THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 267, No. 14, Issue of May 15, pp. 10142-10148,1992

Printed in U. S. A.

Mechanism of Assembly of the RNA Polymerase I1 Preinitiation Complex TRANSCRIPTION FACTORS 6 AND e PROMOTE STABLE BINDING OF THE TRANSCRIPTION APPARATUS TO THE INITIATOR ELEMENT*

(Received for publication, October 25, 1991)

Joan Weliky Conaway, John N. Bradsher, and Ronald C. Conaway From the Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

Assembly of RNA polymerase I1 with the core region of TATA box-containing promoters requires the action of the TATA factor and four transcription factors des- ignated a, Br, 6, and C, which have each been purified to near homogeneity from rat liver. Evidence from previous studies argues that a and play a crucial role in delivering RNA polymerase I1 to the promoter (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sei. U. S. A. 88, 6205-6209). Here we describe the interaction of transcription factor 6 with preinitiation intermediates assembled in the presence of either recombinant yeast TFIID or the high molecular mass, endogenous TATA factor T from rat liver (Conaway, J. W., Hanley, J. P., Garrett, K. P., and Conaway, R. C. (1991) J. Biol. Chern. 266,7804-7811). Results of template challenge experiments argue that 6 enters the preinitiation com- plex through interactions with multiple components of the transcription apparatus. We observe that, in the presence of recombinant TFIID, 6 interacts stably with the preinitiation complex only in the presence of RNA polymerase 11, a, and Br, whereas, in the presence of T ,

6 is capable of interacting stably with the Initial Com- plex independently of RNA polymerase 11. Results of restriction site protection experiments reveal that 6 and c promote binding of the transcription apparatus to the Initiator element and support the model that RNA polymerase I1 assembles at the core promoter in at least two discrete steps, first “touching down” near the TATA element and finally extending its interaction downstream to encompass the cap site.

Initiation of messenger RNA synthesis is a key control point in the expression of many eukaryotic genes. Evidence from biochemical studies indicates that initiation by RNA polymerase I1 from the core region of TATA box-containing promoters is a complex, multistage process requiring the action of at least five accessory initiation factors and an ATP cofactor (1, 2).

In the first committed step in assembly of the active prein- itiation complex, the TATA factor binds stably to the core promoter to form an Initial Complex, which serves as the recognition site for RNA polymerase I1 on the DNA. Although biochemical purification of a “native” or “endogenous” TATA factor from higher eukaryotes has not yet been achieved,

* This work was supported by Grant GM41628 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

TATA factors have been identified in cell extracts from a variety of species including Drosophila melanogaster, rat, and man, and a yeast TATA factor, designated yTFIID or BTFlY, has been purified and cloned (3-10). The recombinant yeast TATA factor was found to substitute for partially purified preparations of the endogenous TATA factor from higher eukaryotes in reconstituted transcription reactions in vitro. cDNA clones encoding homologous transcription factors have been isolated from several higher eukaryotes, including Dro- sophila melanogaster (11, 12), Arabidopsis thulianu (13), and man (14-16). Whereas the recombinant TATA factors range in size from 22 kDa (Arabidopsis) to 38 kDa (Drosophila and human), the partially purified, endogenous TATA factors for which they substitute are considerably larger: the rat factor has an apparent native molecular mass of -750 kDa (17, 18), the Drosophila factor has been reported to be either -100 kDa (11) or larger than 300 kDa (19), and the human factor has been reported to be as small as 120-140 kDa (20) and as large as 17 S (21).

While studies performed with recombinant TATA factors have provided considerable insight into the mechanism of transcription initiation by RNA polymerase 11, a growing body of evidence suggests that the recombinant TATA factors are functionally as well as structurally distinct from their endog- enous counterparts. Results of in vitro transcription (18, 22- 24) and DNAse I footprinting (6, 7, 11, 12, 16, 25, 26) studies argue that the endogenous TATA factors interact with a larger region of the core promoter than the recombinant factors. In addition, several recent reports argue that the endogenous form of the TATA factor includes subunits essential for transcriptional activation by certain DNA-bound regulatory proteins (19, 27, 28).

The molecular mechanism by which RNA polymerase I1 binds productively to the Initial Complex has not yet been firmly established. Until recently, lack of purified, reconsti- tuted transcription systems has hindered detailed biochemical studies of this process. In an effort to overcome this limitation, we have developed a defined transcription system composed of RNA polymerase I1 and a set of accessory transcription factors purified from rat liver. In this system, productive binding of RNA polymerase I1 at the core region of TATA box-containing promoters is controlled by the action of the TATA factor and four additional transcription factors desig- nated a (29), Pr (30), 6 (31), and t (18). Evidence obtained from a combination of restriction site protection and template challenge experiments argues that a and represent the minimal set of factors necessary for selective binding of RNA polymerase I1 to the Initial Complex (17,32-34). In an earlier study, we observed that /?r suppresses nonselective binding of RNA polymerase I1 to free DNA, much as Escherichia coli

10142

Assembly of the RNA Polymerase 11 Preinitiation Complex 10143

d o suppresses binding of E. coli RNA polymerase to non- promoter sites in DNA (33). Br may therefore enhance the specificity with which RNA polymerase I1 recognizes and binds to the Initial Complex, in part by preventing nonpro- ductive interactions of polymerase and free DNA and, in concert with transcription factor a, increasing the affinity of the enzyme for the Initial Complex. a and Pr have each been purified to apparent homogeneity (29,30) and are most likely homologues of human transcription factors designated TFIIB (35) and RAP30/74(TFIIF) (36, 37), respectively.

In addition to a and By, assembly and subsequent ATP- dependent activation of the complete preinitiation complex in the reconstituted liver system requires transcription factors 6 and t. t has been purified to apparent homogeneity from rat liver (18) and is most likely homologous to human transcrip- tion factor IIE (38, 39). 6 , which was previously purified substantially from rat liver nuclear extracts, was found to have a closely associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the core regions of the adeno- virus 2 major late and mouse interleukin-3 promoters (31). Previous findings indicate that 6 and t function directly and stoichiometrically in formation of the complete preinitiation complex and, in concert with a, Br, and the TATA factor, promote formation of stable protein-DNA contacts that an- chor the transcription apparatus at the core promoter (32, 34).' We have extended these observations and, in this report, describe the interaction of 6 with preinitiation intermediates assembled in the presence of either recombinant yeast TFIID or the high molecular mass, endogenous TATA factor T from rat liver. Our results reveal that interaction of 6 with the preinitiation complex depends on the nature of the TATA factor. We observe that, in the absence of RNA polymerase 11, a, and &, 6 interacts stably with Initial Complexes con- taining T but not with those containing recombinant TFIID. Furthermore, our findings indicate that 6 , acting in concert with transcription factor t, is essential for stable interaction of the transcription apparatus with the cap site/Initiator region of the adenovirus 2 major late promoter. We suggest that these results are consistent with the model that RNA polymerase 11 recognizes and binds to the Initial Complex by a two-step mechanism, in which the enzyme, assisted by a and By, first interacts with the Initial Complex at sites up- stream of the cap site near the TATA element and, finally in a step dependent on 6 on t, interacts stably with the cap site/ Initiator region of the promoter to form the complete preini- tiation complex.

EXPERIMENTAL PROCEDURES

Materials-Male Sprague-Dawley rats (200-300 g) were from Sasco. Unlabeled ultrapure ribonucleoside and deoxynucleoside 5'- triphosphates were purchased from Pharmacia LKB Biotechnology Inc. [u-~'P]CTP (>800 Ci/mmol) and [cY-~'P]~ATP (>800 Ci/mmol) were from ICN. Polyvinyl alcohol, type 11, and heparin were obtained from Sigma. Recombinant RNasin was from Promega, and bovine serum albumin (reagent grade) was obtained from ICN Immunobio- logicals. Phenylmethylsulfonyl fluoride was obtained from Sigma and was dissolved in dimethyl sulfoxide to 1 M. Antipain and leupeptin were purchased from Boehringer Mannheim and were dissolved in water to 25 mg/ml and stored frozen. Glycerol (spectranalyzed) was from Fisher. Polyethyleneimine-cellulose sheets were purchased from Brinkman.

Buffers-Phosphocellulose (P11) was obtained from Whatman. Ultrogel AcA 34 was from IBF Biotechnics. 4000SW Spherogel TSK and Spherogel TSK DEAE-5-PW were from Beckman Instruments. Bio-Gel TSK phenyl-5-PW and TSK SP-5-PW were purchased from

J. W. Conaway, unpublished results.

Bio-Rad. All HPLC' was performed using a Beckman System Gold" Chromatograph. Buffer J was 20 mM Hepes-NaOH, pH 7.9, 0.1 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol.

Purification of Transcription Factor &Except for the following modifications, transcription factor 6 was purified from the livers of 200 male Sprague-Dawley rats as previously described (31). Fraction 111 (phosphocellulose fraction) was further purified by Ultrogel AcA 34 gel filtration. Following dilution with an equal volume of 20 mM Hepes-NaOH, pH 7.9, 0.1 mM EDTA, 1 mM DTT, Fraction I11 was precipitated by the slow addition of solid (NH4)'S04 (0.33 g/ml). The resulting precipitate was collected by centrifugation at 15,000 X g for 50 min, dissolved to a final volume of 10 ml in buffer J containing 10 pg/ml antipain and 10 pg/ml leupeptin, and dialyzed against buffer J to a conductivity equivalent to that of 0.5 M (NH4)'S04. The resus- pension was then centrifuged at 15,000 X g for 20 min and applied to a 2.6- X 100-cm Ultrogel AcA 34 column equilibrated in buffer J containing 0.4 M KC1. The column was eluted at -20 ml/h, and 10- ml fractions were collected. The active fractions were pooled (Fraction IV) and further purified on consecutive TSK phenyl-5-PW, TSK DEAE-5-PW, and TSK SP-5-PW HPLC columns as previously described (31).

Preparation of RNA Polymerase II and Transcription Factors- Transcription factors a (29) and /3r (30) were purified from cytosol as previously described. RNA polymerase I1 (33) and transcription factors e (18) and T (17) were purified from nuclear extracts as described. Recombinant yeast TFIID was expressed and purified as described (18) from bacterial strain N5151 containing the plasmid pASY2D (10).

Assay of Runoff Transcription-Except as indicated in the figure legends, assays were performed as described (29) with -200 ng of NdeI-digested pDN-AdML (40) or pN4 (41) and approximately 2 ng of a (fraction V), 10 ng of /3r (fraction V), 40 ng of 6 (fraction VI), 20 ng of e (fraction V), 60 ng of T (fraction V) or 50 ng of recombinant yeast TFIID (AcA 44 fraction), and 0.003 unit of RNA polymerase 11. Transcription was initiated by the addition of 50 p~ ATP, 50 p M UTP, 10 p~ CTP, 10 pCi of [~I-~'P]CTP, and 7 mM MgC12. After 3 min, heparin and GTP were added to 10 pg/ml and 50 p ~ , respec- tively, and reaction mixtures were incubated for 30 min. Runoff transcripts were analyzed by electrophoresis through 6% polyacryl- amide, 7 M urea gels. Transcription was quantitated by densitometry of autoradiograms with an LKB Ultroscan XL laser densitometer. In the experiments of Figs. 3-6 and Fig. 8, the relative molar amounts of full-length runoff transcripts synthesized from the AdML pro- moters on pDN-AdML and pN4 were calculated by determining the area under the appropriate peaks after subtracting an appropriate background. Peak areas were corrected for the lengths of the tran- scripts.

Assay of dATPase-Reaction mixtures (20 pl) contained 40 mM Tris-HC1, pH 7.9, 7 mM MgC12, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mg/ml bovine serum albumin, 2% (v/v) glycerol, 5 p~ dATP, 1 pCi of [a-32P]dATP, and 250 ng of a 60-base pair double- stranded oligonucleotide, designated Ad(-50 to +lo), which contains the core region of the AdML promoter (40). Reactions were performed at 28 "C. dATPase was measured by polyethyleneimine-cellulose thin layer chromatography (42).

Sucrose Gradient Sedimentation-Sedimentation was carried out in 2-ml linear sucrose gradients (15-30% (v/v)) containing 20 mM Hepes-NaOH, pH 7.9, 1 mM EDTA, 1 mM DTT, and 0.4 M KCl. Centrifugation was performed at 55,000 rpm and 4 "C in the TLS55 rotor of a Beckman TLlOO ultracentrifuge. Fractions (2 drops) were collected from the bottom of tubes through a 20-gauge needle.

RESULTS

Properties of Transcription Factor 6-In a previous report, we described the identification and extensive purification of transcription factor 6 from rat liver. The most highly purified preparations of 6 reproducibly contained a set of polypeptides ranging in size from 94 kDa to 35 kDa (31). In addition, we showed that 6 co-purifies closely with DNA-dependent ATP- ase(dATPase) activity when analyzed by hydrophobic inter- action and cation and anion exchange HPLC (31). Using a

' The abbreviations used are: HPLC, high pressure liquid chro- matography; DTT, dithiothreitol; Hepes, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid; SDS, sodium dodecyl sulfate; AdML, ade- novirus 2 major late.

10144 Assembly of the RNA Polymerase I I Preinitiation Complex

modification of the previous purification procedure, we have now confirmed the association of these polypeptides with both transcription and ATPase(dATPase) activities; moreover, we have shown that DNA-dependent ATPase activity co-sedi- ments with the transcription activity of 6 during sucrose gradient analysis of our most highly purified preparations of 6.

6 was assayed by its ability to reconstitute synthesis of accurately initiated runoff transcripts from the core region of the AdML promoter in the presence of saturating amounts of RNA polymerase 11, the TATA factor (either the endogenous TATA factor 7 or recombinant yeast TFIID), and transcrip- tion factors a, By, and t. 6 was purified from a 0.33 M ammonium sulfate extract of crude rat liver nuclei by am- monium sulfate fractionation, followed by chromatography on successive phosphocellulose, Ultrogel AcA 34, TSK phenyl-5-PW, TSK DEAE-5-PW, and TSK SP-5-PW col- umns. As shown in Fig. 1, a set of polypeptides with apparent molecular masses of approximately 94, 85, 68, 46, 43, 40, 38, and 35 kDa co-purifies with transcription and dATPase ac- tivities when 6 is analyzed by TSK SP-5-PW HPLC. As estimated by densitometry of silver-stained SDS-polyacryl- amide gels, these polypeptides comprise 80-90% of the protein in the most active fractions (Fig. 1, p a w l C, see fractions 35 and 36). Thus far, we have been unable to assign transcription and DNA-dependent ATPase(dATPase) activities to specific polypeptides by renaturing and recombining them. Because 6 has not yet been reconstituted from isolated polypeptides, it is not clear whether each polypeptide is unique or whether some are derived from others by proteolysis. Gerard et al. (43)

A - f I TSK SP S-PW

Q I - 4 1

0.5 P

0.3 ’ 0.1 a

10 20 30 L O 50 D

300 f 200 z I

100 - E

o x u

Fraction Number

C

31

FIG. 1. Polypeptide composition of purified 6. Runoff tran- scription assays (with 100 ng of NdeI-digested pDN-AdML as tem- plate and with recombinant yeast TFIID as the TATA factor), dATPase assays, and TSK SP-5-PW HPLC of 6 were all performed as described under “Experimental Procedures.” Panel A, elution profile of 6 during analytical chromatography on TSK SP-5-PW. AdML Runoff Transcript refers to the relative synthesis, per tran- scription reaction, of runoff transcripts synthesized from the AdML promoter in pDN-AdML (expressed in arbitrary units determined by densitometry of autoradiograms). The amount of AdML runoff tran- script synthesized in this experiment was determined by densitometry of the autoradiogram shown in panel B. Panel C, aliquots of TSK SP-5-PW column fractions were analyzed by 8% SDS-polyacrylamide gel electrophoresis, and protein was visualized by silver staining.

have recently reported purification of a HeLa cell transcrip- tion factor, designated BTF2, which is associated with mul- tiple polypeptides ranging in size from 90 to 35 kDa; the possibility that this factor has associated DNA-dependent ATPase activity has not been addressed. I t is noteworthy that yeast RNA polymerase I1 transcription factor b, which also co-purifies with multiple polypeptides in a similar size range (44), is associated with DNA-dependent ATPase activity as well as with kinase activity specific for the C-terminal repeat of RNA polymerase I1 (45).

Purified 6 has a sedimentation coefficient of -12 S when measured by sucrose gradient sedimentation in 0.4 M KC1 (Fig. 2). 6 has a Stokes radius of -80 A when measured by Ultrogel AcA 34 gel filtration in 0.4 M KC1 (data not shown). Assuming a partial specific volume of 0.725 ml/g, 6 has an apparent native molecular mass, calculated from these data by the method of Siege1 and Monty (46), of -390 kDa.

Interaction of 6 with Preinitiation Intermediates Assembled in the Presence of Recombinant Yeast TFIID-We previously used an assay that couples restriction site protection with runoff transcription to probe the interactions of RNA polym- erase 11, recombinant yeast TFIID, and transcription factors a, By, 6, and e with the AdML core promoter during assembly of the complete preinitiation complex (34). Restriction en- zymes that cleave the AdML promoter at sites upstream and downstream of the Initiator element (diagrammed in Fig. 7A) were used to monitor binding of the transcription apparatus to the promoter. Briefly, we observed that, although complete protection of restriction sites a t positions -12 (HinP1) and +15 (XbaI) requires RNA polymerase 11, yeast TFIID, and all four liver factors, a, By, 6, and e , partial protection of the site a t -12 could be achieved in the presence of only RNA polymerase 11, yeast TFIID, a, and By. These and other (17, 32,33) findings were consistent with the model that transcrip- tion factors a and By promote selective binding of RNA polymerase I1 to the Initial Complex and that conversion of this intermediate to the complete preinitiation complex re- quires 6 and t .

Although these results argued that 6 is capable of function- ing a t some stage after RNA polymerase I1 binds to the preinitiation complex, they did not rule out the possibility that 6 can also interact stably with the preinitiation complex at an earlier stage. To establish the requirements for inter-

Thyro c(

- 12 14 16 18 20 2 2 2 4 26 28 30

Fraction Number

7

32

FIG. 2. Sucrose gradient sedimentation of transcription fac- tor 6. Runoff transcription assays (with 100 ng of NdeI-digested pDN-AdML as template and with recombinant yeast TFIID as the TATA factor), dATPase assays, and sucrose gradient sedimentation of 6 were all performed as described under “Experimental Proce- dures.” In this experiment, an 80-pl aliquot of 6 from fraction 34 of the TSK SP-5-PW fractionation shown in Fig. 1 was made 0.5 mg/ ml in bovine serum albumin and sedimented as described. The stand- ards for sedimentation were thyroglobulin, 19.2 S (Thyro), and aldo- lase, 8.3 S (Aldo).

Assembly of the RNA Polymerase 11 Preinitiation Complex 10145

action of 6 with the preinitiation complex, we used a modifi- cation of the template challenge assay originally developed by Kadesch et al. (47). Assays were performed as follows. Two different plasmids, each containing the core region of the AdML promoter, were used as templates for runoff transcrip- tion. As diagrammed in Fig. 3, template I was pDN-AdML (40), linearized with NdeI at a site 254 base pairs downstream of the cap site, and template I1 was pN4 (41), linearized with NdeI at a site 340 base pairs downstream of the cap site. During the first preincubation, templates I and I1 were incu- bated, in separate reaction mixtures, with the recombinant TATA factor and various combinations of 6, RNA polymerase 11, and the other initiation factors. Following this incubation, the two reaction mixtures were combined to begin preincu- bation 2, and, where necessary, additional factors were added to allow formation of complete preinitiation complexes. In the control reaction, 6, RNA polymerase 11, a, and 0-y were all added to reaction mixtures after the two templates were mixed to begin preincubation 2. At the conclusion of prein- cubation 2, transcription was initiated by the addition of ATP, CTP, UTP, and magnesium. After 3 min, heparin was added to prevent further initiation events, and GTP was added to allow synthesis of runoff transcripts. The relative molar amounts of full-length transcripts synthesized from the promoters on each template was determined by densitometry and expressed in the figure as the molar ratio (II/I) of tran- scription from templates I1 and I.

If 6 is capable of interacting stably and stoichiometrically with preinitiation intermediates on template I1 during the first preincubation, the molar ratio (II/I) of runoff transcripts synthesized on the two templates should be greater than that of the control. On the other hand, if 6 interacts catalytically, or not at all, with intermediates on template I1 during the first preincubation, 6 should then be able to interact with or distribute onto both templates during preincubation 2, and the molar ratio of transcription from the two templates should be roughly equivalent to that obtained in the control reaction.

As shown in Fig. 3, in the presence of RNA polymerase 11, a, Pr, and e, 6 interacts stably and stoichiometrically with the preinitiation complex. Stable interactions of 6 with preinitia-

Rotwins

I I Prancubation ! Rancubotion 2

30’ 30’

FIG. 3. Interaction of 8 with the preinitiation complex de- pends on RNA polymerase 11, a, and & when recombinant yeast TFIID serves as the TATA factor. Runoff transcription reactions were performed as described under “Experimental Proce- dures” and as diagrammed in the figure, except that (i) all preincu- bations were carried out at 60 mM KC1 and (ii) a subsaturating amount of 6 (-8.0 ng) was included in reaction mixtures. The relative molar amounts of full-length runoff transcripts synthesized from the promoters on each template is expressed as the molar ratio (II/I) of transcription from templates I1 and I. POL, RNA polymerase 11; yZZD, recombinant yeast TFIID; h p , heparin.

tion intermediates were not detected, however, when either RNA polymerase 11, a and P-y, or all three were omitted from the first preincubation. Since it has been shown previously that a and P-y are required for binding of RNA polymerase I1 to the Initial Complex, these results argue that, in the pres- ence of recombinant yeast TFIID, 6 does not participate in the formation of stable intermediates at the promoter prior to the binding of RNA polymerase 11.

We previously observed that both 6 and t are required for complete protection of transcription intermediates from at- tack by restriction enzymes that cleave the AdML core pro- moter at sites upstream and downstream of the cap site/ Initiator region. The template challenge assay was used to determine whether 6 interacts with preinitiation intermedi- ates containing recombinant yeast TFIID in the absence of t.

As shown in Fig. 4, the molar ratio (II/I) of transcription from templates I1 and I differed significantly from that of the control only when t was present with 6, yeast TFIID, a, P-y, and RNA polymerase I1 during preincubation 1. Thus, an interaction of 6 with the preinitiation complex was detected only when t was present. In addition, we observed that inter- action of t with preinitiation intermediates assembled in the presence of the recombinant TATA factor depends strongly on RNA polymerase 11, a, and P-y (Fig. 5), consistent with results obtained previously in studies of HeLa cell transcrip- tion systems (39,48). As shown in Fig. 6, interaction of e with the preinitiation complex is greatly stabilized by the presence of 6. Thus, in the presence of recombinant yeast TFIID, neither 6 nor t promotes formation of stable intermediates at the promoter prior to the binding of RNA polymerase 11; moreover, stable interactions of both 6 and t with the preini- tiation complex are strongly dependent on the presence of the other factor.

Transcription Factors 6 and t Promote Stable Binding of the

I Reincubalion 1 1 R.insubation 2 I I I n/~ I t , ”

L - I

Proteins

” FIG. 4. Interaction of 8 with the preinitiation complex de-

pends on t when recombinant yeast TFIID serves as the TATA factor. Runoff transcription reactions were performed as described under “Experimental Procedures” and as diagrammed in the figure, except that all preincubations were carried out at 60 mM KC1 and a subsaturating amount of 6 (-8.0 ng) was included in reaction mix- tures. The relative molar amounts of full-length runoff transcripts synthesized from the promoters on each template is expressed as the molar ratio (II/I) of transcription from templates I1 and I. The AdML core promoters in pN, and pDN-AdML are transcribed with similar efficiencies in vitro (22); the apparent difference in promoter strength in this experiment is not reproducible (see Figs. 3, 5, 6, and 8) and most likely results from differences in the concentrations of func- tional templates in the two plasmid preparations used in this set of reactions. pol, RNA polymerase II; yZZD, recombinant yeast TFIID; h p , heparin.

10146 Assembly of the RNA Polymerase 11 Preinitiation Complex

Protoins

Protoim t ,,,

Proincubatin 1 Roincubati 2

3 0 3 0 FIG. 5. Interaction of 6 with the preinitiation complex de-

pends on RNA polymerase 11, a, and By when recombinant yeast TFIID serves as the TATA factor. Runoff transcription reactions were performed as described under “Experimental Proce- dures’’ and as diagrammed in the figure, except that all preincubations were carried out a t 60 mM KC1 and a subsaturating amount of t (-5.0 ng) was included in reaction mixtures. The relative molar amounts of full-length runoff transcripts synthesized from the promoters on each template is expressed as the molar ratio (II/I) of transcription from templates I1 and I. POL, RNA polymerase 11; yIID, recombinant yeast TFIID; hep, heparin.

Roteins

Rotein8

t CTP GTP VTP

R O t . i n 8 w*+

1 m I Roincubotion 1 Roincubotion 2

30 30

FIG. 6. Interaction of t with the preinitiation complex de- pends on 6 when recombinant yeast TFIID serves as the TATA factor. Runoff transcription reactions were performed as described under “Experimental Procedures” and as diagrammed in the figure, except that all preincubations were carried out a t 60 mM KC1 and a subsaturating amount of c (-5.0 ng) was included in reaction mix- tures. The relative molar amounts of full-length runoff transcripts synthesized from the promoters on each template is expressed as the molar ratio (II/I) of transcription from templates I1 and I. POL, RNA polymerase 11; yIID, recombinant yeast TFIID; hep, heparin.

Transcription Apparatus to the Initiator Element-In earlier restriction site protection experiments (34), we observed that transcription factors 6 and t are required for complete protec- tion of sites both upstream and downstream of the AdML Initiator element (49). To determine whether 6 and t are required for interactions of the transcription apparatus with the Initiator element itself, we probed intermediates in assem- bly of the complete preinitiation complex with restriction endonuclease MboII, which has recognition and cleavage sites that fall within the Initiator (Fig. 7A) . As shown in Fig. 7B, synthesis of detectable runoff transcripts from the AdML promoter was abolished by treatment of the template with MboII prior to addition of RNA polymerase I1 and transcrip- tion factors. In contrast, runoff transcripts of the expected length (155 nucleotides; an additional MboII cleavage site is

A

Inrnawuerrsnt

Added btoro MboII digestion

,$I- + + + + ‘ yno L : - + + + + o(

5 - + + + + p r CD

q - + - + + E P - + + + - pol

- a * * ..: 4251 nt

P - + + - + 6

II 4 155 nt

1 2 3 6 5 6

FIG. 7. 6 and c promote binding of the transcription appa- ratus to the Initiator element. Panel A, sequence of AdML core promoter showing positions of recognition and cleavage sites of MboII. Panel B, runoff transcription reactions were performed as described under “Experimental Procedures,” except that 7 mM MgCL was included in all incubations. Each reaction mixture contained 100 ng of NdeI-digested pDN-AdML as template and recombinant yeast TFIID as the TATA factor. The template was incubated with RNA polymerase I1 and factors, as indicated, before digestion for 30 min with 5 units of MboII. After digestion, the remaining components were added, and the reaction was allowed to proceed. The positions of the 254- and 155-nucleotide runoff transcripts from the AdML promoter are indicated by arrows. A weak band with an electropho- retic mobility slightly greater than that of the AdML runoff transcript in lane 1 is seen in lanes 2-6. This approximately 250-nucleotide transcript most likely results from end to end transcription of a small MboII fragment. pol, RNA polymerase II; yZID, recombinant yeast TFIID; nt, nucleotides.

located 155 nucleotides downstream of the AdML start site) were synthesized when complete preinitiation complexes were preassembled at the core promoter prior to treatment with MboII. Both transcription factors 6 and t were required to prevent inhibition by MboII. 6 and t, therefore, play an important role in directing the transcription apparatus to bind tightly at the Initiator element.

Interaction of 6 with Preinitiation Intermediates Assembled in the Presence of the Endogenous TATA Factor ?-AS part of our overall characterization of transcription factor 6, we used the template challenge assay to assess its ability to interact with preinitiation intermediates assembled in the presence of the endogenous TATA factor T. As diagrammed in Fig. 8, Initial Complexes were preassembled (17) on tem- plate I (pDN-AdML) and template I1 (pN4). 6 was then preincubated with Initial Complexes in the presence or ab- sence of RNA polymerase I1 and transcription factors a and 87, the two templates were mixed, and the remaining tran- scription factors and ribonucleoside triphosphates were added to commence RNA synthesis. In contrast to the results of similar experiments performed using recombinant TFIID as the TATA factor (Fig. 3), stable interactions of 6 with tem- plates containing Initial Complexes assembled in the presence of 7 could be detected even in the absence of RNA polymerase 11, a, and 87 (Fig. 8).

The restriction site protection assay was also used to probe the interaction of 6 with preinitiation intermediates when transcription factor 7 serves as the TATA factor. Results obtained under these conditions were very similar to those obtained previously (34) when recombinant TFIID was used as the TATA factor. As shown in Fig. 9, partial protection from inhibition by HinPl was observed when templates were

Assembly of the RNA Polymerase 11 Preinitiation Complex 10147

tl- I b

I a.0 I.DOI P \ 0 . O X . P O l P I

I -

0.5

20 20' t

tenplate I t

7. F pr0tSlns 20 3 30 ,

ATP hep CTP GTP

t t

t t UTP terrp1ate P

T.C Protsinr b"

FIG. 8. 6 interacts with the Initial Complex when transcrip- tion factor T serves as the TATA factor. Runoff transcription reactions were performed as described under "Experimental Proce- dures" and as diagrammed in the figure, except that a subsaturating amount of 6 (-8.0 ng) was included in reaction mixtures. The relative molar amounts of full-length runoff transcripts synthesized from the promoters on each template is expressed as the molar ratio (II/I) of transcription from templates I1 and I. pol ZZ, RNA polymerase 11; hep, heparin.

Addod Boforo RE Digortion

a + - + - € + + + + + + + - I r + + + + + + '

" - p s ' : " + - + + +

P o l l [ + + + + + + 7 H X 'H X"H X ' H X H X H X ."

" ."""

2% ntl

181 nt I

1 2 3 4 5 6 7 0 9 1 0 1 1 1 2

FIG. 9. 6 regulates binding of the transcription apparatus to the core promoter when transcription factor 7 serves as the TATA factor. Runoff transcription reactions were performed as described under "Experimental Procedures," except that 7 mM M&12 was included in all incubations. Each reaction mixture contained 100 ng of NdeI-digested pDN-AdML as template and transcription factor T as the TATA factor. The template was incubated with RNA polymerase I1 and factors, as indicated, before digestion for 30 min with 5 units of the indicated restriction enzyme. After digestion, the remaining components were added, and the reaction was allowed to proceed. RE, restriction enzyme; polZZ, RNA polymerase II; H, HinP1; X , XbaI; nt, nucleotides.

preincubated with 7 , t, RNA polymerase 11, a, and By prior to treatment with restriction enzyme, whereas full protection from inhibition by either HinPl or XbuI also requires 6.

Taken together, the results of template challenge and re- striction site protection experiments suggest that there is no obligatory pathway for entry of 6 into the preinitiation com- plex when 7 directs formation of the Initial Complex. The template challenge data indicate that stable interaction of 6 with preinitiation intermediates formed in the presence of 7

can occur prior to entry of RNA polymerase I1 into the preinitiation complex. Nonetheless, results of restriction site protection experiments clearly indicate that 6 is capable of entering the preinitiation complex after RNA polymerase 11, a, and By have assembled with the Initial Complex to form an intermediate resistant to attack by HinP1.

DISCUSSION

We have investigated steps in assembly of the active RNA polymerase I1 preinitiation complex in a highly purified, re- constituted transcription system from rat liver. In addition to the TATA factor, productive binding of RNA polymerase I1 at the core region of TATA box-containing promoters in this system requires the action of four transcription factors des- ignated a, By, 6, and e. In this report, we have addressed the role of transcription factor 6 in preinitiation complex forma- tion.

In agreement with observations made in HeLa and Dro- sophila K, cell transcription systems, the first committed step in assembly of the complete preinitiation complex is binding of the TATA factor to the core promoter to form an Initial Complex (25, 26, 48, 50-54). Evidence suggests that RNA polymerase 11, with the assistance of a and Py, can locate and bind selectively to the Initial Complex to form an intermedi- ate, referred to as the "site-selected" complex (Fig. 10, inter- mediate I), which partially protects from digestion by restric- tion enzymes a region of the AdML core promoter upstream of the Initiator element (34). In the presence of 6 and e, the site-selected intermediate is converted to the complete prein- itiation complex (34). Formation of the complete complex results in full protection of restriction sites upstream of the Initiator element and in extension of the protected region to include sites downstream of the Initiator element. These results support the model that RNA polymerase I1 and its accessory factors assemble with the Initial Complex in at least two discrete steps, first interacting with the TATA factor and with promoter sequences between the TATA and Initiator elements and, finally, in a step dependent on 6 and t, extend- ing their interactions downstream to encompass the site of transcription initiation.

Exactly how 6 and e promote assembly of the complete preinitiati6n complex is unknown. It is possible, for example, that 6 and e function by an allosteric mechanism, inducing the extended restriction site protection pattern characteristic of the complete preinitiation complex solely through protein- protein contacts that lead to conformational changes in RNA polymerase I1 or other components of the transcription ap- paratus (Fig. 10, intermediate IIA). Alternatively, it is possible

n .POL It .Br I

nrt, Factor

I I

IIA w FIG. 10. Two possible models for interaction of 6 and c with

preinitiation complexes assembled at the AdML core pro- moter. POL ZZ, RNA polymerase 11: Znr, Initiator element. The start site of transcription is indicated by an arrow.

10148 Assembly of the RNA Polymerase 11 Preinitiation Complex

that the altered protection pattern results at least in part from protein-DNA contacts between the core promoter and 6 , c, or both factors (Fig. 10, intermediate IIB). The observa- tion that 6 possesses an associated DNA-dependent ATP- ase(dATPase) activity strongly stimulated by the core region of the AdML promoter (31) is consistent with the possibility that 6 may interact directly with promoter DNA. It seems unlikely, however, that 6 or c is solely responsible for protec- tion of promoter sequences near the cap site, since, presum- ably, RNA polymerase I1 must contact the start site of tran- scription in order to initiate RNA synthesis.

That 6 and c function at least in part through protein- protein interactions with RNA polymerase I1 and the other transcription factors is strongly suggested by the results of the template challenge experiments presented here. When the single subunit yeast TFIID protein is used as the TATA factor, stable interaction of 6 with the preinitiation complex requires RNA polymerase 11, a, By, and e, Likewise, stable interaction of c with the preinitiation complex requires RNA polymerase 11, a, and P-y and is strongly dependent on 6 . In contrast, when the transcription system is reconstituted with the high molecular mass, endogenous TATA factor T, 6 is able to interact stably with intermediates in assembly of the prein- itiation complex even in the absence of RNA polymerase 11, CY, and By.

Acknowledgments-We thank K. P. Garrett, J. P. Hanley, and H. Serizawa for helpful discussions and D. Irish for artwork.

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