INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The TATA binding protein (TBP) is a subunit common to transcription factors required for promoter-dependent transcription by RNA polymerases (Pol) I (SL-1 and TIF-IB), II (TFIID), and III (TFIIIB) (19, 43, 47). Pol-specific polypeptides are associated with TBP in each of these different transcription factors and are referred to as TBP-associated factors (TAFs) (16). TBP binds to the DNA sequence TATAAAA and severely bends DNA through contacts with the minor groove of DNA (32, 33, 39). TBP forms a saddle-like structure, contacts DNA with the underside part of the saddle, and kinks the DNA at two discrete locations. TBP binds slowly to DNA, and removal of the nonconserved N-terminal portion of the protein increases the efficiency of binding to DNA (20-22, 46). In some cases, the transcription factors containing TBP are recruited to DNA through protein-protein interactions and apparently not by direct DNA-protein interactions with TBP. The SUP4 tRNA^Tyr gene from Saccharomyces cerevisiae is an example: TFIIIB is recruited to DNA by protein-protein interactions with TFIIIC (25).

Yeast TFIIIC is a large multisubunit protein that binds to two internal promoter elements of tRNA genes, referred to as box A and box B, and helps position TFIIIB upstream of the start site of transcription (25, 27). The TFIIIB-DNA complex is resistant to polyanions and high salt. TATA-like sequences within the upstream region provide additional criteria for TFIIIB binding site preference and can alter the binding of TFIIIB within an ~20-bp region (24). TFIIIB consists of three subunits: TBP, TFIIB-related factor (BRF), and B" (TFC5) (10, 28, 30). BRF binds directly to TFIIIC-DNA complexes (as shown by a gel shift assay) and facilitates the binding of TBP. The addition of TBP to the BRF-TFIIIC-DNA complex enhances the binding of BRF to DNA upstream of the transcription start site, as shown by DNA photoaffinity labeling (28). TBP-BRF-TFIIIC-DNA complexes are sensitive to high salt and polyanions and cannot mediate Pol III binding or transcription (1, 3). TFIIIB bends DNA to a greater degree than TBP, and the DNA is less bent in the TBP-BRF-TFIIC-DNA complex than in the complete TFIIIB-DNA complex (6, 36).

TFIIIB can also bind to DNA without TFIIIC when a TATA box is present. The yeast U6 snRNA gene contains a TATA box upstream of the start site of transcription and is transcribed in vitro with TFIIIB and Pol III (51). Although TFIIIC is not required for in vitro transcription of the U6 snRNA gene, it is required for in vivo transcription of this gene (7, 13). The in vivo role of TFIIIC in the transcription of the U6 snRNA gene is to alter a repressive chromatin structure and recruit TFIIIB (9, 15). TFIIIB does not usually bind TATA box elements in an orientation-specific manner; thus, transcription can occur in both directions from TATA box elements (51). Rather than trying to adjust the conditions either by deletion of the N-terminal nonconserved region of TBP or by inserting bulges into the DNA template so as to bias the binding of TBP and TFIIIB to DNA (11, 18), we have opted for what we think is the more relevant situation, in which TFIIIB is recruited to DNA in one unique orientation by TFIIIC. We have chosen not to investigate TFIIIB-DNA complexes that are assembled independently of TFIIIC, because although they can form transcriptionally active complexes, they are likely not to be representative of in vivo complexes.

We have focused on characterizing the region upstream of the SUP4 tRNA gene that is involved in TFIIIB binding. Three kinds of DNA photoaffinity probes were used to scan the major and minor grooves of DNA. One set of probes examined primarily the major groove of DNA (AB and DB probes), and another set of probes examined both the major and the minor grooves (S-P probes). The AB and DB probes tethered phenylazide or phenyldiazirine, respectively, to the C-5 of deoxyuridine or the N-4 of deoxycytidine. At the most extended conformation of the tether, the photoreactive site is positioned at the edge of the major groove of DNA, as shown by molecular modeling (results not shown). The DB probes were crucial in ensuring that all potential protein-DNA interactions in the major groove were determined by use of phenyldiazirine to generate the highly reactive carbene, which does not have an amino acid side-chain preference (4, 8, 38). Our cross-linking results indicate that TBP is associated with the TATA-like DNA sequence beginning 30 nucleotides upstream of the start site of transcription. TBP was shown to bind primarily to the minor groove of DNA, and this observation is consistent with the known crystal structure of the TBP-TATA box complex. Significant alteration of the TBP-DNA interface in the TFIIIB-DNA complex from that of the crystal structure of the TBP-TATA box complex was shown by site-specific DNA cross-linking and peptide mapping of photoaffinity-labeled TBP. Evidence is given for the stereospecific binding of the BRF and B" subunits of TFIIIB to the TBP-DNA core, forming a protein clamp around DNA.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Preparation of immobilized DNA. The nontranscribed strand of the SUP4 tRNA^Tyr gene containing an upstream mutant boxB was immobilized as described previously (35). The procedure for the preparation of immobilized transcribed-strand DNA was the same as that for the nontranscribed strand, except for the use of different restriction enzyme sites. pTZ1 DNA was first digested with EcoRI and then biotinylated by filling in the 5' overhangs with Bio-14-dATP and Bio-11-dUTP (GIBCO BRL) by use of the Klenow fragment of DNA Pol I (U.S. Biochemicals). It was subsequently digested with HindIII and PvuII, and the resulting 315- and 44-bp biotinylated fragments were bound to Dynabeads M-280-streptavidin (Dynal).

Coupling of p-azidophenacyl bromide to phosphorothioate-containing oligonucleotides. All procedures with photosensitive reagents were performed with indirect lighting from a 40-W incandescent lamp. Oligonucleotides containing phosphorothioates at the phosphodiester linkage between the third and fourth nucleotides from the 3' end were resuspended in 100 mM triethylammonium bicarbonate (pH 8.0) to obtain a final concentration of 100 pmol/µl. The coupling reaction was carried out by adding 75 µl of the respective oligonucleotide to 75 µl of 100 mM p-azidophenacyl bromide in acetonitrile and incubating the mixture at 25°C for 4 h.

Synthesis of DNA photoaffinity probes. Double-stranded DNA probes containing modified phosphodiesters were prepared by annealing modified oligonucleotides to an immobilized single-stranded DNA template with heating at 70°C for 3 min, followed by cooling at 37°C for 30 min in buffer A (30 mM Tris-HCl [pH 8.0], 50 mM KCl, 7 mM MgCl₂, 0.05% Tween 20). Reducing agents such as dithiothreitol were avoided so as not to reduce the phenylazido moiety (41). The beads were washed three times with buffer A containing 0.1 mg of bovine serum albumin per ml. An -³²P-deoxynucleotide (specific activity, 2,000 to 3,000 Ci/mmol) was enzymatically incorporated with the Klenow fragment of DNA Pol I, and probe synthesis was continued as described above to make DNA double stranded over the entire stretch of the DNA probe (35).

Photoaffinity labeling of the transcription complex. Transcription complexes were formed on probe DNA by use of the 500 mM KCl fraction from BioRex 70 chromatography of the S-100 extract of S. cerevisiae BJ926 (BR500) (35). Alternatively, transcription complexes were assembled by use of recombinant TFIIIB (rTFIIIB) and partially purified TFIIIC as described previously (30). TFIIIC was obtained from the flowthrough fractions of Ni-nitrilotriacetic acid chromatography of His-tagged RNA Pol III as described previously (35).

Cyanogen bromide cleavage of photoaffinity-labeled TBP and preparation of a radiolabeled TBP molecular weight standard. A large photoaffinity labeling reaction mixture (200 µl) was assembled by use of the BR500 fraction that had been treated with heparin at a final concentration of 0.15 mg/ml and placed in a multichannel pipeter basin for irradiation. The conditions for the irradiation and digestion of DNA with DNase I and S1 nuclease were the same as before (35). The protein was concentrated by ultrafiltration (Centricon 10; Millipore). Before the sample was loaded for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), the following were added to the indicated final concentrations: 10% SDS, 0.7 M -mercaptoethanol (BME), 0.44% bromophenol blue, 0.3 M Tris-HCl (pH 6.8), and 16% glycerol. The sample was loaded on an SDS-12% polyacrylamide gel (18 cm by 18 cm by 0.8 mm). After electrophoresis, cross-linked TBP was visualized by autoradiography, and the corresponding gel slice was excised. TBP was electroeluted from the gel slice (Bio-Rad model 422 Electro-Eluter) at 10 mA for 4 h into a volatile buffer consisting of 50 mM ammonium bicarbonate and 0.1% SDS. The eluate obtained was dried by vacuum centrifugation, resuspended in 200 µl of sterile deionized water, and dried again.

Cleavage of photoaffinity-labeled TBP with 2-nitro-5-thiocyanobenzoic acid (NTCB). A large-scale photoaffinity labeling reaction with TFIIIB, six-His-tagged TBP, and partially purified TFIIIC was set up. The cross-linked sample was concentrated approximately 20-fold by ultrafiltration (Centricon 10). The DNA was digested with formic acid-diphenylamine, extracted, and dried as described above for the cleavage with cyanogen bromide. The pellet was resuspended in sample loading buffer, and photoaffinity-labeled TBP was purified by SDS-PAGE as described earlier. The eluted protein was diluted to 2 ml with 8 M urea-0.2 M Tris acetate (pH 8) and concentrated by ultrafiltration (Centricon 10). The sample was diluted and concentrated two more times with the same solution.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

We have examined the protein-DNA contacts of a TFIIIC-dependent TFIIIB-DNA complex to determine the functional and structural features of TBP in a complex where TBP is not the exclusive determinant of the DNA binding specificity. A region spanning ~24 nucleotides (bp 11 to 34) was probed by DNA photoaffinity labeling. Two kinds of DNA photoaffinity probes were constructed to examine protein contacts exclusively in the major groove of DNA (AB and DB probes) or contacts in either the minor or the major groove of DNA (S-P probes). Photoreactive moieties were attached to the phosphate backbone of DNA with a 7-Å tether and the photoreactive moiety directed toward the major and minor grooves of DNA (S-P probes; Fig. 1A). The photoreactive group was attached to DNA by alkylation of a uniquely incorporated phosphorothioate in DNA. Eight and seven positions in the nontranscribed and transcribed strands, respectively, were modified (Fig. 1C). The radioactive label was incorporated 3 nucleotides from the modification site in the 3' direction.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Synthesis of photoreactive DNAs for probing the major and minor grooves of DNA. (A) Modification of the phosphate backbone of DNA. A phosphorothioate incorporated at a unique site in DNA is alkylated with p-azidophenacyl bromide to covalently couple phenylazide to the phosphate backbone. The structure of the chemical modification in DNA shown will position phenylazide in the major and minor grooves of DNA. (B) Phenylazide and phenyldiazirine are tethered to dUTP and dCTP. Phenylazido (X1) and phenyldiazirinyl (X2) groups are positioned in the major groove of DNA by covalent attachment to the C-5 of deoxyuridine or the N-4 of deoxycytidine, respectively. The tether for the attachment of the phenylazido and phenyldiazirinyl groups to the nucleotide base is the same. (C) The positions in the SUP4 tRNA gene containing the photoreactive moiety are shown for the nontranscribed strand (top) and the transcribed strand (bottom). The modified sites are indicated in bold letters: the modified phosphodiester of the S-P probes is positioned between the two nucleotides shown in bold lowercase letters, whereas the modified nucleotide is that shown in bold for the AB and DB probes. The position of the radioactive label is underlined and is located 3 nucleotides away from the modification site for the S-P probes and is immediately adjacent to the modified nucleotide for the AB and DB probes.

Pol III transcription complexes were formed on these modified DNA probes by use of the 500 mM eluate from BioRex 70 chromatography, which contains Pol III, TFIIIB, and TFIIIC. Pol III and TFIIIC were released from DNA by the addition of heparin (100 to 150 µg/ml), and the remaining TFIIIB-DNA complexes were cross-linked by UV irradiation. After enzymatic degradation of the DNA probes, cross-linked proteins were analyzed by SDS-PAGE and autoradiography. TBP (molecular mass, ~27 kDa) was cross-linked to DNA on the 3' side of bp 30, 28, 25, 23, and 20 on the transcribed strand and to DNA on the 3' side of bp 28, 26, and 24 on the nontranscribed strand (Fig. 2A, lanes 3, 5, 7, 9, and 11, and Fig. 2B, lanes 7, 9, and 15). Positions farther upstream, at bp 33 (transcribed strand) and bp 34 and 30 (nontranscribed strand), and downstream, at bp 21, 18, and 13 (nontranscribed strand), did not significantly cross-link TBP. These results are consistent with TBP binding to the TATA-like sequence, TATATGT, 30 bp upstream of the start site of transcription (Fig. 2C). TBP cross-linking was shown to require efficient binding of TFIIIC by competition with specific versus nonspecific DNAs. Equivalent amounts of carrier DNA with or without the SUP4 tRNA gene were added to all samples. When the competitor DNA contained the SUP4 tRNA gene, cross-linking of TBP was eliminated on both strands of DNA (Fig. 2A, lanes 4, 6, 8, 10, and 12, and Fig. 2B, lanes 8, 10, and 16).

View larger version (48K): [in this window] [in a new window]   FIG. 2.   TBP is photocross-linked to a TATA-like sequence of DNA that starts 30 nucleotides upstream of the transcription start site. (A) Interactions of TFIIIB with the major and minor grooves of DNA from nucleotides 33 to 20 (+1 is the transcription start site) on the transcribed strand of the SUP4 tRNA gene were mapped with S-P probes. TFIIIB-DNA complexes were formed with the BR500 fraction, and heparin was added before photocross-linking of the complex. In the odd-numbered lanes, the sample contained 500 ng of pGEM DNA cut with EcoRI as a nonspecific competitor. The samples in the even-numbered lanes contained 500 ng of pTZ1 DNA cut with EcoRI as an effective competitor for TFIIIB and TFIIIC binding to the photoreactive probe DNA. Samples were processed as described in the text and loaded onto an SDS-4 to 20% polyacrylamide gel. Results obtained with a DNA photoaffinity probe containing AB-dCMP at bp 23 are shown in lanes 13 and 14 for comparison. The electrophoretic mobilities of the three subunits of TFIIIB are indicated (in thousands) on the left (labeled with a B), and those of a prestained protein molecular weight marker are indicated on the right. (B) A region from nucleotides 34 to 13 on the nontranscribed strand of the SUP4 tRNA gene was probed. The conditions were the same as those for panel A. (C) The DNA region shown to photocross-link TBP corresponds to a TATA-like sequence positioned like the TATA box of the adenovirus major late promoter with regard to the transcription start site (boxed and in bold letters). The 5' half of the sequence in the SUP4 tRNA gene is identical to that of the TATA box, but the 3' half of the sequence is significantly different.

The BRF (molecular mass, ~67 kDa) subunit was also cross-linked in a TFIIIC-dependent manner at sites within the TATA-like sequence and downstream of this region. BRF was cross-linked on the 3' side of bp 30, 28, and 20 of the transcribed strand and on the 3' side of bp 30, 28, 21, 18, and 13 of the nontranscribed strand (Fig. 2A, lanes 3, 5, and 11, and Fig. 2B, lanes 1, 3, 5, 13, and 15). At sites upstream of the TATA-like sequence, the B" (molecular mass, ~90 kDa) subunit of TFIIIB was efficiently cross-linked at bp 34 and 33 of the nontranscribed and transcribed strands, respectively (Fig. 2A, lane 1, and Fig. 2B, lane 11). Interestingly, B" was also efficiently cross-linked near the center of the TATA-like sequence to the transcribed strand at bp 25 and 23 but not to the nontranscribed strand (compare Fig. 2A, lanes 7 and 9, to Fig. 2B, lanes 7 and 9). Cross-linking on the 3' side of bp 26 and 24 of the nontranscribed strand showed a lack of efficient cross-linking of either of the two largest subunits of TFIIIB (Fig. 2B, lanes 7 and 9).

The S-P (modified-phosphate-backbone) DNAs probed protein-DNA interactions in either the major or the minor groove. In order to distinguish major-groove interactions from minor-groove interactions, we used AB-dCMP and AB-dUMP to place a phenylazido group into the major groove of DNA (Fig. 1B); we compared the results obtained with these probes to those obtained with the S-P probes. The incorporation of modified nucleotides into DNA did not interfere with the formation of transcription complexes or initiation at the normal start site of transcription, as determined by multiple-round transcription assays (data not shown). TBP binds to TATA box DNA through the minor groove of DNA and directs the major groove of DNA in toward itself to create a pocket or core of DNA (32, 33). If TBP is bound to DNA in the TFIIIB-DNA complex as it is to the TATA box, then TBP would not be expected to be cross-linked with the AB probes. Of the 14 different sites in DNA scanned in the nontranscribed and transcribed strands, 12 positions showed no significant amount of photoaffinity labeling of TBP (Fig. 3A and B). TBP was, however, cross-linked to DNA at a very discrete region in the transcribed strand at bp 23 and fivefold less at bp 25 in the preinitiation complex (Fig. 3A, lanes 9 and 10). Cross-linking of TBP, BRF, and B" at these positions was shown by DNA competition to be promoter specific and to require the efficient binding of TFIIIC (results not shown). All the reactions shown in Fig. 3 contained, on a molar basis, 200 times more linearized plasmid DNA (pLNG56) containing one copy of a defective SUP4 tRNA gene than did the photoreactive DNA probe. The SUP4 tRNA gene in pLNG56 has two point mutations in the box B element and binds TFIIIC 300 times less efficiently than does the DNA probe. Cross-linking of TBP, BRF, and B" was eliminated when plasmid DNA (pTZ1) containing the same version of the SUP4 tRNA gene as the DNA photoaffinity probe was substituted for pLNG56 DNA.

View larger version (177K): [in this window] [in a new window]   FIG. 3.   TBP is photocross-linked primarily through the minor groove, except at bp 23 and 25 on the transcribed strand. (A) Photocross-linking experiments with the preinitiation complex were carried out with 14 probes containing AB-dUMP (lanes 1 to 8, 11, 13, and 14) or AB-dCMP (lanes 9, 10, and 12) at the nucleotide positions indicated in the SUP4 tRNA gene and analyzed by electrophoresis on an SDS-4 to 20% polyacrylamide gel. (B) Complexes were formed as in panel A, but heparin (100 µg/ml) was added to remove TFIIIC and Pol III from DNA before photocross-linking (lanes 1 to 14). Lanes 15 and 16 did not contain heparin and are similar to lanes 9 and 10 in panel A. Molecular weight markers in panels A and B are as described in the legend to Fig. 2. (C) TFIIIB-DNA complexes were photocross-linked at bp 23 in the presence of heparin, and TBP was immunoprecipitated with an anti-TBP antibody. The antigen-antibody complex was precipitated with protein A-Sepharose and analyzed by electrophoresis on an SDS-4 to 20% polyacrylamide gel. Lane 1 contains a sample before the addition of antibody to show that BRF and TBP are cross-linked [67 (B) and 27 (B), respectively]. TBP, and not BRF, was immunoprecipitated, as can be seen in a longer exposure (compare lanes 2 and 6 and results not shown). The electrophoretic mobilities of the 120-kDa subunit of TFIIIC (C); the 160-, 128-, and 34-kDa subunits of Pol III (III); and the three subunits of TFIIIB (B) are indicated on the left.

Specific cross-linking of the 34-kDa subunit of Pol III was observed at bp 17 and 16 (Fig. 3A, lanes 6 and 13), and that of the 160-kDa subunit was observed at bp 11 and 12 (lanes 7 and 14), consistent with previous reports (1, 35). Proteins cross-linked at bp 27 were not promoter specific, and the low amount of cross-linking of the ~30- to 50-kDa proteins at bp 22 and 21 was also not specific (results not shown). The 128-kDa subunit of Pol III and the 120-kDa subunit of TFIIIC were most intensely cross-linked at bp 22 and 21 and at bp 17 and 12 (Fig. 3A, lanes 4, 6, and 7). The ~170-kDa protein labeled at bp 22 and 21 appears not to be part of the Pol III transcription complex and is cross-linked independently of TFIIIC. An ~50-kDa protein is labeled at bp 17 and 16 in a promoter-dependent manner (Fig. 3A, lanes 6 and 13, and results not shown).

Pol III and TFIIIC were removed from DNA by the addition of heparin to examine the contacts of TFIIIB alone bound to DNA (31). Cross-linking of TBP was still observed only at bp 23 and 25 on the transcribed strand and with the same efficiency as in the preinitiation complex (Fig. 3B, compare lanes 9 and 10 to lanes 15 and 16). Labeling of the BRF and B" subunits was also heparin resistant and was enhanced at some positions, presumably due to the removal of TFIIIC and Pol III. The photoaffinity-labeled ~27-kDa protein was confirmed to be TBP by immunoprecipitation with anti-TBP antibody (Fig. 3C, lanes 1 and 6).

The influence of B" on TBP interactions with the major groove was analyzed by performing photoaffinity labeling experiments with TFIIIB reconstituted by use of recombinant BRF, TBP, and B". In the presence of B", TBP was cross-linked on the transcribed strand at bp 23 and, to a minor extent, at bp 25 and 18 (Fig. 4A, lanes 3, 5, and 9). In the absence of B", TBP was cross-linked nine times less efficiently on the transcribed strand at bp 23 (Fig. 4A, compare lanes 5 and 6). In the absence of B", TBP was labeled on the nontranscribed strand at bp 33 and 32 and much less intensely at bp 30 and at bp 22 and 21 (Fig. 4B, lanes 4, 6, and 10). Photoaffinity labeling of TBP in the absence of B" is dependent on the prior interaction of TFIIIC and BRF with DNA, as shown by DNA competition experiments with pTZ1 DNA versus pLNG56 DNA (data not shown). The addition of B" enhanced the cross-linking of TBP to DNA at bp 23 and eliminated TBP cross-linking at bp 33 and 32, at bp 30, and at bp 22 and 21 on the nontranscribed strand. As previously noted, B" also enhanced the cross-linking of BRF at bp 38 to 23, while in this same region, the cross-linking of the 120-kDa subunit of TFIIIC was greatly diminished because of direct or indirect displacement or competition by B" (Fig. 4A, lanes 1 to 6, and Fig. 4B, lanes 1 to 8) (3, 26). These data indicate that B" promotes the interaction of TBP with the major groove of DNA at bp 23 on the transcribed strand, whereas the cross-linking of TBP is eliminated at other positions.

View larger version (48K): [in this window] [in a new window]   FIG. 4.   B" helps position TBP in the major groove of DNA at bp 23 on the transcribed strand. Fourteen probes containing AB-dCMP or AB-dUMP (~10-Å tether), seven on the transcribed strand (A) and seven on the nontranscribed strand (B) at the nucleotide positions indicated, were used in photoaffinity labeling experiments. The odd-numbered lanes contained B", BRF, TBP, and TFIIIC, whereas the even-numbered lanes contained BRF, TBP, and TFIIIC only. The cross-linking efficiency of TBP at bp 23 (A, lanes 5 and 6) was seven- to ninefold higher with the addition of B". Molecular weight markers are as described in the legend to Fig. 2.

In order to ensure that all the interactions of TFIIIB with the major groove of DNA were detected, DNA probes containing phenyldiazirine were constructed. Upon photolysis, phenyldiazirine produces highly reactive carbene, which does not have a preference for nucleophilic side chains, as does dehydroazepine formed by the rearrangement of phenylnitrene (4, 8, 14). The photoreactive nucleotides DB-dUMP and DB-dCMP have the same tether as AB-dUMP and AB-dCMP, so that photocross-linking results can be directly compared (Fig. 1B). Cross-linking of the preinitiation complexes revealed some significant differences between the phenyldiazirine- and phenylazide-containing nucleotide analogs (compare Fig. 5A and B to Fig. 3A).

View larger version (43K): [in this window] [in a new window]   FIG. 5.   Photocross-linking with DB probes revealed additional B"-DNA contacts at bp 27 and 23 on the transcribed strand and at bp 30 on the nontranscribed strand. Preinitiation complexes were formed as described in the legend to Fig. 3 with DNA probes containing DB-dUMP or DB-dCMP on the transcribed (A) and nontranscribed (B) strands and photocross-linked as described in the text. In lanes 6 in panel A and 8 in panel B, samples were photocross-linked with DNA probes containing AB-dCMP at bp 23 for comparison. Molecular weight markers are as described in the legend to Fig. 2.

The DB probes showed that the B" subunit interacts with the major groove at bp 27 and 23 on the transcribed strand and at bp 30 on the nontranscribed strand (Fig. 5A, lanes 1 and 5, and Fig. 5B, lane 1). The AB probes inefficiently photoaffinity labeled the transcription complex at bp 30 (transcribed strand) and at bp 27 and 20 (transcribed strand) (Fig. 3A, lanes 1, 8, and 11). Although no proteins were cross-linked at bp 20 by AB-dUMP, the BRF subunit was efficiently cross-linked at this same position by DB-dUMP (compare Fig. 3A, lane 11, to Fig. 5A, lane 2). The difference between DB-dCMP and AB-dCMP cross-linking at bp 23 on the transcribed strand suggests that the efficient cross-linking of TBP by AB-dCMP is due to the proximity of a nucleophilic side chain(s) in TBP. The less efficient cross-linking of TBP by DB-dCMP at bp 23 presumably occurs because DB-dCMP does not have a side-chain preference and the B" subunit is more prevalent (Fig. 5A, compare lanes 5 and 6). TBP was cross-linked at bp 23 on the transcribed strand and, to a minor extent, at bp 24 and at bp 22 and 21 on the nontranscribed strand. Less efficient cross-linking of the B" subunit was also seen at bp 24 and 17 on the transcribed strand (Fig. 5B, lanes 3 and 6).

The largest subunit (160 kDa) of Pol III was cross-linked at bp 11 (transcribed strand) and at bp 12 (nontranscribed strand) with the DB probes, consistent with the cross-linking results obtained with the AB probes (Fig. 5A, lane 4, and Fig. 5B, lane 7). The 34-kDa subunit was cross-linked at bp 16 (transcribed strand) but not at bp 17 (nontranscribed strand) with the DB probes. Efficient cross-linking of an ~50-kDa protein at bp 11 was observed with DB-dUMP but not with AB-dUMP. Photoaffinity labeling of the ~50-kDa protein was TFIIIC dependent and was not changed upon the formation of a stalled elongation complex with the addition of ATP, CTP, and UTP (results not shown). Cross-linking of a similar ~50-kDa protein with AB-dUMP was previously noted at bp 9 and 8 on the nontranscribed strand (35).

A summary of where DNA cross-links to the BRF, B", and TBP subunits of TFIIIB is shown in Fig. 6A. BRF was extensively cross-linked to the nontranscribed strand in the same region in which TBP interacts with DNA, whereas B" was cross-linked most efficiently to the transcribed strand in this same region. TBP was cross-linked mostly in the minor-groove region, except near bp 23 on the transcribed strand. If there is significant structural similarity between the TFIIIB-DNA complex and the TBP-TATA box complex, a more comprehensive interpretation of the data can be made by correlating the cross-linking information to the known crystal structure of TBP bound to a TATA box.

View larger version (119K): [in this window] [in a new window]   FIG. 6.   BRF and B" bind to opposite sides of the TBP-DNA complex. (A) Summary of DNA cross-linking to the BRF, B", and TBP subunits of TFIIIB within the region upstream of the start site of the SUP4 tRNA gene. The thickness of the arrows indicates the relative efficiency of cross-linking at each position. Bases and phosphodiester bonds that were modified are shaded. The BRF and B" cross-links are displayed on the left, and those of TBP are displayed on the right. (B to D) The space-filling model of the TBP-TATA element complex is shown with TBP in magenta and DNA in gray (33). DNA positions that have been shown to be photocross-linked to BRF and B" are highlighted in green and blue, respectively. The DNA structure has been modified from the original in that B-DNA has been added to both ends of DNA and the original DNA hairpin has been removed with the molecular modeling program Midas. One side of TBP is shown in panel B, and the structure is rotated 180° in panel C to visualize the other side of TBP. TBP is shown in ribbon form in panel D to better visualize the upstream or N-terminal stirrup region of TBP, and the structure is oriented such that the stirrup of TBP can be seen interacting with the minor groove of DNA. Illustrations were generated with RasMol version 2.6. TS, transcribed strand; NTS, nontranscribed strand.

Several lines of evidence from results presented here and from work by others (11, 24, 45) indicate that TBP binds to the TATA-like sequence of the tRNA gene in a manner similar to that of the TATA element found in many mRNA genes. Photoaffinity labeling with the S-P probes demonstrates that TBP is positioned at the TATA-like sequence upstream of the start site of transcription. The lack of TBP cross-linking with the AB and DB probes except around bp 23 indicates that throughout most of this region, TBP binds through the minor groove of DNA, consistent with the crystal structure. The cross-linking data from the phosphate backbone and the major groove of the SUP4 tRNA gene were correlated to the known crystal structure of TBP bound to the TATA box of the major late promoter of adenovirus through alignment of the TATA sequence (Fig. 3C and Fig. 6B to D). Corresponding positions in the phosphate backbone and the major groove were highlighted to indicate where BRF (green) and B" (blue) were shown to cross-link DNA.

Correlation of DNA cross-linking data to the crystal structure indicates that BRF most likely interacts with one side of the TBP-TATA box complex (Fig. 6B). The large distortion of the DNA structure due to bending by TBP causes the nontranscribed strand of the TATA box to be located on one side of TBP, whereas the transcribed strand of the TATA box is found on the opposite side of TBP. Examination of the other side of the TBP-TATA box structure shows that the B" subunit most likely interacts extensively with this surface, thus positioning B" on one side of TBP and BRF on the opposite side to clamp TBP in on the surface of the DNA (Fig. 6C). This kind of protein clamp could help explain the exceptionally tight DNA binding of the fully assembled TFIIIB complex. B" appears to extend far into the hole created through positioning of the major groove inward and can be cross-linked deep into this region (Fig. 6C, 30 NTS, 27 TS, and 23 TS). In comparison, BRF appears to extend less into this core region created by the major groove of DNA. On the surface contacted primarily by B", it is significant that the S-P probes cross-link both BRF and B" on the 3' side of bp 30 and 28. The S-P probes consist of two stereoisomers such that the photoreactive group is positioned in the major or minor groove. Consistent with the other positions that were scanned, B" is probably cross-linked by the stereoisomers positioned in the major groove, and BRF is probably cross-linked by those positioned in the minor groove. This interpretation is supported by the observation that only B" is cross-linked in the major groove at bp 27 (transcribed strand) with DB-dUMP. An interaction of BRF near the minor groove of DNA in this region on the transcribed and nontranscribed strands would be consistent with a segment of the BRF subunit contacting one stirrup of TBP, as for TFIIB and TBP (Fig. 6D).

One inconsistency between the cross-linking data and the TBP-TATA box crystal structure is that of TBP cross-linking to the major groove of DNA at bp 23. In order to better understand the structural significance of these cross-linking data and to ascertain the potential orientation of TBP in the complex, it was important to determine which region of TBP is cross-linked to the major groove of DNA at bp 23 in the transcribed strand. Identification of the region of TBP cross-linked to DNA at bp 23 was done by comparing the predicted cleavage products with the photoaffinity-labeled fragments of TBP from single-hit proteolysis. This approach has been successfully applied to the mapping of photoaffinity labeling sites of prokaryotic and eukaryotic RNA polymerases (5, 17, 37, 50, 52). Yeast TBP contains two cysteines at amino acids 78 and 164 and three internal methionines at amino acids 104, 121, and 197 (Fig. 7A).

View larger version (24K): [in this window] [in a new window]   FIG. 7.   The region of TBP centered around  sheet S4 is cross-linked to the major groove of DNA at bp 23 in the TFIIIB-DNA complex. (A) Yeast TBP (yTBP) is schematically depicted with alpha helices marked by bars labeled H1, H1', H2, and H2' and beta sheets depicted by bars labeled S1 to S5 and S1' to S5' (33). CNBr and NTCB cleave at methionine and cysteine residues, respectively, and formic acid (pH 2.5) cleaves at Asp-Pro linkages. The several cleavage sites are indicated above and below TBP by arrows. Proteolytic fragments that would be expected from a single-hit digestion with each of these reagents are shown with their predicted molecular masses. Secondary and less abundant proteolytic fragments that could be obtained by a combination of formic acid cleavage and cyanogen bromide cleavage are indicated by hatched bars. The region of TBP cross-linked to DNA is shown above the TBP sequence; the primary cross-linking site is represented by the box in the center, with flanking regions on each side. (B) Gel-purified, photoaffinity-labeled TBP (lane 1) was modified with NTCB (lane 2) and then cleaved by alkaline hydrolysis (lane 3) to generate predominantly an 18-kDa fragment. (C) Mutant TBP (lanes 1 to 3) with a cyclic AMP-dependent kinase site engineered at amino acid 78 was phosphorylated with heart muscle kinase and [-35S]adenosine--thiotriphosphate. Phosphorylated TBP and photoaffinity-labeled (PAL) TBP were gel purified and cleaved with cyanogen bromide for 10 min (lanes 2 and 4). The cleavage products were analyzed by Tricine-SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Lane 5 contained photoaffinity-labeled TBP that was exhaustively digested with cyanogen bromide after being electroblotted onto a PVDF membrane. The molecular masses (in kilodaltons) of the fragments are indicated to the left and right of the lanes.

Cleavage at cysteine residues was done with NTCB. NTCB modifies sulfhydryl groups in a protein, rendering them susceptible to alkaline hydrolysis (23). Single-hit cleavage with NTCB could potentially generate four fragments, two of which would have molecular masses of ~18 kDa and the other two of which would have molecular masses of ~8.5 kDa. An ~18-kDa labeled fragment would be expected if TBP were photoaffinity labeled between amino acids 79 and 164; an ~8.5-kDa fragment would be expected if either the N-terminal or the C-terminal region were photoaffinity labeled (amino acids 1 to 78 or 165 to 240, respectively). Photoaffinity-labeled TBP was partially cleaved with NTCB, and the predominant proteolytic product obtained (>90%) had a molecular mass of ~18 kDa. This result indicates that TBP was cross-linked in the region from amino acids 79 to 164 (Fig. 7B, lane 3). This region of TBP encompasses helices H1 and H2,  sheets S2 to S5, and most of  sheet S1'.

In order to further delineate the region of TBP that interacts with the major groove of DNA, cleavage at methionine residues was done with cyanogen bromide (Fig. 7A and C). A control sample and a molecular weight standard were created by engineering the cyclic AMP-dependent kinase site, GRRLSL, into wild-type TBP by changing cysteine 78 to arginine and aspartate 81 to serine. The mutant TBP was radiolabeled at serine 81 with [-³⁵S]adenosine--thiotriphosphate and heart muscle kinase under conditions where wild-type TBP was not detectably phosphorylated (data not shown). The proteolytic fragments of ³⁵S-labeled TBP cleaved by cyanogen bromide and separated on a Tricine-SDS-polyacrylamide gel had relative molecular masses consistent with the expected phosphorylation site. These labeled proteolytic fragments of TBP were used as a molecular weight standard for the proteolytic fragments of photoaffinity-labeled TBP (Fig. 7C, lane 2). The presence of all three proteolytic cleavage products in approximately equal amounts indicates that all three internal methionine residues were equally cut by cyanogen bromide. The electrophoretic mobility of the phosphorylated TBP proteolytic fragments is expected to be slightly faster than that of fragments from photoaffinity-labeled TBP. Cleavage of the photoaffinity-labeled protein with formic acid and diphenylamine cleaves DNA at purine residues and leaves two phosphate groups attached to the cross-linked deoxycytidine (42), whereas the phosphorylated TBP proteolytic fragments have a single phosphate group coupled to serine.

The conditions for chemical cleavage of the DNA cross-linked to TBP results in cutting at the Asp-Pro located at amino acids 19 and 20 (Fig. 7A). Chemical cleavage of the DNA with diphenylamine and formic acid resulted in ~17% of the protein being cleaved at the Asp-Pro site and generated a labeled 24.3-kDa fragment of TBP (Fig. 7C, lane 3). Cyanogen bromide cleavage of TBP under these conditions creates two sets of fragments: the primary products from the cyanogen bromide cleavage only and a set of less abundant fragments that are the result of cleavage at Asp-Pro and at a single methionine (Fig. 7A). Photoaffinity-labeled TBP cleaved with cyanogen bromide for 10 min produced three fragments having molecular masses of approximately 21.9, 13.4, and 11.5 kDa (Fig. 7C, lane 4). The lack of labeled proteolytic fragments of ~2.9 kDa (amino acids 1 to 19) and ~4.9 kDa (amino acids 198 to 240) shows that TBP is not cross-linked at the N or C terminus and is consistent with the NTCB cleavage results. The clear identification of the 13.4-kDa fragment was not possible, since it could correspond to proteolytic fragments from amino acids 1 to 121 or 122 to 140. The small amount of labeled 11.5-kDa fragment could be due to less efficient labeling at sites between amino acids 1 and 104 or to fivefold less efficient cutting at the Asp-Pro site, resulting in an 11-kDa fragment (amino acids 21 to 121). A 15.3-kDa labeled fragment could be expected and may be evident migrating slightly more slowly than the 13.4-kDa fragment. The ambiguities mentioned made it necessary to do extensive cyanogen bromide cleavage to better define the cross-linking site on TBP. Three fragments with molecular masses of 11.5 kDa (amino acids 1 to 104), 8.5 kDa (amino acids 122 to 197), and 1.9 kDa (amino acids 104 to 120) could be expected. The primary product obtained was an ~2-kDa fragment that unequivocally corresponds to the region of TBP between amino acids 105 and 120 (Fig. 7C, lane 5). The region of TBP from amino acids 105 to 120 corresponds to  sheet S4 and to edges of the S3 and S5  sheets that are part of the surface of TBP shown to contact the minor groove of the TATA element (33).

The region encompassing  sheet S4 of TBP is thus shown to be in close contact with the major groove of DNA at bp 23 on the transcribed strand as part of the fully assembled TFIIIB-DNA complex. It is evident that at this position in the TFIIIB-DNA complex, the TBP-DNA contacts deviate from those in the TBP-TATA DNA complex. The photoreactive group at bp 23 would be too far from TBP and sterically hindered to cross-link TBP if it were in the same conformation as in the TBP-TATA DNA complex (Fig. 8). Although the exact amino acid cross-linked to DNA at bp 23 has not been determined, it can be inferred because of the strong preference of dehydroazepine (generated from phenylnitrene) for nucleophilic side chains. In the region from amino acids 105 to 120 (Fig. 8, yellow), several nucleophilic amino acids are located on both sides of TBP (nucleophilic groups in black). The region of TBP bound close to the minor groove in the TBP-TATA DNA complex is devoid of exposed nucleophilic side chains. TBP is most likely cross-linked to bp 23 at lysine 120 and/or serine 118, because of steric constraints and the close proximity of these sites to bp 23. If DNA at bp 23 contacted the other side of TBP (Arg 105, Thr 112, or Lys 110), then the DNA (i) would be entering and exiting near the same surface of TBP and (ii) would require a larger deviation from the TBP-TATA DNA structure (Fig. 8).

View larger version (59K): [in this window] [in a new window]   FIG. 8.   The interaction of TBP with the 3' half of the TATA-like sequence in the TFIIIB-DNA complex is not the same as that of the TBP-TATA box complex. The region of DNA at bp 23 on the transcribed strand (TS) that cross-links TBP is highlighted in red by use of the same model as that shown in Fig. 6. The region of TBP cross-linked to bp 23 is highlighted in yellow, and the nucleophilic groups present in this region are shown in black. (A) The TBP-TATA box structure is shown to reveal one side of TBP with DNA projecting down from TBP and toward the transcription start site. The nucleophilic groups on this side of TBP that are within the region cross-linked to DNA are labeled (Lys 120 and Ser 118). (B) The TBP-TATA box structure is shown to reveal the opposite side of TBP (relative to that in panel A) with the upstream portion of DNA pointing directly outward. The nucleophilic side chains on this side of TBP are labeled as in panel A. DNA positions cross-linked to BRF and B" are highlighted in green and blue, respectively, except at bp 23.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The topography of the core region of the TFIIIB-DNA complex was investigated by focusing on a 10- to 15-bp region of DNA centered ~25 bp upstream of the start site of transcription and containing a TATA-like sequence. In this complex, TFIIIB was recruited to DNA through protein-protein interactions with TFIIIC bound to the downstream promoter elements box A and box B. The interactions of all three subunits of TFIIIB (TBP, BRF, and B") with the major and minor grooves of DNA were mapped by DNA photoaffinity labeling. DNA was modified by tethering a photoreactive group either from the phosphate backbone into the major and minor grooves (SP probes) or from the nucleotide base into only the major groove (AB or DB probes). Cross-linking results obtained with the AB and DB probes revealed which proteins interact with the major groove near specific sites; with SP probes, proteins interacting with the minor groove were also determined. In this manner, it was shown that TBP is in close contact with the TATA-like sequence TATATGTG, which starts 30 nucleotides upstream of the start site of transcription of the SUP4 tRNA^Tyr gene. TBP contacts within this segment of DNA are primarily with the minor groove of DNA, as evidenced by cross-linking of TBP by the S-P probes and not by the AB probes at most positions tested in this region. These cross-linking data demonstrate that the interactions of TBP with DNA in a TFIIIB-DNA complex are similar to those of TBP bound to a TATA box, where it primarily contacts the minor groove of DNA.

Other evidence for TBP binding to DNA in the TFIIIB-DNA complex in a manner similar to that with the Pol II TATA box has been shown by Joazeiro and colleagues (24). Although TFIIIC recruits and promotes the binding of TFIIIB to DNA upstream of the start site of transcription, it was shown that TATA sequences in this region influence the binding of TFIIIB within a certain distance from box A. The transcriptional start site and DNase I footprint of TFIIIB were altered by shifting the position of a TATA sequence within the TFIIIB binding region. TFIIIB reconstituted with an altered-specificity mutant of TBP (TBPm3) was properly positioned by use of the mutant TATA sequence TGTAAA, whereas wild-type TFIIIB was unable to bind to this sequence. These data indicate that (i) TBP does bind to DNA in the TFIIIB-DNA complex to help influence its position on DNA and (ii) its DNA binding characteristics are similar to those of TBP alone binding to the TATA element.

Given the evidence of the similarity of the DNA binding properties of TBP alone to those of TFIIIB, our DNA photocross-linking data were correlated to the known crystal structure of TBP bound to the TATA element. This analysis revealed that BRF and B" form a protein clamp on the TBP-DNA complex with BRF binding to one side of the TBP-DNA complex and B" binding to the opposite side. The major groove of the ~8-bp TATA sequence is pushed in toward itself to create a highly condensed region of DNA that serves as a scaffold for the interactions of BRF and B". Mapping of the DNA contact sites of BRF indicates that BRF binds to the outer layer of one side of this core region. Additional BRF contacts are found to encompass the C-terminal stirrup of TBP, the same stirrup region that interacts with the core region of TFIIB, as shown in the TFIIB-TBP-TATA crystal structure (40).

The region surrounding the N-terminal stirrup of TBP had been shown in two separate studies to be important for BRF binding (11, 45). One approach was to make radical amino acid changes on the surface of TBP by site-directed mutagenesis to determine which residues of TBP are critical for the binding of BRF. A second approach was to show that TFIIA competes for the binding of BRF to the TBP-DNA complex, whereas TFIIB does not compete for binding (11). The crystal structure of TFIIA-TBP-DNA has shown that TFIIA interacts with the N-terminal stirrup of TBP, the stirrup opposite that contacted by TFIIB (48). At first glance, these data and the results discussed earlier indicating that BRF is bound close to the C-terminal stirrup may appear to be conflicting. First, it is important to note that the interactions being examined in these studies are those of TBP alone bound to a TATA box with BRF. The N-terminal stirrup of TBP is therefore important for the preliminary contact between BRF and TBP, but additional interactions between BRF and TBP are likely to exist in the TFIIIB-DNA complex. We and others have obtained cross-linking data showing that the region of BRF showing sequence homology to TFIIB is cross-linked to DNA near the 5' end of the TATA-like region (reference 29 and results not shown). The BRF contacts with DNA between bp 26 and 30 are dependent on the presence of B" and suggest that the interactions of BRF with the C-terminal stirrup of TBP are B" dependent. B" makes extensive contacts within the core region of the major groove of DNA as it extends into this area from the side of the TBP-DNA complex opposite that bound by BRF. A TBP mutant (E93R) that has been shown to be defective only in the binding of B" is located on the same side of TBP as that shown to make contact with B" by DNA photocross-linking (11). The surface of B" making contact with the core region of DNA appears to be fairly devoid of nucleophilic side chains, probably entails a more hydrophobic region of the protein, as evidenced by cross-linking preference, and may help account for the nonionic binding properties of TFIIIB. At bp 23 in the transcribed strand, B" was cross-linked with phenyldiazirine-containing probes (DB probes) and not with phenylazide-containing probes (AB probes). Although not as pronounced, the same cross-linking preference was also observed at bp 27 (transcribed strand) and bp 30 (nontranscribed strand) for B". Aryl azides have a strong preference for nucleophilic side chains, whereas phenyldiazirines are more reactive, with no obvious side-chain preference (4, 8, 49). The protein clamp formed by sandwiching TBP on one side with BRF and on the other side with B" is likely to be responsible for the stability of the TFIIIB-DNA complex in the presence of high salt and polyanions.

Most of the DNA photocross-linking data are consistent with the canonical TBP-TATA element crystal structure, except for the very strong cross-linking of TBP in the major groove at bp 23. The highly efficient cross-linking of TBP with AB-dCMP and not with DB-dCMP at bp 23 indicates that a nucleophilic surface of TBP is in close contact with the major groove of DNA at this position. Peptide mapping of TBP photoaffinity labeled at bp 23 revealed that the region of TBP from amino acids 105 to 120 is close to the major groove of DNA at bp 23. The implications of this data are twofold. First, the demonstration of this region of TBP contacting the 3' side of the TATA-like sequence shows that TBP is oriented in the same direction on a tRNA gene as on a TATA-containing gene transcribed by Pol II. The orientation of TBP on the DNA is important to determine, because TBP appears not to have a strong orientation specificity, as the TBP homolog of Pyrococcus woesei has an orientation opposite that of the TBP-TATA box structure in eukaryotes (12, 34, 51). Second, the trajectory of DNA as it exits the downstream half of the TBP-DNA complex must be different from that in the TBP-TATA box complex. The distance from bp 23 to the cross-linked region of TBP is too large (15 Å) and there is too much steric interference for this cross-linking to occur if the structure of the core DNA with TBP were the same as the TBP-TATA box crystal structure.

We can postulate two models that would be consistent with the TBP-DNA cross-linking data at bp 23 and that differ by which side of TBP the DNA contacts as it exits from the 3' end of the complex. The region of TBP from amino acids 105 to 120 includes nucleophilic amino acids that are found on both sides of TBP, as shown in Fig. 8, and are the putative cross-linking sites. Changes in the trajectory of the DNA could be due to changes in the kinking of DNA near bp 23. TBP bends the TATA element by kinking DNA at two positions near the ends of the complex by inserting pairs of phenylalanines into the minor groove (33). One of these two positions is located between bp 7 and 8 in the TATA element and corresponds to bp 24 and 23 in the TATA-like sequence of the SUP4 tRNA gene. Elimination of the kink between bp 24 and 23 could help bring the major groove of DNA at bp 23 into closer proximity to Lys 120 and Ser 118 of TBP. This model would position the downstream DNA on the side of TBP that would be consistent with BRF binding to one side of TBP. Another model, with the B" side of TBP contacting the major groove at bp 23, is less likely because of (i) the drastic change in the trajectory of DNA required to bring bp 23 close to Arg 105, Thr 112, and Lys 110 and (ii) the potential steric clash of the DNA entering and exiting TBP on the same side of TBP.

B" was shown to have an important role in the formation of TBP contacts with the major groove of DNA at bp 23. TBP cross-linking at bp 23 is enhanced ninefold by the addition of B", while at other positions, the cross-linking of TBP is eliminated. The reduction of TBP cross-linking at these positions could be caused by efficient competition for the photoreactive group by BRF and B" bound at these regions or by more site-selective placement of TBP in the presence of B". Another indication that B" is involved in changing DNA contacts at bp 23 is the observation that B" also makes close contact with DNA at this position. B" is efficiently cross-linked by the S-P probes at bp 25 and 23 and, when the phenylazide is replaced with a phenyldiazirine, B" is the most efficiently cross-linked protein at bp 23 (transcribed strand). B" is less efficiently cross-linked at bp 24 (nontranscribed strand) with DB-dUMP, consistent with B" binding to DNA at this site.

Our evidence for the change in DNA trajectory in the TFIIIB complex from that of TBP bound to a TATA box is consistent with the DNA bending data for TFIIIB (6, 36). TFIIIB bound either to a tRNA gene or to the 5S rRNA gene bent DNA to a larger degree than TBP bound to the TATA box (46). After adjustment for the intrinsic bend in the SUP4 tRNA gene, the bend angle for TFIIIB was ~115 to 135°; with the 5S rRNA gene, it was ~105 to 140°. In comparison, the bend angle for TBP on the adenovirus major late promoter was ~95°. The observation for the 5S rRNA gene that the TFIIIB-DNA complex devoid of B" was not as bent as the complete TFIIIB-DNA complex coincides with our results indicating that the altered path of the 3' half of the TATA element DNA on TBP required the B" subunit.

DNA photoaffinity labeling continues to provide more details on the topography of Pol III transcription as different kinds of photoreagents are used and the data are correlated to the known X-ray crystallographic structures of some of the components of the transcription complex. Together, topological mapping by site-specific cross-linking and X-ray crystallographic analysis of subcomponents of the complex make it possible to obtain a comprehensive view of large protein assemblies such as that of the eukaryotic transcription complex. This study has also shown the need to use several different kinds of photoreagents to obtain complementary information. Finally, the kind of information obtained from DNA cross-linking is substantially enhanced when the region of the protein cross-linked is identified to better define the protein region interacting with DNA.