Localization of subunits of transcription factors IIE and IIF immediately upstream of the transcriptional initiation site of the adenovirus major late promoter.

The assembly of a preinitiation complex containing RNA polymerase II on promoter DNA is a complex process that involves several general transcription factors. Using 5-[N-(p-azidobenzoyl)-3-aminoallyl] photocross-linking, we previously determined the locations of the two large subunits of transcription factor (TF) IIA (A35 and A21), TATA box-binding protein (TBP), RNA polymerase II-associated protein (RAP) 30, and TFIIB along the Ad2 ML promoter. We have now localized TFIIE34 and RAP74 just upstream of the transcription start site. The two subunits of TFIIF, RAP74 and RAP30, cross-linked to nucleotides that probed adjacent spaces on the same face of the DNA helix beginning just downstream of TBP at −19 and extending to −5. Specific photocross-linking of TFIIE34 required the presence TFIIE56. In addition, TFIIE and RAP74 strongly stimulated cross-linking of RAP30 and the large subunits of RNA polymerase II to position −19. Our topological data support the idea that RAP74 and TFIIE34 may be involved in melting of the promoter DNA upstream of the initiation site.

the TBP-promoter complex (4 -7). Recruitment of RNA polymerase II is mediated by TFIIB and RAP30, the small subunit of TFIIF, which can directly associate with TFIIB (7)(8)(9)(10)(11). Direct protein-protein interactions between RNA pol II and TBP have also been reported (12). RAP74, the large subunit of TFIIF, can bind to RAP30 and RNA pol II (13) and stabilize the association of a TBP⅐TFIIB⅐RAP30⅐RNA pol II complex with promoter DNA (14). TFIIE and TFIIH can then sequentially bind to this TBP⅐TFIIB⅐TFIIF⅐pol II⅐promoter complex (15,16). TFIIE may bind to several general transcription factors, including TFIIF (17) and TFIIH (17), as well as RNA pol II (15).
For most promoters, a minimal preinitiation complex containing TBP, TFIIB, TFIIF, and RNA pol II can initiate transcription in vitro and synthesize a transcript if the template DNA is supercoiled (18 -22). Initiation of transcription at the IgH promoter can even occur in the absence of TFIIF (18,21). For linear promoters, TFIIE and TFIIH are required for the production of an accurately initiated run-off transcript (20,22). These two factors are involved in promoter clearance, probably because they mediate ATP-dependent melting of the template DNA (20,22). Recently, Pan and Greenblatt (23) reported that, under certain circumstances, initiation of transcription is limited by melting of the promoter immediately upstream of the transcription start site, and they suggested that efficient melting of the promoter upstream of the position ϩ1 involves an unidentified factor, which may well be TFIIE and/or IIH.
Although several protein-protein interactions involved in preinitiation complex formation have been described, little is known concerning the topology of complexes assembled onto promoter DNA. Using N 3 R photocross-linking, we recently began to address the physical structure of preinitiation complexes containing various sets of general transcription factors and RNA pol II. We localized the relative positions of two subunits of TFIIA (A35 and A21) as well as those of TBP, TFIIB, RAP30, and the two largest subunits of RNA pol II on the Ad2ML promoter (14). TFIIA cross-links to the coding strand opposite TBP at the TATA box and cross-links upstream of TBP around position Ϫ40. RAP30 cross-links strongly and TBP and TFIIB weakly to the coding strand just downstream of TBP at position Ϫ19 (14).
We have now localized two additional polypeptides of the general transcription machinery, RAP74 and TFIIE34, along the Ad2ML promoter. Our photocross-linking experiments also provide information on the enzymology of preinitiation complex assembly.

EXPERIMENTAL PROCEDURES
Protein Factors-Recombinant yeast TBP (24), human TFIIB (25), human RAP30 (10,26), human RAP74 (26), and human TFIIE56/34 (27)(28)(29), as well as calf thymus RNA pol II (30), were prepared as described previously. N 3 R Photocross-linking-The synthesis of N 3 R-dUMP, the preparation of the probes, and the conditions for binding reactions were as described previously (14). A schematic representation of our various photoprobes is shown in Fig. 1. Binding reactions were as we previously described (14). For each photoprobe, the concentration of poly(dG⅐dC)-(dG⅐dC) in binding reactions was optimized in order to favor specific binding over nonspecific binding, and was between 0.1 and 1 g/binding reaction. A typical reaction with all the factors contained 200 ng each of TBP, TFIIB, RAP30, RAP74, TFIIE56, TFIIE34, and purified RNA pol II. Ultraviolet irradiation, nuclease treatment, and SDS-PAGE analysis of radiolabeled photocross-linking products were as we previously described (14). Immunoprecipitations-The photocross-linking procedure was exactly as described above. Instead of submitting the radiolabeled photocross-linking products to SDS-PAGE, they were immunoprecipitated using antibodies directed against various subunits of the general transcription factors. Immunoprecipitation using affinity-purified antibodies raised against TBP, TFIIB, RAP30, RAP74, TFIIE56, and TFIIE34 were performed as we previously described (14). Immunoprecipitated polypeptides were resolved using SDS-PAGE (14).

RESULTS AND DISCUSSION
Probes for Photocross-linking-Incorporation of the photoreactive nucleotide N 3 R-dUMP into DNA probes can be achieved only where T residues are present in the sequence. In this study, we prepared photocross-linking probes that placed the photoreactive nucleotide at positions Ϫ19, Ϫ9, Ϫ8, Ϫ5, and Ϫ2 on the coding strand using the wild type Ad2 ML promoter (see Fig. 1). In order to probe the space along promoter regions that lacks T residues, we also constructed mutant promoters that contain single T residues at various additional positions, including Ϫ14 on the coding strand and Ϫ15, Ϫ10, and Ϫ5 on the non-coding strand. The design of each probe is shown schematically in Fig. 1.
Specific Photocross-linking of TFIIE34 in a Region between Ϫ14 and Ϫ2 in the Presence of TFIIE56 -In order to map the location of additional GTFs along the Ad2 ML promoter, we first synthesized probe Ϫ14/Ϫ2, which places 5 photoreactive nucleotides at positions Ϫ14, Ϫ9, Ϫ8, Ϫ5, and Ϫ2 on the coding strand (see Fig. 1). Using this probe, we observed photo-linking of two large polypeptides of approximately 145-180 kDa and two additional polypeptides of approximately 25-40 kDa in the presence of TBP, TFIIB, RAP30/74, RNA pol II, and TFIIE56/34 ( Fig. 2A, lane 1). To assess whether photocrosslinking was promoter-specific, we compared cross-linking reactions performed in the presence or in the absence of TBP. In the conditions that we used (e.g. particular concentrations of salt and nonspecific competitor DNA), we always observed that assembling a reaction in the absence of TBP had the same effect as using a photoprobe with a mutated TATA element (see Ref. 14 and below). As expected for promoter-specific crosslinking events, cross-linking of the various polypeptides to probe Ϫ14/Ϫ2 was abolished in the absence of TBP ( Fig. 2A,  compare lanes 1 and 2) or when we used a photoprobe with a mutated TATA element (TAGAGAA instead of TATAAAA) ( Fig. 2A, compare lanes 1 and 3). The two large cross-linked polypeptides are almost certainly the two largest subunits of RNA pol II because (i) their apparent M r values correspond to those of the two largest subunits of calf thymus RNA pol II (30), (ii) the two largest subunits of our highly purified RNA pol II preparation are the only polypeptides in the range of 145-180 kDa that we used in our photocross-linking experiments, and (iii) photocross-linking of these two large polypeptides was obtained only in the presence of RNA pol II in the reactions (data not shown). The smaller of the two polypeptides in the 25-40-kDa range has not been identified, but may be a subunit of RNA pol II. The larger of the polypeptides in the 25-40-kDa range was immunoprecipitated with a specific antibody directed against TFIIE34 (Fig. 2B, lane 5), but not with a control antibody (Fig. 2B, lane 6). Cross-linking of TFIIE34 to probe Ϫ14/Ϫ2 required the presence of TBP in the reactions (Fig. 2B,  lane 2). In the absence of TFIIE56, we still observed crosslinking of TFIIE34 (Fig. 2B, lane 3), but this cross-linking was not specific since it occurred in the absence of TBP (lane 4). Longer exposure of our cross-linking gels also revealed specific cross-linking of RAP74 to the Ϫ14/Ϫ2 photoprobe (data not shown; see below for a more accurate localization of RAP74). Photocross-linking of RAP74 to probe Ϫ14/Ϫ2 was also abolished when we used a photoprobe with a mutated TATA element (data not shown), indicating that cross-linking of RAP74 is promoter-specific as well.
Surprisingly, we have not yet been able to cross-link TFIIE56 to any of our photoprobes in the presence of TBP, TFIIB, RAP30/74, RNA pol II, and TFIIE56/34. This was particularly surprising since TFIIE is a heterotetramer containing 2 molecules of TFIIE56 (31). Our photocross-linking data suggest that the large subunit of TFIIE may be located quite far away from the promoter DNA or in a region where the chemical arm of N 3 R-dUMP cannot reach. Alternatively, TFIIE56 may act as a molecular chaperone in catalyzing the assembly of the preinitiation complex in the presence of TBP, TFIIB, TFIIF, RNA pol II, and TFIIE34. If this is true, TFIIE56 may become stably bound to the preinitiation complex only in the presence of TFIIH.
Photocross-linking of RAP74 to Positions Ϫ15 and Ϫ5, but Not to Position Ϫ10 -We and others have shown previously that RAP74 is not essential for the recruitment of RNA pol II into a TBP-TFIIB-RAP30 complex (9, 10), but increases the photocross-linking of RAP30 and the large subunits of RNA pol II to position Ϫ19 just downstream of the TATA box (see Ref. 14 and below). In order to localize RAP74 along promoter DNA, we used three individual probes, which placed a photoreactive nucleotide at positions Ϫ15, Ϫ10, and Ϫ5, respectively, on the non-coding strand (see Fig. 1). We obtained specific photocrosslinking of the 145-kDa subunit of RNA pol II to positions Ϫ15 and Ϫ5 in the presence of TBP, TFIIB, RAP30/74, RNA pol II, and TFIIE56/34 (Fig. 3, A and C, lanes 1). This cross-linking of RNA pol II was strongly stimulated by the presence of RAP74 (compare lanes 1 and 2). The presence of RAP74 in the reactions also resulted in cross-linking of a polypeptide of approximately 74 kDa to positions Ϫ15 and Ϫ5 (Fig. 3, A and C,  compare lanes 1 and 2), but not to position Ϫ10 (Fig. 3B, lane  1). The 74-kDa radiolabeled polypeptide was immunoprecipitated using an antibody raised against RAP74 (Fig. 3, A and C,  lanes 4), but not with antibodies directed against any other GTF subunits (data not shown). Photocross-linking of RAP74 and the 145-kDa RNA pol II subunit mostly depended on the presence of TBP (Fig. 3, A and C, lanes 3 and 5). Therefore RAP74 associates with the preinitiation complex in a region located approximately between positions Ϫ15 and Ϫ5 along promoter DNA. This would place RAP74 adjacent to RAP30, which cross-links strongly to position Ϫ19 on the coding strand (see Ref. 14). Interestingly, positions Ϫ15 and Ϫ5 are separated by exactly one turn of the DNA helix. Taken together with the observation that RAP74 does not cross-link to positions Ϫ10 (Fig. 3B) and Ϫ20 (data not shown) on the non-coding strand, our data suggest that RAP74 occupies a space restricted to one face of the helix. In addition, the incorporation of N 3 R-dUMP at position Ϫ19 on the coding strand, where we obtained crosslinking of RAP30, and positions Ϫ15 and Ϫ5 on the non-coding strand, where we obtained cross-linking of RAP74, all place the nitrene-reactive group on the same face of the DNA helix. These data suggest that the two subunits of TFIIF occupy adjacent spaces on one face of promoter DNA. These topological interpretations of our photocross-linking data are represented schematically in Fig. 5.
RAP74 and TFIIE Strongly Stimulate Cross-linking of RAP30 and RNA Pol II to Position Ϫ19 -We previously showed that several polypeptides cross-link to position Ϫ19 on the coding strand of the Ad2 ML promoter (14). These include TBP, TFIIB, RAP30, and the two large subunits of RNA pol II. We reported that TBP cross-links to that position in the context of a TBP⅐TFIIA⅐promoter (TATA) complex, while RAP30 is the main cross-linked polypeptide in the context of a TBP⅐TFIIB⅐RAP30⅐pol II⅐promoter (TATA) complex (14). In such a complex, we also obtained weak cross-linking of TFIIB to position Ϫ19 (14). This position of TFIIB agrees with that recently determined by hydroxyl-radical footprinting (32) and x-ray crystallography (33). These observations indicate that position Ϫ19 delimits the spatial interface where TBP directs the recruitment of RNA pol II to the preinitiation complex through protein-protein interactions with the bridging factors RAP30 and TFIIB. In addition, we showed that RAP74 in-  1 to lanes 3 and  4). Both subunits of TFIIE were required to promote efficient cross-linking of RAP30 and RNA pol II subunits (data not shown). Photocross-linking of RNA pol II and RAP30 to this position also required the presence of TBP (Fig. 4A, lane 2). Immunoprecipitations using antibodies raised against various subunits of GTFs, including RAP30 (Fig. 4B, lanes 3 and 4), TFIIB (lanes 5 and 6), TBP (lanes 7 and 8), and TFIIE34 (lanes 9 and 10) revealed that RAP30 and RNA pol II are the only proteins that cross-linked specifically to position Ϫ19 in the context of a TBP-TFIIB-RAP74/30⅐RNA pol II⅐TFIIE56/34⅐promoter (TATA) complex. Photocross-linking of TFIIE34 to position Ϫ19 was also observed, but it was not specific since it occurred in the absence of TBP (compare lane 9 to lane 10). Specific photocross-linking of TFIIE34 was only obtained using probes Ϫ14/Ϫ2 (see Fig. 2) and Ϫ10 (data not shown). Long exposure of the gel shown in Fig. 4B revealed only nonspecific cross-linking of TFIIB (data not shown). These observations suggest that RAP74 and TFIIE help tether RAP30 and RNA pol II to our photoreactive nucleotide placed at position Ϫ19. There may also be a conformational change in a preinitiation complex that contains RNA pol II that has the effect of making TFIIB inaccessible to the chemical arm of the photoreactive nucleotide. Such a conformational change has already been proposed for TFIIB (34). Fig. 5 shows a schematic representation of our photocrosslinking data in the context of a molecular structure for the TBP-promoter complex (35,36). We propose that position Ϫ19 delimits the space where RAP30 and TFIIB stabilize the interaction between TBP and RNA pol II along the promoter DNA.
The position shown for TFIIB is based on our cross-linking experiments performed in the absence of TFIIE (14). RAP30 and RAP74 are placed along one face of the DNA between Ϫ19 and Ϫ5, and TFIIE34 is located between Ϫ14 and Ϫ2.
Recently, Pan and Greenblatt (23) reported that initiation of transcription on closed, linear templates is limited by melting of the promoter immediately upstream of the transcription start site. Synthesis of a promoter-specific trinucleotide on linear DNA requires TFIIF and is stimulated by TFIIE (23). Artificial melting of the promoter DNA between Ϫ9 and Ϫ1 on such a template obviates the requirement for TFIIF, suggesting that TFIIF may be involved in melting of the promoter upstream of ϩ1 (23). Photocross-linking of RAP74 and TFIIE34 to nucleotides located between Ϫ15 and Ϫ2 suggests that these factors may be involved in melting of the DNA helix during transcription initiation.