COM M E N TA RY

NATURE MEDICINE VOLUME 9 | NUMBER 10 | OCTOBER 2003 1239

LASKER BASIC MEDICAL RESEARCH AWARD

The temporal and spatial expression of spe-cific genes is central to processes such asdevelopment, differentiation and home-ostasis in eukaryotes, and is regulated pri-marily at the level of transcription. Anunderstanding of the molecular basis forthis regulation has presented a major chal-lenge for the past 40 years. After earlyinsights into genetic control mechanisms inprokaryotes, elegantly elaborated in theclassic 1961 paper of Jacob and Monod, itwas anticipated by many that similar prin-ciples would apply in eukaryotes. However,early studies in animal cells faced the prob-lem of genomic complexity (including reit-erated DNA sequences) and the lack oftractable genetic approaches. By the time Ientered graduate school in the mid 1960s,the general RNA classes—ribosomal(rRNA), transfer (tRNA), messenger(mRNA) and the enigmatic heterogeneousnuclear (hnRNA)—had been recognized,and variations in the levels of these RNAclasses were being defined during variousgrowth, developmental, hormonal and viralresponses. However, except for the earlywork from the laboratories of Max Birnstieland Don Brown on the purification andstructural analysis of the amplified rRNAgenes of Xenopus laevis, essentially nothingwas known about gene structure, transcrip-tional regulatory mechanisms or the basictranscriptional machinery in animal cells.Thus, the field was ripe for discovery ofeven the most fundamental principles ofeukaryotic transcription, and a major ques-tion was how similar they would be to thoseof prokaryotes.

My interest in transcription was stimu-lated, as a Wabash College undergraduatein 1963, by a biochemistry course that covered recent work in bacterial geneticregulatory mechanisms. This interest was further heightened by a course taught

by Sol Spiegelman in 1964, my first year ofgraduate studies in biochemistry at theUniversity of Illinois. Although researchopportunities in eukaryotic transcriptionwere limited, I was able to join the labora-tory of Bill Rutter just as he was moving tothe University of Washington. Though notyet studying transcription per se, Rutterhad begun a biochemical analysis of cell-specific protein synthesis during pancreaticdevelopment and had visions of extendingthis to the regulation of RNA synthesis. Butbecause the pancreas was not yet amenableto transcription studies, I was forced toexplore other model systems.

Nuclear RNA polymerases: multiplicity,structure and general functionsMy early graduate work at the University ofWashington was spent analyzing RNA synthesis by endogenous RNA polymerasein nuclei isolated from rat liver during hormonal responses and from developingsea urchin embryos. But although thesestudies allowed quantitation of RNA syn-thesis, they were not especially satisfyingand provided little insight into transcrip-tion mechanisms or their regulation. Itherefore opted to exploit the transcrip-tional machinery as an entry point into thisarea, with the ultimate goal of reconstruct-ing specific transcription events usingpurified components. At that time, andafter Sam Weiss’ original description in1959 of RNA polymerase activity in a crudenucleoprotein preparation from rat livernuclei, the abundant (and soluble) bacter-ial RNA polymerase was being activelystudied, and trace amounts of solubleactivity from mammalian nuclei also hadbeen reported. Studies by Widnell and Tataof the effects of salt and divalent metal ionson the base composition of RNA synthe-sized in isolated nuclei also raised the

intriguing possibility of two differentenzyme activities, but did not exclude tem-plate-related effects1.

After realizing from initial studies thatmost of the mammalian RNA polymerasewas tightly bound to chromatin as a result ofits engagement in active transcription, I firsthad to develop new methods to quantita-tively solubilize the RNA polymerase andremove the interfering DNA and histonecomponents. A number of trials and someinsight from a 1942 paper by Mirsky andPollister on nucleohistone propertiesresulted in a relatively simple procedure:nuclear disruption and histone dissociationfrom DNA at high salt concentration, fol-lowed by DNA breakage and dissociation ofRNA polymerase by sonication, and finallyselective precipitation of DNA-histone com-plexes by rapid dilution to a low salt concen-tration. The resulting soluble enzymepreparation then was amenable to conven-tional purification. Remarkably, the initialchromatographic step on DEAE-Sephadex inFebruary 1969 yielded three peaks of activity(designated RNA polymerases I, II and III)that showed template-dependent incorpora-tion of all four ribonucleoside triphosphates,distinct salt and metal ion responses andreproducibly distinct chromatographicbehavior2 (Fig. 1). Seeing these three peaks ofactivity in the middle of the night on the ini-tial run was one of the truly exhilarating“Eureka!” moments in my scientific career,and I slept little in the ensuing weeks as I fur-ther characterized the enzymes.

I first discovered RNA polymerases I, IIand III in nuclei from sea urchin embryos—afortunate choice because of the relative abun-dance of all three RNA polymerases in thatorganism, and because it afforded me oppor-tunities for rare excursions from the labora-tory (Fig. 2). I subsequently found similaractivities in rat liver nuclei and in yeast.

The eukaryotic transcriptional machinery:complexities and mechanisms unforeseenRobert G Roeder

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

COM M E N TA RY

1240 VOLUME 9 | NUMBER 10 | OCTOBER 2003 NATURE MEDICINE

(Disappointingly, none of the RNA synthe-sis–deficient yeast mutants of Lee Hartwellshowed discernable defects in any of the solu-bilized RNA polymerases!) I also found thatPol I was localized within nucleoli, the sites ofrRNA gene transcription, whereas Pol II andPol III were restricted to the nucleoplasm3.This further suggested specific roles for eachof the enzymes in the transcription of dis-tinct classes of genes. With some trepidation,both for prematurely releasing my results andfor giving my first public scientific presenta-tion, I reported these findings at the April1969 meeting of the Federation of AmericanSocieties for Experimental Biology. Needlessto say, my findings were received with someacclaim, especially as a symposium speaker atthe same meeting had reviewed current workand concluded that animal cells most likelycontained a single enzyme! Coincidentally,my discovery of the three RNA polymerasesoccurred within weeks of publication of thelandmark Burgess, Travers, Dunn and Bautzpaper describing bacterial sigma factor andits effect on selective transcription byEscherichia coli RNA polymerase4, furtherenhancing my excitement over future studies.

During a subsequent postdoctoral stintat the Carnegie Institution of Washingtonwith Donald Brown, a pioneer in studies ofgene structure and function in animal cells,Ron Reeder and I attempted to show that

purified RNA polymerase I could accu-rately transcribe purified rRNA genes. Tomy great surprise and disappointment, thiscould not be demonstrated and I began towonder about the possibility of other

essential factors or even the necessity of achromatin context for specific transcrip-tion. Despite the failure of this key experi-ment, I learned from Brown about theadvantages of Xenopus as a model systemand the necessity for good assays to askspecific questions about gene control.

In my subsequent position in the depart-ment of biological chemistry at WashingtonUniversity School of Medicine, and as aprelude to further functional studies withpurified genes and enzymes, I set out witheager young colleagues to purify all threeenzymes to homogeneity and to rigorouslyestablish their specific functions. In the lat-ter case, Roberto Weinmann showed thatpurified RNA polymerases I, II and III couldbe distinguished on the basis of differentialsensitivities to the mushroom toxin α-amanitin. By monitoring the α-amanitinsensitivities of specific transcription eventsby endogenous (engaged) RNA polymerasesin isolated nuclei, he documented the syn-thesis of rRNA by Pol I, the synthesis of ade-novirus pre-mRNA by Pol II and thesynthesis of cellular 5S and tRNA and aden-ovirus VA RNA by Pol III5,6. These resultsstrengthened the case for global regulationof the major classes of RNA through distinctenzymes and, perhaps more importantly,gave us important insights into model genesystems to reconstitute specific transcrip-tion events with specific RNA polymerases.

Meanwhile, to ascertain the structural

Figure 1 Chromatographic resolution on Sephadex A25 of the three nuclear RNA polymerases fromsea urchin embryos. Original graph from chromatographic analysis (18 February 1969). Red, RNApolymerase activity; blue, total protein; black, ammonium sulfate gradient. Pol II activity isunderrepresented because of suboptimal (low-Mg2+) assay conditions.

Figure 2 Robert Roeder collecting sea urchins in the frigid waters of the Strait of Juan de Fuca (1968).

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

COM M E N TA RY

NATURE MEDICINE VOLUME 9 | NUMBER 10 | OCTOBER 2003 1241

basis for the functional differences betweenthe RNA polymerases, Lawrence Schwartzand Virgil Sklar rigorously purified substan-tial amounts of all three enzymes frommouse plasmacytomas. Comparative analy-sis clearly showed complex and distinct sub-unit structures for all three enzymes,although there seemed to be at least twocommon subunits7. These results confirmedand extended preliminary studies from theChambon and Rutter labs on Pol I and PolII. Remarkably, the phylogenetically con-served eukaryotic enzymes are now knownto contain 14 (Pol I), 12 (Pol II) and 17 (PolIII) subunits (the products of 31 distinctgenes), including 5 shared subunits andenzyme-specific homologs to the β-, β’- andα-subunits of E. coli RNA polymerase.Clearly, the different mammalian enzymeswere not derived from subtle modificationsof a single enzyme, as would prove to be thecase for the bacterial enzyme, whichacquires different initiation specificitieswith distinct sigma factors. Thus, the studiesof the mammalian enzymes provided thefirst indication, in eukaryotes or prokary-otes, of transcriptional regulatory mecha-nisms involving gene-specific structuralvariations in the general transcriptionmachinery, as well as early indications oflevels of control and complexity far beyondthose existing in prokaryotes.

Reconstitution of accurate transcription:general initiation factors and mechanismsWith purified enzymes in hand and theirgeneral functions established, the nextchallenge was to achieve specific initiation.Focusing on the small and highly reiterated5S RNA genes, which had been isolated byDon Brown and shown to be actively tran-scribed in oocytes, Carl Parker showed in1976 that purified Pol III, but not Pol I, PolII or E. coli RNA polymerase, could accu-rately transcribe 5S RNA genes in purifiedchromatin from immature oocytes but notin total cellular or cloned 5S DNA tem-plates8. This long-awaited result—the firstdemonstration of accurate transcription bya eukaryotic RNA polymerase in a reconsti-tuted cell-free system—greatly excited usand was significant in several respects. Itsuggested the presence of essential (chro-matin-bound) RNA polymerase–specificaccessory factors, cast doubt on the signifi-cance of the very popular studies elsewhereof chromatin transcription by bacterialRNA polymerases, validated our biochemi-cal approach and stimulated studies withpurified DNA templates. By 1978 we wereable to show accurate transcription of

cloned 5S RNA, tRNA and adenovirus VARNA genes by purified Pol III in conjunc-tion with soluble subcellular fractions fromXenopus oocytes and human HeLa cells9,10.Shortly thereafter, and with new informa-tion from the laboratory of Jim Darnell onthe location of transcription start sites forpre-mRNA synthesis in the adenovirusgenome, Tony Weil was able to show accu-rate transcription initiation (at the naturalstart site) on an adenovirus DNA fragmentby purified Pol II and a crude subcellularfraction11. Don Luse also showed accuratetranscription initiation from the β-globinpromoter using the same assay. These veryexciting findings immediately prompted abiochemical analysis of general and gene-specific transcription initiation factors fora variety of genes, and provided assays fordetermining DNA sequence requirementsfor transcription initiation. However, thepermissive transcription in HeLa cellextracts of promoters that are naturallyactive only in adenovirus-infected or ery-thropoietic cells led us to believe that wewere dealing with general initiation factorsand left unexplained the basis for cell-restricted transcription.

Although a multitude of labs focused onthe determination of DNA sequencerequirements for initiation, using cell-freetranscription and transfection assays, wefelt that the unknown protein factors werethe most important (and challenging) partof the problem and returned to the coldroom to fractionate HeLa extracts. In 1980,Jacki Segall, Takashi Matsui and Tony Weil

were able to distinguish two factors, TFIIICand TFIIIB, that are generally required fortranscription of class III genes and severalfactors, including TFIID, that wererequired for transcription of class IIgenes12,13. In early 1983 my laboratorymoved to The Rockefeller University, wherewe continued the purification of generalinitiation factors. Work from my own andother laboratories over the next decade ormore established that: (i) the two Pol IIIfactors contain at least nine distinctpolypeptides; (ii) there are six Pol II factors(TFIID, TFIIA, TFIIB, TFIIE, TFIIF andTFIIH) with a total of about 32 distinctpolypeptides; (iii) there are several Pol Ifactors; (iv) the Pol I, Pol II and Pol III factors are structurally and functionallydistinct; and (v) the general initiation fac-tors are highly conserved from yeast tohumans, thus paving the way for geneticanalyses of the factors in yeast. The struc-tural complexity of the individual, enzyme-specific classes of initiation factors wasquite surprising, especially in light of theircommon usage by most or all genes withina given class, and the earlier establishedcomplexity of the cognate RNA poly-merases. However, these accessory factorsclearly offered the possibility for yetanother layer of gene control through vari-ations in the their levels or activities orthrough selective interactions with gene-specific regulatory factors.

In an attempt to make sense of this complexity, and anticipating that specificDNA-protein and protein-protein interac-

Figure 3 General initiationfactors and PIC assemblypathways for class III geneswith internal promoterelements, and activation bya gene-specific factor. Onthe tRNA gene promoter,assembly of a functional PIC containing Pol III andgeneral initiation factors(yellow) is nucleated bystable binding of TFIIIC to the BoxA and BoxBelements. In contrast,assembly of a PIC on thedivergent promoter of the 5S RNA gene is more highlyregulated and requires priorbinding to BoxA and BoxC of the 5S gene–specificactivator TFIIIA, whichinteracts with and stabilizesTFIIIC binding. For both genes, TFIIIB is recruited by TFIIIC and Pol III is recruited by TFIIIB and TFIIIC. Solid black bars indicate interactions between Pol III and the various factors. NTP,ribonucleoside triphosphates. Adapted from ref. 23.

5S RNA gene

IIIA

IIICIIIB

Pol III

BoxA BoxC

tRNA gene

IIIC

Pol III

BoxA BoxB

≥ 25 polypeptides

IIIB

TFIIIA

TFIIIC

TFIIIB

Pol III

5S RNA

PICNTP

TFIIIC

TFIIIB

Pol III

tRNA

PICNTP

(Core promoter

recognition)

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

COM M E N TA RY

1242 VOLUME 9 | NUMBER 10 | OCTOBER 2003 NATURE MEDICINE

tions would serve as points of control byother factors, we undertook mechanisticstudies with purified RNA polymerasesand partially purified initiation factors.Studies by Andrew Lassar, Jim Bieker,Noboru Nakajima, Mike Van Dyke andMichele Sawadogo led to the definition ofcore promoter recognition factors andpathways for the ordered assembly of func-tional preinitiation complexes (PICs)14–16

for class III genes (tRNA, VA RNA) con-taining the most common class III pro-moter elements (A and B boxes) and forclass II genes containing the most commonclass II core promoter element (TATA box),as shown in Figures 3 (left) and 4 (right).These pathways emphasize primary corepromoter recognition by TFIIIC and byTFIID, respectively, forming complexesthat nucleate the assembly of the remainingcognate factors and RNA polymerases intothe PIC.

Beyond our initial studies in the mid1980s, many laboratories have contributedto the elaboration of these pathways. Ofspecial note are contributions of the labo-ratories of Phil Sharp and LennyGuarente17, as well as Danny Reinberg, toelucidation of the later steps in the Pol II

pathway18. A yeast TATA-binding polypep-tide (TBP) that could substitute for humanTFIIID in PIC assembly and function wasdescribed in 1988 by Sharp and Guarenteand by Chambon and Sentenac, and provedcrucial not only for the more detailedmechanistic studies, but also for the isola-tion and characterization by several groupsof metazoan TBPs and TBP-associated fac-tors within the >1 MDa TFIID complexes.Masami Horikoshi and Alex Hoffmannspearheaded our efforts on this front, pro-ceeding necessarily through an orthologwalk (based on conserved sequences) fromSaccharomyces cerevisiae TBP, throughSchizosaccharomyces pombe TBP, to humanTBP and associated factors. X-ray struc-tural analyses of TBP and TBP-TATA andhigher-order complexes by the laboratoriesof Stephen Burley and Paul Sigler also pro-vided important insights into early steps inPIC assembly19. More recently, the remark-able work of Roger Kornberg on the X-raystructure of RNA polymerase II has givenus further insights and hope for a struc-tural analysis of the complete PIC20. Giventhat RNA polymerases and general initia-tion factors are the ultimate targets of reg-ulatory factors, these basic assembly

pathways revealed many potential points ofcontact for receiving signals from interact-ing regulatory factors or cofactors.

Regulation of transcription: sequence-specific factorsBy 1971, genetic and biochemical studieshad firmly established transcriptional reg-ulation by sequence-specific DNA-bindingproteins (activators and repressors) inprokaryotes, and later mechanistic studiesshowed direct interactions with RNA poly-merase. In the absence of documented cell-and promoter-specific binding factors, thepermissiveness of our cell-free systems andthe observations by others of short-livednuclear RNAs, post-transcriptional RNAprocessing pathways and global chromatinactivation domains left me wonderingabout the possibility of somewhat diver-gent regulatory mechanisms in eukaryotes.Nevertheless, the notion of regulation bygene- and cell-specific transcription fac-tors remained most appealing to me, and Ibelieved that in the absence of tractablegenetic approaches in animal cells, ourreconstituted cell-free systems provided anecessary and powerful approach to thisproblem. In 1979 (ref. 21), this approachled to David Engelke’s identification andpurification of the 5S gene–specific activa-tor TFIIIA, and to our demonstration ofsite-specific binding to the internal 5S pro-moter that had just been mapped by DonBrown. The primary sequence of TFIIIAwas revealed through cognate cDNAcloning by Ann Ginsberg in 1984 (ref. 22).

TFIIIA was the first of many sequence-spe-cific DNA-binding transcription factors to beidentified, purified and cloned in eukaryotes,and holds the additional distinction of beingthe prototype zinc finger protein. This struc-ture, first proposed by Aaron Klug, is the pre-dominant DNA-binding motif in eukaryoticregulatory factors. Mechanistic studies alsoshowed that TFIIIA binding to the 5S pro-moter precedes and facilitates the sequentialbinding of TFIIIC, TFIIIB and PolIII14 (Fig.3, right). These studies with TFIIIA were thefirst to show a primary role for a gene-spe-cific regulatory factor in RNA polymerasefunction in eukaryotes. However, in distinctcontrast to the bacterial mechanism involv-ing direct RNA polymerase–regulatory factorinteractions, the effect on eukaryotic RNApolymerase is mediated indirectly through ahost of other initiation factors. This morecomplicated mechanism, which allows formore subtle regulation through multipleinputs, has proved to be a dominant theme ineukaryotes.

Chromatin template Accessible

distal regulatory element(s)

TFIID

TFIIA

TFIIB

TFIIF–Pol II

Accessiblecore promoter

TFIIE

TFIIH

Chromatin remodeling factors

Mediator

Cell-specific cofactors

Negative cofactors

Regulatory factors

CTD

Regulatory factors IIA IIB IIF

IIHPol II IIDTAFs

TBP IIE

PIC (44 polypeptides)

Core promoterrecognition( )

DRE TATA

Figure 4 General initiation factors and PIC assembly pathway for class II genes with a TATA-containing core promoter, and regulation by gene-specific factors and interacting cofactors. Assemblyof a PIC containing Pol II and general initiation factors (yellow) is nucleated by binding of TFIID to theTATA element of the core promoter. A model for the regulation of PIC assembly and function involves,sequentially: (i) binding of regulatory factors to distal control elements; (ii) regulatory factorinteractions with cofactors that modify chromatin structure to facilitate additional factor interactions;and (iii) regulatory factor interactions with cofactors that act after chromatin remodeling to facilitate,through direct interactions, recruitment or function of the general transcription machinery. TAFs, TBP-associated factors. Adapted from ref. 23.

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

COM M E N TA RY

NATURE MEDICINE VOLUME 9 | NUMBER 10 | OCTOBER 2003 1243

Figure 5 Comparison oftranscription factors andactivation mechanismsin prokaryotes andeukaryotes. In eachcase, the ultimatepurpose of DNA-binding activators is tomediate RNApolymerase recruitmentand function. Butwhereas activatorsinteract directly withRNA polymerasesubunits (primarily αand σ) in prokaryotes,the effects of activatorson RNA polymerase IIin eukaryotes aremostly indirect andinvolve interactionswith diverse cofactorsthat modify chromatinstructure and facilitaterecruitment of RNApolymerase II andgeneral initiationfactors. Chromatinremodeling factors(pink) include ATP-dependent factors, histone acetyltransferases (HATs) and histone methyltransferases (HMTs),examples of which are indicated in parentheses. Cofactors (blue) that act at the level of DNA includethe Mediator, general positive cofactors such as PC3 and PC4, cell-specific factors (X) such as OCA-Band OCA-S, and negative cofactors such as the TBP-interacting NC2. CTD, C-terminal domain of largesubunit of Pol II. For further details see text and ref. 23.

Our success with TFIIIA gave me moreconfidence than ever about the validity ofmy views on gene- and cell-specific tran-scription factors and led my group and oth-ers to search, using a similar functionalapproach, for such factors for class II genes.The earliest factors that were so identifiedand shown to act through promoter-specificelements were Sp1 (discovered by Bob Tjianin 1983), HSF (by Carl Parker in 1984) andUSF (by us and others in 1985). Along withearlier studies of the glucocorticoid receptorby Jan-Åke Gustafsson and Keith Yamamotoin 1981 and later studies of genetically iden-tified yeast regulatory factors in 1985, thesestudies provided compelling evidence forfactors that regulate RNA polymerase func-tion by binding to distal DNA control ele-ments. In keeping with the paradigmestablished for TFIIIA, the earliest in vitromechanistic studies implicated TFIID, theprimary core promoter-recognition factor,as a presumptive activator target15 and stim-ulated intense efforts to further characterizethis factor. At present, dozens (perhaps hun-dreds) of sequence-specific DNA-bindingfactors, both ubiquitous and cell specific,have been identified and implicated in vari-ous genetic regulatory pathways.

Yet another layer of complexity:diverse transcriptional coactivatorsBecause regulated expression of the diverseprotein-coding genes in the nucleus under-lies essentially all aspects of physiology,studies of transcriptional regulationbecame increasingly focused on PolII–related regulatory mechanisms. By theearly 1990s, the availability of cell-free sys-tems reconstituted with highly purifiedpreparations of Pol II and general initiationfactors made it possible to analyze anincreasingly broad spectrum of regulatoryfactor functions in defined systems. Giventhe specificity intrinsic to the DNA-bindingregulatory factors, the structural complex-ity of the general transcription machinery(their ultimate target) and the documentedinteractions between regulatory factors andseveral general initiation factors, it wasthought that the essential components werein hand and that detailed regulatory factormechanisms could be elucidated. Quite sur-prisingly, however, sequence-specific DNA-binding activators were unable to functionin systems reconstituted with these compo-nents and we were forced, once again, topostulate and search for other factors.Further biochemical studies led to the dis-

covery in several laboratories of variouscoactivators that were essential for activatorfunction23. The most significant of these,the so-called ‘Mediator’, is a ∼ 25-subunitcomplex that has proven to be the mainconduit for communication between DNA-bound activators and the general transcrip-tional machinery (Fig. 4). Mediatoractivities were first discovered in 1991 inextracts from yeast by Roger Kornberg24

and in extracts from HeLa cells by MichaelMeisterernst in my lab25. The yeastMediator was first purified by the Kornberglab in 1994 (ref. 26), and the humanMediator was first purified by Joe Fondellin my lab in 1996, as the thyroid hormonereceptor–associated protein complex27.Earlier studies by Rick Young had identifiedessential yeast Mediator components, SRBs,that interact genetically with Pol II andreside in a Pol II holoenzyme28, presagingthe soon-to-be-described mechanismsinvolving direct Mediator interactions withPol II. To date, a number of site-specificDNA-binding regulatory factors have beenshown to function through direct interac-tions with distinct Mediator subunits, andthe huge complex is viewed as a multivalentcontrol panel that integrates diverse signalsfrom both activators and repressors29.

Several more highly regulated and highlyselective coactivators are now known toexist. Two that were first identified by YanLuo in my laboratory through biochemicalcomplementation assays include the B-cell-specific OCT coactivator (OCA)-B (ref. 30)and, most recently, the S-phase-specificOCA-S (ref. 31). These factors interact withthe ubiquitous DNA-bound OCT-1 onimmunoglobulin and histone-2B promot-ers, respectively, and provide a remarkableexample of selective cell-specific promoteractivation through distinct coactivators thatact, in part, through a common DNA bind-ing factor. OCA-B is notable because its dis-covery in 1992 provided a new paradigm fortissue-specific gene regulation, and OCA-Sis notable because it contains a nuclear formof the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase as a key compo-nent and is regulated by NAD+ and NADH.Emphasizing the continuing importance ofthe biochemical complementation assaysthat we developed, it seems likely that OCA-S, because of the roles of its componentparts in other essential cellular pathways,would not easily have been identified byother means such as genetics.

Studies in many other laboratories haveidentified another important group ofcofactors—coactivators and corepressors—

MEDIATOR

PC4

x

IIEIIF

PC3

IIH

Activators (~200)

4 polypeptides ( α ββ′σ)

Prokaryotes

EukaryotesActivators (>2,000)

Coactivators

Pol II

CTD

TAFs IID

NC2

>100 polypeptides

HMTs(SETs, PRMTs)

TBP

IIBIIA

POL

σ

HATs(p300, STAGA)

ATP-dependent(SWI-SNF, NURF)

Nucleosome

2

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

COM M E N TA RY

1244 VOLUME 9 | NUMBER 10 | OCTOBER 2003 NATURE MEDICINE

that act through modifications of chro-matin structure. These include ATP-dependent factors that alter the structure orposition of nucleosomes, and factors thateffect covalent modifications of the histonetails. The resulting chromatin alterations arethought to facilitate or prevent PIC forma-tion or proper function. This suggests thesequential use of a complex (and potentiallyvariable) set of cofactors in the activation ofany given gene by one or more (usually agroup of) DNA-bound regulatory factors(Fig. 4).

Conclusions and perspectivesSince the discovery of the three nuclearRNA polymerases over 34 years ago, wehave come to appreciate, largely throughbiochemical approaches, the remarkablecomplexity of the general transcriptionalmachinery and the increasingly large set ofcofactors needed for regulatory factorfunction. This complexity far exceeds thatobserved for the prokaryotic transcrip-tional machinery (Fig. 5) and was not evenremotely anticipated three or four decadesago. Why all this complexity? We now knowthat the human genome contains some35,000 genetic loci, many with multipleinitiation and splice sites, and (based onsequence motifs) that more than 2,000 ofthese loci are likely to encode DNA-bind-ing regulatory factors. We also know thatgene expression must be exquisitely regu-lated and finely tuned during development,differentiation and homeostatic responses.To this end, cells seem to have evolved notonly the expected highly complex array ofDNA-binding regulatory factors, but also acomplex machinery through which theseregulatory factors act, thus affording agalaxy of possible outcomes from a limitednumber of signaling pathways. Morespecifically, this complex machineryallows: (i) global control mechanisms forthe major classes of RNA; (ii) mechanismsto activate or repress genes packagedwithin natural chromatin structures; (iii)multiple targets for the cooperative func-tions of combinations of DNA-bound reg-ulatory factors; (iv) mechanisms for theintegration of multiple signaling pathways;and (v) a basis for alternative (redundant)gene activation and repression pathways.

Major challenges still lie ahead. One willbe to integrate the functions of the relevantactivators and coactivators and the generaltranscription machinery on natural chro-matin templates in systems reconstitutedwith completely defined components, andto detail, for individual genes, the mecha-

nisms involved. These studies can be com-plemented and, in part, guided by variousgenomic, proteomic and genetic studies;they should ultimately be directed towardan understanding of the function of com-plex and far-distal enhancers, as well as theapparent coupling of various events oftranscription (initiation, elongation andtermination) and RNA processing andtransport. Another challenge will be todelineate the structure and dynamics of thePIC and its interacting activator-cofactorcomplexes. Finally, there is hope that whilelearning how the cell uses its transcriptionmachinery for normal gene regulation, wemight also deduce ways to selectively regu-late and control aberrant gene expression.

ACKNOWLEDGMENTSI thank my mentors Bill Rutter and Don Brown for their inspiration and friendship and for thefreedom to pursue my specific goals in theirlaboratories; my students and postdocs for theirdedication and many contributions; my family for their unwavering support and understanding;and the many institutions that have supported my research over four decades.

1. Widnell, C.C. & Tata, J.R. Studies on the stimula-tion by ammonium sulphate of the DNA-dependentRNA polymerase of isolated rat-liver nuclei.Biochim. Biophys. Acta 3, 478–492 (1966).

2. Roeder, R.G. & Rutter, W.J. Multiple forms of DNA-dependent RNA polymerase in eukaryotic organ-isms. Nature 224, 234–237 (1969).

3. Roeder, R.G. & Rutter, W.J. Specific nucleolar andnucleoplasmic RNA polymerases. Proc. Natl. Acad.Sci. USA 65, 675–682 (1970).

4. Burgess, R.R., Travers, A.A., Dunn, J.J. & Bautz,E.K.F. Factor stimulating transcription by RNA poly-merase. Nature 221, 43–46 (1969).

5. Weinmann, R. & Roeder, R.G. Role of DNA-depend-ent RNA polymerase III in the transcription of thetRNA and 5S RNA genes. Proc. Natl. Acad. Sci.USA 71, 1790–1794 (1974).

6. Weinmann, R., Raskas, H.J. & Roeder, R.G. Role ofDNA-dependent RNA polymerases II and III in tran-scription of the adenovirus genome late in produc-tive infection. Proc. Natl. Acad. Sci. USA 71,3426–3430 (1974).

7. Sklar, V.E.F., Schwartz, L.B. & Roeder, R.G. Distinctmolecular structures of nuclear class I, II and IIIDNA-dependent RNA polymerases. Proc. Natl.Acad. Sci. USA 72, 348–352 (1975).

8. Parker, C.S. & Roeder, R.G. Selective and accuratetranscription of the Xenopus laevis 5S RNA genes inisolated chromatin by purifed RNA polymerase III.Proc. Natl. Acad. Sci. USA 74, 44–48 (1977).

9. Ng, S.-Y., Parker, C.S. & Roeder, R.G. Transcriptionof cloned Xenopus 5S RNA genes by X. laevis RNApolymerase III in reconstituted systems. Proc. Natl.Acad. Sci. USA 76, 136–140 (1979).

10. Weil, P.A., Segall, J., Harris, B., Ng, S.-Y. & Roeder,R.G. Faithful transcription of eukaryotic genes byRNA polymerase III in systems reconstituted withpurified DNA templates. J. Biol. Chem. 254,6163–6173 (1979).

11. Weil, P.A., Luse, D.S., Segall J. & Roeder, R.G.Selective and accurate initiation of transcription atthe Ad2 major late promoter in a soluble systemdependent on purified RNA polymerase II and DNA.Cell 18, 469–484 (1979).

12. Segall, J., Matsui, T. & Roeder, R.G. Multiple fac-tors are required for the accurate transcription ofpurified genes by RNA polymerase III. J. Biol.Chem. 255, 11986–11991 (1980).

13. Matsui, T., Segall, J., Weil, P.A. & Roeder, R.G.Multiple factors required for accurate initiation oftranscription by purified RNA polymerase II. J. Biol.Chem. 255, 11992–11996 (1980).

14. Lassar, A.B., Martin, P.L. & Roeder, R.G.Transcription of class III genes: formation of preini-tiation complexes. Science 222, 740–748 (1983).

15. Sawadogo, M. & Roeder, R.G. Interaction of a gene-specific transcription factor with the adenovirusmajor late promoter upstream of the TATA boxregion. Cell 43, 165–175 (1985).

16. Van Dyke, M.W., Roeder, R.G. & Sawadogo, M.Physical analysis of transcription preinitiation com-plex assembly on a class II gene promoter. Science241, 1335–1338 (1988).

17. Buratowski, S., Hahn, S., Guarente, L. & Sharp,P.A. Five intermediate complexes in transcriptioninitiation by RNA polymerase II. Cell 4, 549–561(1989).

18. Roeder, R.G. The role of general initiation factors intranscription by RNA polymerase II. TrendsBiochem. Sci. 21, 327–335 (1996).

19. Nikolov, D.B. & Burley, S.K. RNA polymerase IItranscription initiation: a structural view. Proc.Natl. Acad. Sci. USA 1, 15–22 (1997).

20. Cramer, P., Bushnell, D.A. & Kornberg, R.D.Structural basis of transcription: RNA polymerase IIat 2.8 angstrom resolution. Science 5523,1863–1876 (2001).

21. Engelke, D.R., Ng, S.-Y., Shastry, B.S. & Roeder,R.G. Specific interaction of a purified transcriptionfactor with an internal control region of 5S RNAgenes. Cell 19, 717–728 (1980).

22. Ginsberg, A.M., King, B.O. & Roeder, R.G. Xenopus5S gene transcription factor, TFIIIA: characteriza-tion of a cDNA clone and measurement of RNA lev-els throughout development. Cell 39, 479–489(1984).

23. Roeder, R.G. The role of general and gene-specificcofactors in the regulation of eukaryotic transcrip-tion. Cold Spr. Harb. Symp. Quant. Biol. LXIII,201–218 (1998).

24. Flanagan, P.M., Kelleher, R.J. III, Sayre, M.H.,Tschochner, H. & Kornberg, R.D. A mediatorrequired for activation of RNA polymerase II tran-scription in vitro. Nature 6317, 436–438 (1991).

25. Meisterernst, M., Roy, A.L., Lieu, H.M. & Roeder,R.G. Activation of class II gene transcription by reg-ulatory factors is potentiated by a novel activity. Cell66, 981–993 (1991).

26. Kim, Y.J., Bjorklund, S., Li, Y., Sayre, M.H. &Kornberg, R.D. A multiprotein mediator of tran-scriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell4, 599–608 (1994).

27. Fondell, J.D., Ge, H. & Roeder, R.G. Ligand induc-tion of a transcriptionally active thyroid hormonereceptor coactivator complex. Proc. Natl. Acad. Sci.USA 93, 8329–8333 (1996).

28. Thompson, C.M., Koleske, A.J., Chao, D.M. &Young, R.A. A multisubunit complex associated withthe RNA polymerase II CTD and TATA-binding pro-tein in yeast. Cell 7, 1361–1375 (1993).

29. Malik, S. & Roeder, R.G. Transcriptional regulationthrough mediator-like complexes in yeast and meta-zoan cells. Trends Biochem. Sci. 25, 277–283(2000).

30. Luo, Y., Fujii, H., Gerster, T. & Roeder, R.G. A novelB cell-derived coactivator potentiates the activationof immunoglobulin promoters by octamer-bindingtranscription factors. Cell 71, 231–241 (1992).

31. Zheng, L., Roeder, R.G. & Luo, Y. S phase activationof the histone H2B promoter by OCA-S, a coactiva-tor complex that contains GAPDH as a key compo-nent. Cell 114, 255–266 (2003).

Robert G. Roeder heads the Laboratory ofBiochemistry and Molecular Biology, TheRockefeller University, 1230 York Ave., NewYork, New York 10021, USA. e-mail:roeder@rockefeller.edu

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine