Proc. Natl. Acad. Sci. USAVol. 92, pp. 11864-11868, December 1995Biochemistry

Core promoter-specific function of a mutant transcription factorTFIID defective in TATA-box bindingERNEST MARTINEZ*, QIANG ZHOUtt, NOELLE D. L'ETOILEt, THOMAS OELGESCHLAGER*, ARNOLD J. BERKt,AND ROBERT G. ROEDER*§*Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021; and tMolecular Biology Institute andDepartment of Microbiology and Molecular Genetics, University of California, Los Angeles, CA 90024-1570

Contributed by Robert G. Roeder, September 12, 1995

ABSTRACT In conjunction with other general initiationfactors, the TATA box-binding protein (TBP) can direct basaltranscription by RNA polymerase II from TATA-containingpromoters, but its stable interaction with TBP-associatedfactors (TAFs) in the TFIID complex is required both foractivator-dependent transcription and for basal transcriptiondirected by an initiator element. We have generated a TATA-binding-defective TFIID complex containing an amino acidsubstitution in the DNA-binding surface of its TBP subunit.This mutated TFIID is defective in both basal and activatedtranscription from core promoters containing only a TATAbox but supports transcription from initiator-containing pro-moters independently of the presence or absence of a TATAsequence. Our results show that a functional initiator elementis needed to bypass the requirement for an active TATADNA-binding surface in TFIID and imply that gene-specifictranscription can be achieved by modulating distinct corepromoter-specific TFIID functions-e.g., TBP-TATA versusTAF-initiator interactions.

The general class II transcription initiation factor TFIID is amultisubunit complex composed of the TATA box-bindingprotein (TBP) and several TBP-associated factors (TAFs) (1).For TATA-containing class II genes, the binding ofTBP to theTATA element recruits TFIID to the promoter and representsthe first step in preinitiation complex assembly (2-4) that canbe influenced by upstream activators (5-13). Although TBP, inthe absence of TAFs, suffices to direct basal TATA-dependenttranscription, TAFs are required for the stimulation by up-stream activators (for review, see ref. 1). Since specific TBP-TATA interactions dramatically distort DNA (14, 15), TBPmay also control the architecture, and hence the function, ofthe preinitiation complex on TATA-containing promoters. Incontrast to the situation in TATA-containing promoters, themechanisms of transcription initiation at promoters that lacka TATA box, and in particular the role of TBP in transcriptioninitiation at TATA-less genes, are still poorly characterized.Earlier studies showed that an initiator DNA sequence canfunction as an independent core promoter element whichspecifies the position of the transcription start site in TATA-less promoters and that it also can stimulate basal transcriptionfrom TATA-containing promoters when located about 25 bpdownstream of a TATA box (reviewed in ref. 16). Recently,TAFs (in association with TBP) in the human TFIID complexhave been found to be essential not only for activator-dependent transcription but also for the basal (activator-independent) transcription function of an initiator element,regardless of the presence or absence of a TATA box (17). Thisnovel initiator-dependent function of TAFs in basal transcrip-tion was subsequently confirmed for Drosophila TFIID as well,albeit in the context of a TATA-containing promoter (18). In

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addition, evidence suggests that TBP binding to DNA is not arate-limiting step for the initial stages of TFIID recruitment toseveral initiator-dependent TATA-less promoters (17). To-gether, these observations raise the intriguing possibility thatTBP-DNA interactions, especially as observed in the TBP-TATA cocrystal structure (14, 15), may play an important roleonly on certain class II promoters. By using a mutant TFIIDcontaining a TBP subunit defective in TATA DNA binding, weshow that TBP-DNA interactions are largely dispensable forspecific transcription of initiator-dependent TATA-less class IIgenes. Our results thus demonstrate that TBP. like TAFs,differentially contributes to basal transcription in differentcore promoter contexts.

MATERIALS AND METHODSPlasmids. The 70-kDa heat shock protein (Hsp7O) and

terminal deoxynucleotidyltransferase (TdT) core promotertemplates were respectively plasmids pHsp70(-33/ +99)-CATand pTdT(-41/+59) (17). Linear templates G5-TdT andG5-E1B were, respectively, plasmids pG5TdT(-41/+59) andpG5-ElB-CAT (19) cut with Nde I. pG5TdT(-41/+59) wasobtained by digestion of pTdT(-41/+59) with EcoRI at aunique site, filling in the ends with Klenow DNA polymerase,and blunt-end ligation to the Pvu II-Xba I Klenow-filled DNAfragment containing five Gal 4 sites from pG5-ElB-CAT.T- I+, T+I+, and T+I are, respectively, plasmids pG5-TdT(-41/+33), pG5TdT(-41TATA+/ +33), and pG5TdT(-41TATA+/Inr-+33) obtained as described in Fig. 5 andpreviously (17). The ,3-Pol template is plasmid p,BP10 (20). TheG5,B-Pol template is plasmid pG5j3Pol(-41/+58)CAT andwas obtained by replacing, in pG5-ElB-CAT, the XbaI-BamHI fragment containing the E1B TATAboxwith anXbaI-BamHI PCR DNA fragment containing the human ,B-poly-merase core promoter sequences from -41 to +58. PCRmutagenesis was used to replace the threonine-210 codon witha lysine codon in the gene encoding the influenza hemagglu-tinin (HA)-epitope-tagged human TBP in plasmid pLTReTBP(21), creating pLTReTBPT21OK.

Cell Lines and Protein Purification. A HeLa cell line stablyexpressing mutant HA-TBPT2l0K (clone 12 cells) was estab-lished by retrovirus-mediated transformation (21) using thevector pLTReTBPT2l0K. Nuclear extracts from wild-type HA-TBP-expressing cells (LTRct3 cells) (21) and clone 12 cellswere fractionated on Whatman phosphocellulose P11. TheHA-tagged TFIIIB present in the P11 B fraction (0.1-0.3 MKCl) was immunoaffinity-purified on a monoclonal antibody12CA5 resin as described (21). The P11 D fraction (0.5-0.85

Abbreviations: TBP, TATA box-binding protein; TAF, TBP-associatedfactor; Hsp, heat shock protein; TdT, terminal deoxynucleotidyltrans-ferase; HA, hemagglutinin; EMSA, electrophoretic mobility-shift assay.tPresent address: Center for Cancer Research and Department ofBiology, Massachusetts Institute of Technology, Cambridge, MA02139.§To whom reprint requests should be addressed.

11864

Proc. Natl. Acad. Sci. USA 92 (1995) 11865

N

T21 0

K

208 R T T A L I F214

160 1hTBPNF

- 1. ++++ -N

FIG. 1. Structure of Arabidopsis thaliana TBP a-carbon chainbound to a TATA element (15). The DNA filling the interior concavesurface is not shown. Threonine-70, corresponding to threonine-210(T210) in human TBP, is shown as a side chain and in space-fillingmode. The position of the threonine-210 -> lysine substitution(T210K) in the first repeat (arrow) of the human TBP core domain(residues 160-339) is schematically indicated below the structure.

M KCl) was loaded on a Whatman DE52 column, and the0.1-0.3 M KCl fraction was used to immunopurify HA-taggedTFIID as above. Recombinant hexahistidine-tagged humanTBP was expressed in bacteria and purified on Ni2+_nitrilotriacetate (NTA) agarose as described (22) and thenfractionated on heparin-Sepharose with step elution at 0.2-0.5M KCl. Bacterially expressed Flag-tagged Gal4-VP16 waspurified as reported (23).

In Vitro Transcription and Electrophoretic Mobility-ShiftAssay (EMSA). RNA polymerase III transcription reactionsusing pVA1 as template (24) contained 18 ,ug of TBP-immu-nodepleted P11 B fraction, 6 ,ug of P11 C fraction (0.3-0.6M KCl), and 3 ,A (3 ng of TBP) of HA-TFIIIB complex. Heat

inactivation of TFIID in HeLa nuclear extracts, RNA poly-merase II transcription reactions, and primer extensionanalyses were performed as described (17, 25). EMSA wasperformed (10) with a 200-bp PCR probe containing theE1B TATA box from pG5-EIB-CAT (19) and with equalamounts of each TFIID complex as determined by silverstaining and Western blotting.

RESULTS AND DISCUSSIONTo address directly the contribution ofTBP-DNA interactionsin the context of a complete TFIID complex during transcrip-tion of different class II genes, we have introduced a pointmutation (T210K) that converts threonine-210 in the firstrepeat of the human TBP core domain to lysine (Fig. 1). Thecorresponding mutation in Saccharomyces cerevisiae TBP(Ti 12K) is dominant negative, prevents TBP from binding toa TATA element in vitro (26), and impairs transcription ofTATA-containing class II but not TATA-less class I or class IIIgenes (27). Threonine-210 directly contacts the DNA sugar-phosphate backbone near the middle of the lower strand of theTATA element (14, 15). Thus, the larger lysine side chain in themutant probably sterically interferes with stable DNA binding. Agene encoding a HA epitope-tagged human TBP carrying theT210K mutation was stably introduced into human HeLa cells.Nuclear extracts were prepared from cells expressing wild-typeHA-TBP and mutant HA-TBPT2l0K and were used to immuno-purify, respectively, the wild-type and mutant epitope-taggedTBP-TAF complexes TFIID and TFIIIB.We first ascertained that the human HA-TBPT2l0K mutant

protein expressed in HeLa cells is correctly folded by showingthat (i) the mutant TFIIIB complex can restore RNA poly-merase III transcription of the tRNA-like adenovirus VAIgene in a reconstituted TBP-dependent transcription system tolevels similar to those obtained with wild-type TFIIIB (Fig. 2A,compare lanes 2 and 3), (ii) all the major TAFs can associatewith TBPT210K into a stable TFIID complex (Fig. 2B), and (iii)TBPT2L0K binds TFIIA and TFIIB like wild-type TBP (data notshown).We verified that this mutation impairs TATA element

binding by human TBP in association with TAFs in a stableTFIID complex by using an EMSA. Wild-type (Fig. 2C, lanes2 and 3) but not mutant (lanes 4 and 5) TFIID generated a

BTFIID wt TFIID mt

0 1e 2 3' 0 1e 2 3°250 -

135115 -

TFIIIB 80

- mt wt 55_HA-TBPvwtImt: it ~~~43>

3130.

28

20 -

18 -

CTFIID

wt mt

_ -.__-O.t#

1 2 3 4 5

FIG. 2. TFIIIB and TFIID complexes containing TBPT210K, a mutant protein defective in TATA-box binding. (A) RNA polymerase IIItranscription of the adenovirus VAI gene was analyzed in a TBP-dependent system in the absence (lane 1) or presence of equal amounts of eitherwild-type (wt; lane 3) or mutant T210K (mt; lane 2) HA-tagged TFIIIB complex. VAI RNA is indicated. (B) Subunit composition ofimmunoaffinity-purified TFIID complexes containing wild-type HA-TBP (TFIID wt) and mutant HA-TBPT210K (TFIID mt). A silver-stained6-20% gradient SDS/polyacrylamide gel is shown. Lanes 0, 8 ,ul of the last buffer wash of the resin before TFIID elution; lanes 10, 2°, and 30, 8,ul each of consecutive TFIID elutions with the HA peptide. TAFs and their molecular mass (in kilodaltons) are indicated. A star indicates weaklystained or substoichiometric TAFs. (C) EMSA with increasing amounts of wild-type (wt; lanes 2 and 3) and T210K mutant (mt; lanes 4 and 5)TFIID complexes. A probe containing the adenovirus E1B TATA box was used. Arrow indicates the specific protein-DNA complex.

A

VAl -1

1 2 3

mI m m I AI

Biochemistry: Martinez et al..

I'm

.

11866 Biochemistry: Martinez et al..

A

Hsp7O -_

(TATAi)

B

TFIID wtC -

TFIID mtA

TFIIDwlt mt

1-Pol -_.

(TATA-)

I 2 J 4

1 2 3 4 5 6 B7 8 9

TFIID wt TFIID mt

TdT-

(TATA-)

1 2 3 4 5 6 7 8 9

- + Gal-VP1 6

TFIID TFIIDwt mt TBPwt mt TBP .

G5-TdT-i_

(TATA-)

G5-El B -_-

(TATA )

1 2 3 4 5 6 7 8

FIG. 3. Mutant TFIID(TBPT2I0K) supports basal and activator-mediated transcription from TATA-less but not TATA-dependentpromoters. (A) Transcription from a supercoiled human Hsp7O corepromoter was analyzed in normal (control, C; lane 1) and TFIID-inactivated (lanes 2-9) HeLa nuclear extracts. Reactions (lanes 2-9)were complemented with either bovine serum albumin (2 jig; lane 2),HA peptide (4 jig; lane 6), or increasing amounts of wild-type (wt)TFIID (4, 6, and 8 ng of TBP; lanes 3-5) or mutant (mt) TFIID (2, 3,and 4 ng of TBPT21oK; lanes 7-9). Specific transcripts (Hsp7O arrow)were detected by primer extension. (B) Transcription from a super-coiled TATA-less mouse TdT core promoter was analyzed as in A.Specific transcripts are indicated (TdT arrow). A bracket indicatesweak initiations upstream of the main + 1 start site. (C) Transcriptionfrom linear templates containing five Gal4 binding sites upstream ofthe TdT core promoter (G5-TdT) or the ElB TATA box (G5-ElB)was analyzed in a TFIID-inactivated nuclear extract complementedwith either bovine serum albumin (1 jig; lanes 1 and 8), wild-typeTFIID (2.5 ng of TBP; lanes 2 and 5), mutant TFIID (2.5 ng ofTBPT2lOK; lanes 3 and 6), or recombinant human hexahistidine-taggedTBP (20 ng of TBP; lanes 4 and 7). Reactions were performed in theabsence (lanes 1-4) or presence (lanes 5-8) of a 1 pmol of Flag-taggedGal4-VP16 (Gal-VP16). Specific transcripts are indicated by arrowsfor G5-TdT and G5-EIB. The bracket is as in B.

protein-DNA complex with a DNA probe containing theTATA box of the adenovirus E1B promoter. Similar results

+ Gal-VP16

TFIID TFIID- TBP wt mt - TBP wt mt

G51-Pol -_.

(TATA-)

1 .2.3456 7 8

FIG. 4. Activator-dependent function of mutant TFIID(TBPo210K)on the TATA-less ,B-polymerase promoter. (A) Basal transcriptionfrom the human ,B-polymerase core promoter (region -41 to +62) wasanalyzed as in Fig. 2, in the absence (lane 2) or presence of equivalentamounts (4 ng of TBP) of either wild-type (wt; lane 3) or mutant (mt,lane 4) TFIID. Control (C; lane 1), as in Fig. 2, shows the position ofspecific transcripts (,B-Pol arrow). (B) Activator-dependent transcrip-tion from a template containing five Gal4 sites upstream of the13-polymerase core promoter (region -41 to +59) was performed asabove, in the presence of either bovine serum albumin (0.5 jig; lanes1 and 5), hexahistidine-tagged human TBP (4 ng of TBP; lanes 2 and6), wild-type TFIID (wt, 1 ng of TBP; lanes 3 and 7), or mutant TFIID(mt, 1 ng of TBPT21OK; lanes 4 and 8) in the absence (lanes 1-4) orpresence (lanes 5-8) of 1 pmol of Flag-tagged Gal4-VP16. Position ofcorrectly initiated transcripts is shown (G513-Pol arrow and arrow-head). Transcription in the absence of activator was not detected withthis template and at the low level of. TFIID employed; the weaknonspecific band in lanes 1-6 is located above the position of thespecific transcripts and is resistant to low concentrations of a-amanitin(data not shown).

were obtained in DNase I footprinting assays in which wild-type but not mutant TFIID interacted with a TATA-containing promoter DNA fragment (T+I+ in Fig. 5) both inthe absence and in the presence of TFIIA (data not shown).We next examined the ability of mutant TFIID to support

transcription from TATA-containing and TATA-less class IIpromoters in a TFIID-inactivated nuclear extract (Fig. 3).Consistent with its deficiency in TATA-box binding, themutant TFIID complex did not support basal transcriptionfrom the TATA-dependent natural human Hsp7O core pro-moter (Fig. 3A, lanes 7-9) and the synthetic G5-E1B promoterthat contains the adenovirus ElB TATA box (Fig. 3C, lane 3).These results demonstrate that TFIID interactions with thegeneral transcription machinery cannot compensate for theDNA-binding deficiency of mutant TBPT21oK on these twoTATA-containing promoters. In contrast, mutant TFIID di-rected specific basal transcription from the TATA-less mouseTdT core promoter to levels similar to those obtained withequivalent amounts of wild-type TFIID (Fig. 3B, lanes 3-5 and7-9; Fig. 3C, lanes 2 and 3). Moreover, transcription initiationdirected by mutant TFIID was more specific for the + 1position of the TdT initiator element (Fig. 3 B and C, TdTarrow) as compared with transcription directed by wild-typeTFIID, which also directed weak initiation from TdT promoterpositions just upstream of the main + 1 initiation site (Fig. 3 Band C, brackets) and from flanking plasmid sequences fartherupstream (data not shown).

Because upstream activators can influence the binding ofTFIID to TATA-containing promoters (5-13), we tested

Proc. Natl. Acad. Sci. USA 92 (1995)

Proc. Natl. Acad. Sci. USA 92 (1995) 11867

TT-wt T+lwI T+-w T

TFIID : . wt mt . wt mt wt mt TXN

1 2 3 4 5 6 7 8 9

GAL4 SITES

T - I+INR

TATA INR

T TIIiTATA

FIG. 5. The activity of mutant TFIID(TBPT210K) is independent ofthe -30 DNA sequence but requires a functional initiator element.Transcription activities mediated by TFIID(TBPT2l0K) in the presenceof Gal4-VP16 of the templates T-I+, T+I+, and T+I- are compared.T-I+ contains five Gal4 sites upstream of the natural TATA-lessinitiator (INR)-containing region (-41 to +33) of the TdT promoter.T+I+ differs from T-I+ by the substitution of nucleotides in thenatural TdT -30 region to create a consensus TATAAAA element.T+I- differs from T+I+ by six base-pair substitutions in the initiatorelement that abolish initiator activity (17). Transcription reactionmixtures (as in Fig. 2) contained 1 pmol of Flag-tagged Gal4-VP16 andeither bovine serum albumin (0.5 ,tg; lanes 1, 4, and 7) or equalamounts (1.5 ng of TBP) of wild-type TFIID (wt, lanes 2, 5, and 8) ormutant TFIID (mt, lanes 3, 6, and 9). Specific transcripts (arrow) weredetected by primer extension using the same end-labeled primer for allthree constructs.

whether a functional TATA-binding domain in TFIID isrequired for the response to upstream activators in differentcore promoter contexts. We first analyzed activation by thechimeric Gal4-VP16 activator from templates containing fiveGal4 sites upstream of the EiB TATA box (G5-E1B) and ofthe TATA-less TdT core promoter (G5-TdT) in the presenceof either the wild-type or the TATA-binding-deficient TFIID(Fig. 3C). Whereas wild-type TFIID supported activation byGal4-VP16 from both templates (lane 2 versus lane 5), mutantTFIID mediated activation only of the G5-TdT promoter andto a level comparable to that of wild-type TFIID (lanes 5 and6). These results demonstrate that, as for basal transcription,activation by Gal4-VP16 from the G5-E1B template requiresa functional TATA-binding domain within TFIID. In contrast,neither a TATA box nor a functional TATA-binding domainis needed for TFIID to stimulate basal and Gal4-VP16 acti-vator-dependent transcription from the TdT promoter. Equiv-alent results were obtained with Gal4-Spl activator (data notshown). In addition, because mutant TFIID is required forbasal and activated transcription from both supercoiled (Fig.3B) and linear (Fig. 3C) TdT templates, the mechanismsinvolved differ from the recently described TBP-independentbasal RNA polymerase II transcription that requires the use ofsupercoiled promoters (28).We analyzed similarly the activity of mutant TFIID on the

TATA-less human f3-polymerase gene promoter (Fig. 4). Incontrast to the results obtained with the TdT promoter, themutant TFIID did not support basal transcription from the,B-polymerase core promoter (Fig. 4A, lane 4). This suggeststhat nonspecific TBP-DNA interactions contribute to theformation of a productive basal transcription initiation com-plex on the f3-polymerase promoter. Interestingly, however, ona template with five Gal4 sites located upstream of thef3-polymerase core promoter, wild-type and mutant TFIID

2

TBPmt

GTF

POLUA TA~~~~~~~~~~~~~~~~~~....... ... . _v

~~~~~~~~~~~~~~~~~~~~~~..............-T7 GTF

UA

TAT INR_ +++

FIG. 6. Summary and model for core promoter-specific functionof TATA-binding-defective TFIID. Line 1: On a TATA-containinginitiator-less promoter, wild-type TFIID (TBP wt) binds to theTATA element and interacts both with upstream activators, throughTAFs (21, 23, 29-33) and/or additional cofactors (33), and withcomponents of the general transcription machinery-i.e., generaltranscription factors (GTF) and RNA polymerase II (POL), todirect specific transcription (TXN). Depending on the promoter,TAFs may or may not interact (schematized with a double-headedarrow) with the initiatorless region downstream of the TATA box(10, 33, 34) in a manner that can be influenced by upstreamactivators (8-10). This is consistent with the ability of TAFs todirectly contact two DNA regions downstream of the initiator insome genes (35). Line 2: On this initiatorless promoter the TATA-binding-defective mutant TFIID (TBP mt) does not support tran-scription, suggesting that interactions of mutant TFIID with acti-vators and the rest of the transcription machinery are not sufficientfor its functional recruitment to this type of promoter. Lines 3 and4: In the presence of a functional initiator element, TATA-binding-defective TFIID directs a similar level of transcription in thepresence (line 4) or absence (line 3) of a consensus TATA element.This indicates that specific TBP mt-DNA interactions are largelydispensable (symbolized by a TBP mt that does not touch thepromoter) and are most likely compensated by the concertedinteractions of different TAFs with activators, the initiator, com-ponents of the general transcription machinery (GTF and POL), andpossibly other factors that recognize this complex and/or theinitiator element (X?); the latter may include previously describedinitiator-binding proteins (reviewed in ref. 17). The present data donot exclude the possibility of nonspecific low-affinity contactsbetween TBP mt and DNA in the preinitiation complex. Line 5: Inthe presence of wild-type TFIID (TBP wt), TATA and initiatorelements synergize to give maximal levels of transcription.

mediated similar levels of Gal4-VP16-dependent transcription(Fig. 4B, lanes 7 and 8; see also figure legend). This indicates

1-J1

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11868 Biochemistry: Martinez et al..

that upstream activators can compensate for the TATA-binding deficiency of mutant TFIID on the 13-polymerasepromoter but not on the G5-E1B promoter (Fig. 3C). To-gether, these results suggest that differences in core promotersequences are responsible for the differential activity of mu-tant TFIID on the TATA-containing Hsp7O and E1B andTATA-less TdT ,3-polymerase templates.To identify the core elements that bypass the requirement

for a functional TATA-binding surface in TFIID, we tested thecontribution of the TdT -30 and initiator regions for Gal4-VP16-activated transcription in the presence of the TATA-binding-deficient TFIID. Substituting a consensus TATA el-ement for the natural -30 region of the TdT core promoter(T+I+) strongly stimulated activated transcription mediated bywild-type TFIID (Fig. 5; lane 2 versus lane 5) but did not affecttranscription directed by mutant TFIID (lane 3 versus lane 6).This further confirms that mutant TFIID has no residualTATA DNA-binding activity and that the T210K mutation inTBP does not create a new DNA-binding surface specific forthe natural TdT -30 region. Significantly, in the presence ofa TATA box and of Gal4-VP16, mutation of the initiatorelement (T+I-) abolished transcription directed by mutantTFIID (lane 9 versus lane 6). These results demonstrate thatthe transcription activity of mutant TFIID is independent ofthe DNA sequence in the -30 region but requires a functionalinitiator element. Differences in TdT and ,B-polymerase initi-ator regions could thus explain the requirement of an activatorfor transcription mediated by mutant TFIID from the f3-poly-merase promoter (see above).We have not been able to detect significant binding of

wild-type or mutant TFIID to the TATA-less TdT promoter(T-I+) by.Nase I footprinting assays (data not shown). Incontrast, wild-type (but not mutant) TFIID binds efficiently tothe initiator-less T+I- template, producing a footprint thatextends from the TATA element to sequences downstream ofthe initiation site (data not shown). Thus, the similar tran-scription activity of T-I+ and T+I- templates (Fig. 5) does notcorrelate with the differential affinity of TFIID for thesepromoters. This suggests that the function of the initiator mayalso involve its recognition by other components of the tran-scription machinery (see legend of Fig. 6).

In conclusion, we have demonstrated that a functionalTATA-binding domain in TFIID is required for the activity ofTATA-containing initiatorless promoters but dispensable forspecific transcription from promoters containing an initiatorelement (Fig. 6). Since TAFs are essential for basal initiatorfunction (17, 18, 34) but dispensable for basal transcriptionfrom TATA-dependent promoters (2-4), TFIID thus containsat least two separable core promoter-specific functions-i.e.,TBP-TATA and TAF-initiator interactions (see legend of Fig.6)-that are variably required or dispensable depending on thecore promoter structure. Our results thus suggest the possi-bility of selective gene regulation by modulating distinct corepromoter-specific TFIID functions. This is consistent with theobserved core promoter-specific activity of some activatorsand repressors (36-41). Finally, since TBP binding to theTATA element induces a sharp DNA bend (14, 15), our resultsraise the possibility either that core promoter bending is notessential for transcription from some promoters or that bend-ing is achieved by a different mechanism and/or requiresadditional factors at TATA-less genes.

We thank S. Burley for comments on the manuscript and J. Huang andX. Zhang for excellent technical assistance. This work was supported byNational Institutes of Health grants to R.G.R. and A.J.B. and by general

support from the Pew Trusts to the Rockefeller University. E.M. wassupported by a postdoctoral fellowship from the Swiss National ScienceFoundation; Q.Z. and N.D.L. were supported by a National Institutes ofHealth grant to A.J.B., and T.O. was supported by a postdoctoralfellowship from the Deutsche Forschungsgemeinschaft.

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