subunit interactions in the the in interactions subunit of representation Schematic Supplementary Fig. 5d Fig. Supplementary 18 20 upeetr Fg 5d Fig. Supplementary Fig. Fig. Supplementary Fig. 5d Fig. Supplementary upeetr Tbe 2 Table Supplementary 5 ). The intramodular cross-linking data are in good good in are data cross-linking intramodular The ). 11 22 8 Head Supplementary Fig. 5d ) ) and applied high-resolution MS (CX-MS) Supplementary Table 2 Supplementary Supplementary Fig. 5a Supplementary Supplementary Fig. 4b Supplementary and and 30 17 6 2 ), which we used to build a spatial spatial a build to used we which ), , lane 3 versus lanes 4 and 5) and and 5) and 4 lanes versus 3 lane , 14 , lanes 6, 7), between MED11, MED11, between 7), 6, lanes , Fig. Fig. , lane 4), between MED8 and and MED8 between 4), lane , 10 7 ). In agreement with their their with agreement In ). 5 Supplementary Supplementary Fig. 5 ). Remarkably, the cross- the Remarkably, ). Supplementary TableSupplementary 2 , lane 6), between MED6 31

). ). Alternatively, this – advance online publication online advance Middle , c c 21 ) and detected an ) and detected ). ). The data reveal Supplementary Supplementary Supplementary Supplementary 26 4

22– ). ). We 20

,

2 2 4 5 5 ), ), 3 - - - , .

general architecture of architecture the general human core Mediator complex from these the and subunits the among interactions deduced the level, gross a in approaches various from deduced complex Mediator main for network the human three core interaction subunit (composite modules its bridges that Mediator the of backbone essential the ( reconstitutions our in included not were however, which, module, tail the of subunits MED16 and MED24 the with interacted MED14 6a independ Fig. could ( MED14 modules middle and head that the with associate ently established we subunits, module to the head-middle interaction ( an additional interaction (between MED17 and MED7) that contributes head and middle modules. Relatedly, this series of ( analyses also identified 2b Fig. ( MED17 of presence the on dependent was mation 5d Fig. that is heterodimer anchored to the head via MED8 ( lane 5). Importantly, as they do in yeast, MED18 and MED20 formed a ( MED18 and MED8 between relates to the dissociable kinase module. kinase dissociable the to relates our present study nor the study by Tsai Neither modules. and head middle of tail, the subunits with contacts of Mediator the yeast length the complex natural and multiple makes Tsai of analysis cryo-EM recent the with agreement good in is complex Mediator the of backbone architectural an as MED14 of model This three separate modules of the Mediator into a entity.single functional for integrating features necessary the appears to architectural furnish MED14 Thus, subunits. MED10) and (MED7 middle-module and MED17) and MED8 (MED6, head-module as well as MED24) and CX-MS data establish that MED14 interacts with tail subunits (MED16 complex bulk the to tail the factors. nuclear of complement natural more a containing extract nuclear a versus system purified and of roles in a Mediator subunits mechanisms minimal underlying the understand to and complex Mediator core active the of ponents com minimal the uniquely, us, to identify allowed has our approach Thus, extract. nuclear a in function Mediator core for MED14) with Mediator core in function an for assay with factors, purified is (along essential required not although MED26, that show also results Our activity. transcription (p53)-dependent activator selective and basal both of very acquisition the a with correlates that facilitating interaction for II Pol strong critical most one the is subunit MED14 the complex, this In complex. Mediator core 14-subunit active tionally association with these two modules is necessary to reconstituteMED14 a complex, func stable a yield interactions head-middle Although activity. and architecture Mediator in MED14 for role critical a light high to converge studies functional and structural These subunits. selected of analyses interaction pairwise and CX-MS through plex com Mediator core active the of map connectivity spatial detailed various a display Mediator. We generated to also ascribed that previously functionalities subcomplexes Mediator of generation aimed the approach at reconstitution-based a describe we paper, this In DISCUSSION Tsaiof data the with agreement good in are data Supplementary Fig. 5f Supplementary Fig. 6c Fig. Supplementary Notably, through coexpression of MED14 with either head- or middle- for M + H MED17, for data cross-linking the with agreement In MED14 MED14 has been viewed as a tail component, albeit one that bridges et et al. ). Moreover, MED17 also copurified with the middle module middle the with copurified Moreover, also ). MED17 , lane 8 and lane 3 versus lane 4). lane versus 3 lane and 8 lane , , b 2 ), in agreement with the cross-linking data. Also of note, note, of Also data. cross-linking the with agreement in ),

7 , which revealed that density attributable to that attributable spans MED14 density , revealed which ), thus identifying it as a major link between the ). These results further implicate MED14 as MED14 implicate further results These ). 3 5 . Our protein-protein–interaction and and protein-protein–interaction Our . Supplementary Fig. 5d Fig. Supplementary Supplementary Fig. 5g et et al. 2 7 addressed how MED14 et al. et s e l c i t r a Supplementary Supplementary Supplementary Supplementary Supplementary 2 ). , lane 3 versus versus 3 lane , 7 . Fig. Fig. 6 ). At ).  ------

© 2014 Nature America, Inc. All rights reserved. leads to abrogation of transcription activity transcription of abrogation to leads tion of the total Mediator, its frac depletion from HeLa cell nuclear extract small very a constitutes (PC2) Mediator the of subpopulation MED26-containing the though even that observation prior our with for this subunit in negative counteracting cofactors. This is consistent a role suggests transcription and activator-dependent basal both late MED26-containing complex can function in nuclear extract to stimu DSIF include may (ref. that cofactors acting negatively of effects the overcome to is Mediator the of role important an milieu absolute is extracts in ment acts Mediator whereas its require systems, in the purified transcription to mainly stimulate that shown have studies Previous extract. factors, the H + M + 14 complex failed to function in HeLa cell nuclear pure with reconstituted assays in transcription to its contrast activity that cofactors impinge machinery upon the transcriptional mediating numerous and of action the coordinate can transcription Mediator signals, basal activation stimulating of functions core the acquiring of own. its on conformation incapable necessary is contacts, II Pol responsible the be of yet majority a may for which complex, head–middle stable wise binding II Pol upon occur that movements intermodule documented the to remains that possibility MED14 effectsalternative the on PolTherefore, II interactions. binding II Pol are in indirectMED14 and are related implicated have studies other nor studies genetic yeast prior neither images published the in II Pol with contact direct in be to seem not does backbone MED14 the as identified now density the contacts, of delineation precise allow not did modules analyses EM the tail Although and complex. Mediator remodeled middle the of head, the by formed cavity a within rest to comes then and module head the with interacts first CTD II Pol Tsai formation, holoenzyme for responsible are complex Mediator yeast cryo-EM study by nished However, the backbone. MED14 most their recent whereas fur surface large the and II Pol between interaction physical direct Most a from simply, result could this of transcription? to stimulation transcription. core role in Mediator–enhanced no additional with recruitment, Mediator for site target activator an as principally serve may module tail the that suggest results the Reciprocally, assembly. itating transduction of the signals within necessary the Mediator–PIC backbone of the Mediator complex but also is a critical subunit in facil the head subunit MED17. Thus, MED14 is not simply an architectural with interacts for which p53, function coactivator a selective exhibits with MED26) for forming a Mediator complex (H + M + 14 + 26) that and acquire basal transcription activity. MED14 is also required (along II Pol with interact complex the did coexpression through assembly this into incorporated was MED14 when Only hands. our in II Pol with interact to failed alone) complex head the as well (as complex of stimulating basal transcription. Indeed, the metazoan head–middle activity Mediator rudimentary most the even support to unable was complex bimodular head–middle reconstituted our that surprising activity basal and to stimulate the yeast Mediator was to reported interact with Pol II (via MED17) offunction the core Mediator. Previously, the isolated head module of Tsai s e l c i t r a  With its structural complexity, and in addition to its overlapping overlapping its to addition in and complexity, structural its With and ultimately to Pol II contribute interaction How MED14 might In a major extension of the solely architectural focus of the study by et et al. t al. et 4 7 ) and potentially NC2 (ref. (ref. NC2 potentially and ) 45 2 , 7 4 4 , , we for required that show is MED14 the further critically 5 6 previously proposed a multistep model in which the the which in model multistep a proposed previously . It is likely that in the absence of MED14, the other the MED14, of absence the in that likely is It . 2 7 did not shed additional light on which features of the 3 4 , 1 1 0 . Therefore, it initially was somewhat was somewhat it . initially Therefore, 4 . This suggests that in the cellular cellular the in that suggests This . 5 . Furthermore, to our knowledge, knowledge, our to Furthermore, . 4 8 . hs or idn ta a that finding our Thus, ). 4 . 1 0 , Gdown1 Gdown1 , 2 advance online publication online advance . . Thus, in 4 4 ------

ment is stage specific ment is stage role. essential and distinct cally mechanisti its with agreement in persists, requirement MED14 the Of context, in note, even this interference. cofactor negative disallow and interactions II Pol favor that conformations in Mediator of ing MED26-dependent include recruitment of activities Possibilities that neutralize the negative II. cofactors or freez Pol with interactions rela module middle cavity II–binding the Pol the from in distant tively localization its However, unclear. is Mediator MED26-containing overcomes the of effect negative factors whereby mechanism precise The stages. elongation the or transition role for additional subsequent atMED26 the initiation-to-elongation a preclude however, not, does It machinery. elongation the of ment involve precede which process, transcription the of stages earliest the at MED26 for function a suggests observation This restriction. the overcomes Mediator MED26-containing that and promoter the extract to of at Pol level the the II recruitment restriction earlier in an even impose cofactors the collectively, that, reveals dissection tic machinery the from elongation the handoff to a in initiation functions subunit this which in model a to P-TEFb- and super ELL-containing elongation complex, thus leading reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial complexes (in experiments. D.L. helped M.A.C. in the generation of head-module partial preparations, reconstitutions, manuscript. M.A.C. outcarried biochemical experiments including cDNA Y.S. M.A.C., S.M., R.G.R., and B.T.C. the designed experiments and wrote the Research Fund Postdoctoral Fellowship. supported by an American Eastern Division–New Cancer Society York Cancer GM103511 (B.T.C.), GM109824 (B.T.C.) and GM103314 (B.T.C.). M.A.C. was Institute of Health and grants GM090929 (R.G.R. S.M.), CA129325 (R.G.R.), Department of Defense grantand W81XWH-13-1-0172 (R.G.R.) by US National Molecular forBiology) discussion. Funding for this work was provided by US system and M. Guermah (Rockefeller University, Laboratory ofand Biochemistry of Cellular and forBiology) Structural assistance with the Agilentoffline HPLC system, J. Fernandez-Martinez and M.P. Rout (Rockefeller University, Laboratory Eidgenössische Technische Hochschule Zurich) for the MultiBac baculovirus We thank T. Richmond (Institute of Molecular andBiology Biophysics, version of the pape Note: Any Supplementary Information and Source Data files are available in the the in vers available are references associated any and Methods M states. disease in awry go that those including functions, Mediator complex, we hope to obtain a better understanding of the full range of core Mediator of the derivatives larger and reconstitute studies these of As scope the we expand Mediator derivatives. ever-larger building lating increasingly complex metazoan-specific regulatory functions by of recapitu feasibility the illustrates Mediator’snicely core functions compositionally generate over and above to out functions carry that complexes Mediator defined ability our Nonetheless, (HeLa) type-specific regulation. cell or requirement MED26 general a reflect C AUTH A c O ethods A A recent study in the and TFIID with interactions in implicated been has MED26 know MP ion of the pape the of ion O E TI R R C N l e ON G G FI dgm Supplementary Supplementary Fig. 5d TRIBUTI N r l . . A en N

r CIAL CIAL I Drosophila ts . nature structural & molecular biology molecular & structural nature 4 9 ON . Thus, it remains unclear whether our results our results whether unclear . it Thus, remains in vitro N S T E R has suggested that the MED26 require transcriptions and coimmunoprecipitation E ). ). Y.S. outcarried the CX-MS experiments. STS 1 2 . However, our mechanis our However, . 2 7 argues against direct direct against argues http://www.nature.c online online onl om/ ine ------

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© 2014 Nature America, Inc. All rights reserved. Reactions took place for 50 min at 30 °C and then were processed and analyzed reaction mixes, which contained [ response element. Reactions were initiated by addition of protein factors to the response element, and p53 the 5×TREML a templateof copies contained five five containedcopies of further a thyroid 5×p53REML template The promoter. containedG-less cassettes downstream ofadenovirus the major late (ML)core Transcription reactions typically previously contained described 50 ngas of essentially test templates.performed were All templatesextract nuclear or tors vitro In expresses that Flag-taggedline MED26 (ref. cell a from extract nuclear of fraction M 0.85 P11 lulose (ref. MED10 subunit core the expresses stably that line cell HeLa a from purified affinity similarly also was Mediator use, routineFor subunits. epitope-tagged and TFIID were purified from corresponding HeLa cell lines that stably express as were the various transcriptional activators (p53, TR as described before Recombinantpurifiedand werebacteria expressed TFIIFandTFIIB, TFIIEin described previously as essentially was factors transcription general the of Purification of transcription factors, activators and coactivators. 500 ml of infectedfrom cells complexwas 100 Mediator core pure of yield typical A cocktail. virus the with in Sf9 cells. For protein production in Hi5 cells, scaled-up cultures were infected two different bacmids to form the third and fourth viruses, which were amplifiedsecondpFBDM-MED17virus. and pFBDM-Flag-MED14 were integrated into andMED9MED26) and integrated into another forbacmid production ofthe and pUCDM transfer vectors (HA-MED7, MED4, MED21, pFBDMcDNAsintotheinserted were also moduleHis-MED10,forsubunits middle the of MED31, fer vectors were integrated into a single bacmid for virus generation. Individual inserted into the pFBDM and pUCDM transfer vectors, and the resulting trans (MED6, MED8, MED11, MED18, MED19, MED20, MED22 and MED30) were reported here. The individual cDNAs for subunits of the Mediator head module summarizes our reconstitution protocol for one of the largest Mediator variants as well as identification, by trial and error, of which subunit to tag. The following elution was with 0.5 mg/ml of the corresponding peptide. beads, anti-HAand chromatography.M2 6) both (SuperoseFor gel-filtration and Flag-tagged HA-tagged forSP-Sepharose)ion-exchange subunits,respectively),(typically and agarose anti-HA and agarose M2 (anti-Flag affinity of combinationsvarious through purified then was extract TheKCl. mM 300 to (20,000 r.p.m. in a Beckman Type 45 Ti rotor for 30 min), the lysate was diluted (0.5 pepstatin β mM EDTA,3.5 mM 0.1glycerol, 20% 7.9,Tris-Cl, pHmM 10 KCl, mM (500 infectedwith theamplified Infected viruses. cells were homogenized inBC500 were cells Hi5proteinproduction,For cells. Sf9 in amplifiedwere viruses ing transposition and Cre-Lox recombination) for generation of viruses. The result cation. The transfer vectors were integrated into a single bacmid (through both inserted into different subunits of the Mediator to facilitate downstream purifi and pUCDM transfer vectors stoichiometriccomplexes, Mediator subunitcDNAs wereclonedintopFBDM complexes. Mediator human of Reconstitution for expression. each cDNAsof variantshortest the We selected MED22. and MED8 for seen MED22, MED26, MED30 and MED31. Interestingly, at least two variants were PCR primers to generate individual clones for MED8, MED9, MED11, MED19, tion with oligo-dT primers. The resulting cDNA was amplified transcrip with reverse byappropriate preparedcDNA and purified was cells HeLa from RNA Totalcells. HeLa from clonescDNA new isolated we subunits,remaining the MED10, MED7, MED6, MED14, MED16, MED18, MED4, MED20, MED21 and MED24 (refs. for cDNAs existing used we vectors, sion cDNAcloning of Mediator subunits. ONLINE nature structural & molecular biology molecular & structural nature autoradiography.andgelsurea 50%electrophoresis bypolyacrylamide, 5% on -mercaptoethanol and 0.1 mM PMSF supplemented-mercaptoethanolPMSF mM protease0.1withinhibitors and Optimization of the reconstitution protocol entailed extensive viral titrations, 3 4 ). The PC2 form of Mediator was affinity purified from the phosphocel transcription assays. transcription

METHODS µ g/ml) and leupeptin (0.5 (0.5 leupeptin and g/ml) 3 4 . Baculovirus-expressed TFIIA was purified from insect cells, 4 ). 3 0

. Various tags (histidine, myc, HA or Flag) were In vitro In µ g. α - 32 P]UTP or [ For subcloning into baculovirus expres transcription assays with purified fac purified with assays transcription µ g/ml). After ultracentrifugation ultracentrifugation After g/ml). α - 32 n re t oti near- obtain to order In α P]CTP as the labeled NTP. and RXR α 4 ). Pol II, TFIIH , 29 Purification , 38 , 5 0 ). For 3 3 4 4 ------. .

all the primary-antibody dilutions were 1:1,000. Antibody to MED30, which has 1:100, atmanufacturer’s on used was which anti-MED18, forwebsite).Except (sc-107739) and MED14 (sc-9419) were purchased from Santa Cruz (validation blotting laboratory’s previously published collection and have been validated for Western Antibodies. immunoblotting. material was eluted by boiling in SDS-PAGE sample buffer and characterized by up ten-fold, as previously described tions. The reaction mixes were set up as for depleted nuclear extracts in the presence or absence of variant Mediator preparamanufacturer. The beads were incubated with either mock-depleted orDynabeads M-280 Streptavidin Mediator- (Invitrogen 11205-D), as recommended by the novirus major late (Ad ML) promoter–containing DNA fragment was bound to Immobilized template recruitment assays. NP-40 and eluted. The immunoprecipitates were analyzed by western blotting. TFIID. After incubation for 2 h, the beads were washed again in BC200to plusbinding reactions0.1% containing various Mediator derivatives and either Pol II or toProtein A–Sepharose wereThe beads beads. andwashed addedwith BC200 Immunoprecipitation assays. previously described antigen-purifiedimmunodepletedanti-MED30aswith (below),antibodywas For reactions with Mediator-depleted nuclear extract, HeLa cell nuclear extract both in solution and in gel with trypsin to identify cross-linked peptides disulfide reduction and cysteine alkylation, the cross-linked complextion. The reaction was wasthen quenched digestedin 50 mM ammonium bicarbonate. After (d0:d12 with 1:1 ratio, Creative Molecules) for 45 min atsuberatedisuccinimidyl labeled isotopically4 mM °C1 bycross-linked with chemically constant agita spectrometry. mass and cross-linking Chemical purified by chromatography against bacterially expressed antigen validatedbeen for immunodepletion and coimmunoprecipitation and 7,500 for MS2. Ions (370–1,700 m/z) with charge state of >3 wereanalyzed selectedin the fororbitrap mass analyzer. The target resolution was 60,000 for MS1 collisional dissociation/HCD (HCD energy 27–33, 0.1-ms activation time) and mode, where the top eight most abundant ions were fragmentedVelos by S higher-energylenses was set at 35%. The instrument wastransmissionon ion and °C, operatedkV.275 1.9–2.2temperature atwas capillary The in the data-dependent eter (Thermo Fisher). The flow rate was ~200 nl/min. The spray voltage was set system (Agilent) and analyzed with an LTQ Velos Orbitrap Pro mass spectrom 46–100% B, 118–139 min, equilibrated with 100% A until 150 min) with a HPLC wereeluted 150-mina LCin gradient toB,46%B(8% 0–118min,followed by acetic acid and mobile phase B of 70% ACN with 0.5% acetic acid. The peptides 0.5% consistedof A Dr. Mobilephasesize,GmbH).Maisch pore 200-Å silica, The column was packed with 8 cm of reverse-phase C18 material (3- sprayionization emitter tip (360 O.D, 75I.D., with 15- FA)andontoloaded self-packeda PicoFrit column withanintegrated electro and analyzed by LC/MS. SEC fractions in the molecular-mass range of ~2.5 kDa to 8 kDa were collected with offline HPLC separation with an auto sampler (Agilent Technologies).andfractionated by peptide SEC (Superdex Peptide PC 3.2/30, GE Healthcare) Three dissolved in 20 After extraction and purification, the resulting proteolytic peptide mixture was trypsin. bygel in digestedand pieces small intocrushed sliced, was kDa ~220 aboveregion gelPAGE TheSDS gel.4–12% a electrophoresisin by separated buffer.The sample was cooled at room temperature for cysteine alkylation and tion,~50 and fractionated by peptide size-exclusion chromatography peptide mixture was purified with a C18 cartridge (Sep-Pak, Waters), lyophilizedadded to the digest and was incubated for a further 4 h. The resulting proteolytic (Waters) at 37 °C. After 12–16 h of incubation, an additional 1–2 (Promega)trypsin in 1M urea with ~2% acetonitrile (ACN) and 0.1% Rapigest in-solution digestion, ~50–100 Purified peptides were dissolved in the sample loading buffer (5% MeOH, 0.2% 4 . Antibodies to MED18 (sc-161835), MED8 (sc-103619), MED22 MED22 (sc-103619), MED8 (sc-161835), MED18 to Antibodies . µ g purified complexpurified g resuspendedwas loading andLDSheated 2× in Antibodies against most of the Mediator subunits were from ourMediator fromsubunitswere the of mostagainst Antibodies µ l of a solution containing 30% ACN and 0.2% formic acid (FA) 3 4 . Antigen-purified MED30 antibody was coupled µ g of purified complex was digested with 2 5 2 . After incubation and washing, the bound in vitro A PCR-generated biotinylated ade transcription but were scaled The purified complex was complex purified The µ doi: m tip,m New Objective). 5 10.1038/nsmb.2914 5 . For in-gel diges µ 3 g trypsin was 3 4 was affinity . µ m porous 53 , 5 4 µ . For g of 5 1 ------

© 2014 Nature America, Inc. All rights reserved. fied fied as a result. generally expected and C terminal to aspartate and glutamate for arginine-containing peptides was mentations.appearanceTheprolinedominant toof terminal fragmentions N be assigned and must follow a pattern that contains a continuous stretch of frag acids, and for both peptide chains the major MS/MS fragmentation peaks must for positive identifications, both peptide chains must contain at least five amino verification of the resulting MS/MSmanual spectra to on subjected the were basis and of (FDR) the ratefollowing discovery criteria: false 5% at filtered were variable modification, and a maximum of two trypsin miscleavages. The results cysteine carboxymethylation as a fixed modification, methionine oxidation as a accuracy of MS1 of human Mediator complex and BSA. Other search parameters included: mass by pLink software orbitrap was 500 ms for full scan and 500–700 ms for MS2. the for time ion-injectionmaximum The ms. 200 at set LTQwas the for time ion-injectionmaximumorbitrap. andThe trap ion linear respectively,the for 10 of limits accumulation ion-trap and event, MS/MS an trigger to parameters include: ‘lock mass’ at 371.1012 Da, the minimal threshold of 5,000 fragmentation. A dynamic exclusion of (15 s/2/55 s) was used. Other instrumental doi: The raw data were transformed to mascot generic format (MGF) and searched 10.1038/nsmb.2914 ≤ 5 6 57 10 p.p.m. and MS2 with a database containing sequences of the protein subunits , 5 8 . A total of 277 unique cross-linked peptides were identi ≤ 20 p.p.m. for the initial database search, 5 and 10 and 6 - - , 58. 57. 56. 55. 54. 53. 52. 51. 50.

Michalski, A., Neuhauser, N., Cox, J. & Mann, M. A systematic investigation into investigation systematic A M. Mann, & J. Cox, N., Neuhauser, A., Michalski, spectrometry: mass trap ion desorption laser Matrix-assisted B.T. Chait, & J. Qin, B. Yang, Leitner, A. Algret, R. Y. Shi, TRAP/SMCC/mediator- R.G. Roeder, & Y.K. Kang, A.E., Wallberg, S., Malik, by initiation transcription Accurate R.G. Roeder, & R.M. Lebovitz, J.D., Dignam, Gu, W. the nature of tryptic HCD spectra. HCD tryptic of nature the ions. (1996). ofpeptide 2108–2112 fragmentation effective and isolation efficient Methods Nat. 11 chromatography.exclusion size by enrichment and proteases 2014). July (29 pathway. TORC1 the of pore nuclear the 2014). from module heptameric complex. coatomer-related a of architecture 4. (2002). factor nuclear hepatocyte receptor nuclear orphan by templates chromatin and DNA from activation transcriptional dependent Res. nuclei. Acids mammalian isolated from extract soluble a in II polymerase RNA regulation. transcription in M111.014126 (2012). M111.014126 et al. t al. et et al. t al. et o. el Proteomics Cell. Mol.

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