Alignment of the B" Subunit of RNA Polymerase III Transcription Factor IIIB in Its Promoter Complex*

TFIIIB, the central transcription initiation factor of the eukaryotic nuclear RNA polymerase (pol) III is composed of three subunits: the TATA-binding protein; Brf, the TFIIB-related subunit; and B", the Saccharomyces cerevisiae,TFC5 gene product. The orientation of the B" subunit within the TFIIIB-DNA complex has been analyzed at two promoters by two approaches that involve site-specific photochemical protein-DNA cross-linking: a collection of B" internal and external deletion proteins has been surveyed for those deletions that alter the interaction of B" with DNA or change the orientation of B" relative to DNA; a method for regionally mapping cross-links between specific DNA sites and 32P-end-labeled protein has also been applied. The results map an N-proximal segment of B" to the upstream end of the TFIIIB-DNA complex and amino acids 299–315 to the principal DNA-contact site, approximately 8 base pairs upstream of the TATA box. The analysis also indicates that a segment comprising amino acids 316–434 loops away from DNA, and locates the C-proximal 170 amino acids of B" downstream of the TATA box. Examination of two-cross-link products formed by DNA with adjacent and nearby photoactive nucleotides supports the conclusion that Brf and B" share an extended interface along the length of the TFIIIB-DNA complex.

TFIIIB, the central transcription initiation factor of the eukaryotic nuclear RNA polymerase (pol) III is composed of three subunits: the TATA-binding protein; Brf, the TFIIB-related subunit; and B؆, the Saccharomyces cerevisiae, TFC5 gene product. The orientation of the B؆ subunit within the TFIIIB-DNA complex has been analyzed at two promoters by two approaches that involve site-specific photochemical protein-DNA cross-linking: a collection of B؆ internal and external deletion proteins has been surveyed for those deletions that alter the interaction of B؆ with DNA or change the orientation of B؆ relative to DNA; a method for regionally mapping crosslinks between specific DNA sites and 32 P-end-labeled protein has also been applied. The results map an Nproximal segment of B؆ to the upstream end of the TFIIIB-DNA complex and amino acids 299 -315 to the principal DNA-contact site, approximately 8 base pairs upstream of the TATA box. The analysis also indicates that a segment comprising amino acids 316 -434 loops away from DNA, and locates the C-proximal 170 amino acids of B؆ downstream of the TATA box. Examination of two-cross-link products formed by DNA with adjacent and nearby photoactive nucleotides supports the conclusion that Brf and B؆ share an extended interface along the length of the TFIIIB-DNA complex.
The eukaryotic (nuclear) RNA polymerases are brought to their promoters by relatively complex core transcription apparati. In the RNA polymerase (pol) 1 III transcription system of Saccharomyces cerevisiae, which is the focus of this work, the polymerase recruitment function is executed by the core transcription factor (TF) IIIB. TFIIIC and TFIIIA, the other components of the core transcription apparatus, bind DNA and serve as assembly factors for TFIIIB; the six-subunit TFIIIC interacts directly with TFIIIB, and TFIIIA serves as a 5 S rRNA gene-specific platform for TFIIIC. TFIIIB is composed of three subunits: the TATA-binding protein (TBP), Brf, and BЉ (Tfc5). TBP co-directs binding of TFIIIB to very strong TATA boxes. At promoters that lack these intrinsic TBP-binding sites, TFIIIC functions as an assembly factor that deposits TFIIIB on its upstream DNA site (reviewed in Ref. 1). In both kinds of situations, TBP is located close to DNA (2).
The 596-amino acid Brf is joined from two evolutionarily distinct parts. Its designation as the TFIIB-related factor derives from the homology of its N-proximal half to TFIIB (3)(4)(5). Just as TFIIB is able to recruit pol II to the transcriptional start site, so the principal polymerase recruitment capacity of TFIIIB resides in the corresponding, N-proximal, half of Brf. The C-proximal half of Brf is pol III-specific (6), and has no sequence-homologous counterparts in the pol I and pol II transcription apparati. The principal TBP and BЉ affinities of Brf, and also its TFIIIC affinity, reside in this C-proximal half (7). However, the N-proximal half of Brf alone has sufficient affinity for TBP and BЉ to form a TFIIIB-DNA complex at a strong TATA box that is able to recruit pol III to accurately initiated transcription (7).
Since TFIIB and the N-proximal half of Brf sit in corresponding locations in their respective DNA complexes (7,8), and exercise similar functions, it is surprising that Brf and TBP are not by themselves competent to direct transcriptional initiation by pol III. In fact, the 594-amino acid BЉ is absolutely required for transcription by pol III, in duplex DNA or chromatin, at TATA-containing and TATA-less promoters, in vivo as well as in vitro. It is also BЉ that makes the TFIIIB-DNA complex extraordinarily stable (9).
In the experiments that are reported here, we have mapped BЉ along its DNA site in the TFIIIB-DNA complex; BЉ external and internal deletion proteins that form stable TFIIIB-DNA complexes have been examined by photochemical protein-DNA cross-linking along the extended DNA site in order to find out which of these deletions alter the interaction of BЉ with DNA or change the orientation of BЉ relative to DNA. A relatively simple and highly sensitive method has also been developed to map cross-links from specific DNA sites to their targeted region on 32 P-end-labeled protein.
Eluted proteins were subjected to partial cleavage with 100 mM CNBr or 10 mM NTCB. For CNBr partial cleavage, samples were brought to low pH with 1% SDS and 0.05 N HCl. CNBr (100 mM) was then added for 20 min at 21°C. Further reaction was quenched by adding 0.25 volumes of 100 mg/ml dithiothreitol in 1 M NaHepes (pH 7.8). For NTCB partial cleavage, samples were treated with 10 mM NTCB for 30 min at 37°C for cyanylation of cysteine residues. The pH was then adjusted to 9.0 with Tris base, and samples were incubated overnight at 37°C for the subsequent proteolytic cleavage. The 32 Plabeled fragments generated by NTCB or CNBr cleavage were resolved on 11-15% SDS-polyacrylamide gels. Comparisons of cleavage patterns of free and DNA-cross-linked 32 P-labeled BЉ were made on phosphoimage profiles.

Photochemical Cross-linking of BЉ Deletion Mutants-Previ-
ous examination of the internal structure of a TFIIIB-DNA complex by photochemical cross-linking revealed that BЉ crosslinks to DNA between 43 and 2 bp upstream of the transcriptional start site (16,17). In order to map the locations of individual domains of BЉ relative to DNA, we have analyzed the photochemical cross-linking patterns of BЉ with internal and external deletions, using ABdUMP incorporated at specific sites along the TFIIIB-binding sites of the SUP4 and SNR6 genes ( Fig. 1). Each DNA photoprobe also has a radioactive nucleotide incorporated next to, or in close vicinity to, its photoactive nucleotide(s). TFIIIB complexes containing either fulllength or truncated BЉ were assembled on each of these DNA photoprobes; reaction mixtures were then UV-irradiated, digested with nucleases, and analyzed by SDS-PAGE. The efficiency of TFIIIB-DNA complex formation was monitored in parallel by electrophoretic mobility-shift analysis of an UVirradiated, but not nuclease-treated, portion of each reaction mixture. If deletion of a specific segment of BЉ results in the loss of cross-linking at a particular DNA site, this suggests localization of that peptide segment in the vicinity of that DNA site (so long as complex formation is not correspondingly diminished). In the case of the SNR6 gene promoter, unidirectional assembly of TFIIIB-DNA complexes is specified by a modified TATA-box (TGTAAATA) in conjunction with the mutant TBPm3 (15). At the SUP4 tRNA promoter, with its very weak TATA box, TFIIIB-DNA complex assembly requires the transcription factor TFIIIC, and is unidirectional (15).
The SUP4 Promoter-Efficiencies of forming heparin-stable TFIIIB-DNA complexes and photochemical cross-linking of each BЉ deletion mutant and wild type BЉ were compared for the photoactive DNA probes shown in Fig. 1A. BЉ lacking its N-terminal 262 amino acids or its C-terminal 130 amino acids remains competent to assemble into a heparin-resistant TFIIIB-DNA complex via the TFIIIC-dependent assembly pathway on the SUP4 gene (but does so relatively inefficiently; Ref. 11). Full-length BЉ cross-linked most efficiently to AB-dUMP positioned between bp Ϫ38 and bp Ϫ32 on the SUP4 gene ( Fig. 2A; cf. Ref. 16). Removal of 223 or 262 amino acids from the N terminus of BЉ increased the efficiency of crosslinking between bp Ϫ38 and bp Ϫ30 more than 2-fold compared with full-length BЉ (adjusted for differences in formation of heparin-resistant complexes; Table I, part A, and Fig. 2A). Further downstream, between bp Ϫ26 and Ϫ2, the cross-linking efficiencies of BЉ-(224 -594) and full-length BЉ did not differ significantly.
The combination of low level formation of heparin-resistant TFIIIB-DNA complexes with BЉ-(263-594) and low efficiency cross-linking of BЉ between bp Ϫ26 and bp Ϫ2, resulted in cross-linking signals too close to background to be reliably quantified, but the observed levels were consistent with unimpaired cross-linking of BЉ-(263-594) at these positions (Table I,  part A). C-terminally truncated BЉ-(1-464) and BЉ-(1-487) were not sufficiently well resolved from Brf in SDS-PAGE to quantify reliably. It was, however, possible to assess that BЉ-(1-487) was not significantly impaired in cross-linking to bp Ϫ38/Ϫ37, Ϫ33/Ϫ32, and Ϫ30, where BЉ cross-links efficiently (Table I, part A). N-and-C-terminally truncated BЉ-(40 -487) did resolve well from Brf, and its cross-linking to all probes tested was unimpaired (Table I, part A, and Fig. 2B). These results indicate that the cross-links of BЉ to the SUP4 gene (more specifically to the bp Ϫ38/Ϫ2 segment of this gene) primarily involve its amino acid 224 -487 segment; this is also the active core of BЉ for SUP4 transcription (11). Internal deletion mutants of BЉ, which span much of the region of BЉ not covered by the N-and C-terminal truncations, and which are capable of being assembled into heparin-resistant DNA complexes, were also examined. Only BЉ with amino acids 291-310 deleted displayed a defect in cross-linking, predominantly with probe Ϫ38/Ϫ37 for which cross-linking efficiency is reduced more than 10-fold (Fig. 2C, lanes 3 and 5) and, to a lesser extent, with probes Ϫ33/Ϫ32 and Ϫ30 (Table I, part A). Fig. 2C shows two other aspects of this defect. 1) Heparin did not diminish cross-linking of intact BЉ (lanes 2 and 3), but substantially reduced cross-linking of BЉ⌬291-310 without substantially reducing the cross-linking of Brf (lanes 4 and 5).
The SNR6 Promoter-On the SNR6 gene (18,19), BЉ truncated by N-terminal deletion to amino acid 186 or by C-terminal deletion to amino acid 487 was not deficient in cross-linking (Table I, part B). N-terminal truncation to amino acid 224 or 263 somewhat lowered cross-linking at bp Ϫ13/Ϫ12 and Ϫ5 (20 -40% of that obtained with intact BЉ; Table I, part B), but a comparable depression of cross-linking was not observed when TFIIIB complexes with the SUP4 gene were probed at the close-by Ϫ14/Ϫ12 and Ϫ3/Ϫ2 sites (Table I, part A). Since no single region of BЉ is required for TFIIIB-SNR6 DNA complex formation, a more complete set of internal deletions, spanning regions not covered by N-and C-terminal truncations, could be examined at this promoter. BЉ cross-links most efficiently to bp Ϫ39/Ϫ38 on the SNR6 gene (7); only at bp Ϫ33 and Ϫ13/Ϫ12 do cross-linking efficiencies exceed 10% of cross-linking to bp Ϫ39/ Ϫ38. Only BЉ⌬272-292 was deficient in cross-linking to probe Ϫ39/Ϫ38 (20% of the efficiency of cross-linking to intact BЉ; Table I, part B, and Fig. 2D). Curiously, BЉ⌬291-310, which was deficient for cross-linking at a similar location on the SUP4 gene, was not significantly impaired for cross-linking on the SNR6 gene (Table I and Fig. 2; BЉ⌬272-292 was not examined on the SUP4 gene because it does not assemble into a stable, heparin-resistant complex; Ref. 11). BЉ⌬424 -438 displayed somewhat lower cross-linking to bp Ϫ28 and Ϫ13/Ϫ12 (20 -40% of the cross-linking efficiency with intact BЉ) and BЉ⌬409 -421 was somewhat impaired in cross-linking to bp Ϫ5 (40% efficiency relative to intact BЉ).
Comparisons of cross-linking to the SUP4 and SNR6 genes are complicated by the fact that TFIIIC positions TFIIIB somewhat heterogeneously onto the AT-rich upstream sequence of the SUP4 gene (20), whereas assembly on the SNR6 gene directed by TBPm3 is fixed at the TGTA box. One would therefore expect a "blurring" in the BЉ cross-linking pattern to the SUP4 gene relative to SNR6 (i.e. efficient cross-linking be- tween bp Ϫ38 and Ϫ30 on the SUP4 gene, but predominantly at bp Ϫ39/Ϫ38 on the SNR6 gene). Conversely, even if as few as 2% of TFIIIB complexes on the SNR6 gene were bound in the reverse orientation (cf. Ref. 15), they might make a significant contribution to cross-linking at bp Ϫ13/Ϫ12 (through the BЉ segment that cross-links efficiently to bp Ϫ39/Ϫ38 in the opposite orientation of TFIIIB).
Split BЉ-BЉ assembly into a TFIIIB-DNA complex buries two regions that are more surface-exposed in the free protein (as judged by hydroxyl radical footprinting): region I, covering amino acids ϳ390 -470; and region II, covering amino acids ϳ270 -305 (11). The above experiments clearly suggest that region II lies close to the major sites of BЉ cross-linking on the SNR6 and SUP4 genes (since BЉ⌬291-310 depressed crosslinking to the SUP4 gene and BЉ⌬272-292 to the SNR6 gene).
In order to gain further information about the location of region II relative to DNA, BЉ was split at amino acid 370 (which is highly accessible to hydroxyl radical cleavage in TFIIIB-DNA complexes; Ref. 11). Heparin-resistant TFIIIB-DNA complexes can be formed on both the SUP4 and SNR6 genes only when both parts of this split BЉ (amino acids 1-370 and 371-594) are provided together. The transcriptional activity of these TFIIIB-DNA complexes is, however, diminished (data not shown). Fig.  3 compares the cross-linking profiles of wild type and split BЉ on the SUP4 and SNR6 genes. Cross-linking to the SUP4 gene at bp Ϫ38/Ϫ37, Ϫ33/Ϫ32, and Ϫ30 is contributed almost entirely by amino acids 1-370 of BЉ, but there is some crosslinking of the C-terminal amino acids 371-594 to bp Ϫ33/Ϫ32 (Fig. 3A). The weak cross-linking of intact BЉ to sites downstream of bp Ϫ30 on the SUP4 gene appears to involve solely its C-terminal half. Similarly, BЉ-(1-370) dominates cross-linking at bp Ϫ39/Ϫ38 of the SNR6 gene (Fig. 3B), but some crosslinking of the C-terminal segment is also apparent at this site. In contrast to the cross-linking pattern at bp Ϫ33/Ϫ32 of the SUP4 promoter, most of the cross-linking of BЉ at bp Ϫ33 of SNR6 is contributed by its C-terminal segment. As on the SUP4 gene, most of the C-terminal BЉ segment cross-links also to bp Ϫ28, Ϫ13/Ϫ12, and Ϫ5, but low level cross-linking of the N-terminal segment at these sites also persists.
A High Molecular Weight Photoadduct-TFIIIB-DNA complexes with the Ϫ38/Ϫ37 SUP4 probe, which contains two ABdUMP residues, yielded a high molecular weight material cross-linking adduct, whose mobility depended on the BЉ external deletion mutant used (Fig. 4, lanes 1 and 3). Such high molecular weight bands have been characterized as the products of multiple cross-linking events (21). That this particular multiply cross-linked product contains both BЉ and Brf was demonstrated in the following way. The mobility of the high molecular weight band increased when BЉ was truncated, indicating that this DNA-protein adduct contains BЉ (Fig. 4, lane 3). That it also contains Brf was shown by using split Brf. The two Brf fragments, Brf-(1-282) and Brf-(284 -596), together form a stable and transcriptionally fully competent TFIIIB complex on the SUP4 gene (7). The cross-linked products of the TFIIIB-SUP4 gene complex formed with intact and split Brf also yielded high molecular weight bands with different electrophoretic mobilities (Fig. 4, lane 2). Since the high molecular weight Brf-BЉ-DNA photochemical adduct has also been observed in UV-irradiated TFIIIB complexes formed on SUP4 photoprobes Ϫ33/Ϫ32, Ϫ22/Ϫ21, Ϫ19/Ϫ17, Ϫ14/Ϫ12, and Ϫ3/Ϫ2 (data not shown), we conclude that Brf and BЉ share an extended interface with DNA.
Partial CNBr Cleavage of BЉ-DNA Photoadducts-Use of internal deletions to map the orientation of a protein along its DNA site is subject to the restriction that those deletions should not drastically change the protein-DNA alignment. A second approach, which is free of that restriction, was developed for mapping specific BЉ segments to the vicinity of specific sites on the SUP4 and SNR6 genes. An example of the mapping procedure is shown in Fig. 5. N-proximally 32 P-labeled BЉ-(138 -594) was used to form TFIIIB-DNA complexes with unlabeled Ϫ39/Ϫ38 SNR6 photoprobe and subjected to UV-irra-   c Close proximity to Brf on the separation gel prevented quantification for DNA sites that weakly cross-link to full-length BЉ. d Cross-linking efficiency normalized by comparing BЉ/Brf cross-linking ratios to those obtained with full-length BЉ. diation (Fig. 5A). The [ 32 P]BЉ-DNA adduct was recovered from a 6% polyacrylamide-SDS gel (Fig. 5B, lane 3) and subjected to partial CNBr cleavage. The cleavage products were then resolved on a 13% polyacrylamide-SDS gel (Fig. 5C) . This specifies that the major site of cross-linking of BЉ to the SNR6 gene at bp Ϫ39/Ϫ38 is situated between amino acids 299 and 315. An identical site between Met-298 and Met-315 was identified for the multiply crosslinked products identified in Fig. 5B (data not shown). Figs. 6 and 7 summarize the results of this method of mapping BЉ cross-links to the SNR6 gene at bp Ϫ42, Ϫ39/Ϫ38, Ϫ33, Ϫ23/Ϫ22, and Ϫ13/Ϫ12 by partial CNBr and NTCB (cysteinespecific) cleavage, respectively. Panel A of Fig. 6 shows that, when BЉ-(138 -594) is cross-linked to the SNR6 Ϫ42 probe, products of CNBr cleavage C-terminal of Met-222 are shifted to lower mobility, implying that the BЉ-DNA link is located between amino acids 223 and 276. Panel B displays the profile of the experiment with probe Ϫ39/Ϫ38 shown in Fig. 5. The Met-298 peak was consistently somewhat reduced relative to the Met-276 peak (in seven out of seven experiments), suggesting that a subsidiary cross-linking site of BЉ is located between amino acids 277 and 297. Panel A of Fig. 7 confirms that the bulk of the cross-linking to the Ϫ39/Ϫ38 probe occurs C-terminal to Cys-280.
Panel C of Fig. 6 shows that shifting the site of DNA crosslinking downstream by only 5 bp, to bp Ϫ33, shifts the protein side of the cross-link far toward the C-end of BЉ; only CNBr cleavage products to the carboxyl side of Met-435 are shifted to lower electrophoretic mobility (the products of cleavage at Met-425 and Met-434 fail to separate on these gels). The corresponding NTCB cleavage pattern (Fig. 7, panel B) shows that the point of DNA attachment is to the amino side of Cys-485. Thus, the cross-link to bp Ϫ33 is located between amino acids 425 and 485 of BЉ. The result supports the evidence presented in Fig. 3B that the amino acid 371-594 segment of BЉ crosslinks to bp Ϫ33 of the SNR6 gene.
Cross-linking to the SNR6 gene at bp Ϫ23/Ϫ22 and Ϫ13/Ϫ12 has also been examined in this manner. In these cases, the level of cross-linking was low enough that the background of free BЉ (see Fig. 5B, lane 2) makes the outcome of the analysis more tentative. Panels D and E of Fig. 6 display the CNBr cleavage profiles of probes Ϫ23/Ϫ22 and Ϫ13/Ϫ12, respectively. Both profiles imply that the cross-linking occurs at the Cterminal side of Met-425 and/or Met-434. Partial cleavage at cysteine indicates that at least part of the cross-linking to bp Ϫ13/Ϫ12 occurs within the amino acid 426 -485 segment (Fig.  7, panel C).
The same method was used to map segments of BЉ that are located in vicinity to bp Ϫ38/Ϫ37, Ϫ33/Ϫ32, Ϫ30, and Ϫ26 of the SUP4 gene (in TFIIIB-DNA complexes assembled with TFIIIC and subsequently stripped of the latter with heparin). The three upstream-lying sites of cross-linking generated nearly identical partial CNBr digestion profiles (Fig. 8, panels  A-C) indicating that the segment of BЉ that cross-links to these three sites lies between amino acids 299 and 314. (There is an indication, at or near the limits of reliability, that the amino acid 277-297 segment makes a small contribution to DNA cross-linking at bp Ϫ38/Ϫ37, but not at bp Ϫ30.) It is feasible for the amino acid 299 -314 segment to span 8 base pairs. However, as proposed above, a more likely explanation is that TFIIIC positions TFIIIB heterogeneously over the AT-rich upstream region of the SUP4 gene promoter (20), and that the amino acid 299 -314 segment generates the highest levels of cross-linking, wherever it is located. Heterogeneous placement of TFIIIB by TFIIIC may contribute to the less complete shift to high molecular size of partial CNBr products with C termini between Met-315 and Met-425/M434 with probe Ϫ30 (panel C). Panel C indicates that the photo-adduct at bp Ϫ30 primarily links amino acids 299 to 314 and, with much lower efficiency, amino acids 435 to 512; however, the background of free BЉ was not insignificant with this probe. Shifting the DNA end of the cross-link another 4 bp downstream, to Ϫ26, changed the CNBr cleavage pattern, with the BЉ cross-link now completely partitioned to the C-terminal side of Met-434. DISCUSSION The Alignment of BЉ in the TFIIIB-DNA Complex-BЉ is, to varying degree, accessible to cross-linking by ABdUMP placed site-specifically along the length of the TFIIIB-DNA complex (16). However, it is most efficiently cross-linked to DNA upstream of the TATA box (7). BЉ binding to the TBP-Brf-DNA complex extends the DNA footprint ϳ10 bp upstream of the TATA box, and is stabilized by an additional 15-20 bp DNA stretch upstream of the TATA box (see Ref. 22 and Footnote 2). Thus, the DNA segment that is additionally covered when BЉ adds to the TBP-Brf-DNA complex harbors a site of efficient BЉ cross-linking and contributes to the stability of the TFIIIB-DNA complex.
A central ϳ225-amino acid segment of BЉ appears to encompass its functional core. Two domains, one at each end of this core region (amino acids 272-292 and 424 -449), are required for TFIIIC-dependent transcription in vitro, and one or the other of these segments is required for TFIIIC-independent transcription (11). An additional and partly overlapping ϳ65amino acid segment (amino acids 355-421) is required for transcription of linear DNA (17).
The principal target for DNA cross-linking of BЉ lies between amino acids 277 and 315, i.e. it encompasses the N-proximal "essential" amino acid 272-292 segment. This segment of BЉ faces the upstream part of the DNA site occupied by TFIIIB. Thus, the DNase I footprint extension that is generated when BЉ enters the BЈ-DNA complex is at least partly due to a direct BЉ-DNA interaction.
The N-proximal 223 amino acids of BЉ are not essential for transcription of bare DNA (11). This segment of BЉ diminishes DNA-protein cross-linking upstream of the TATA box at least 2-fold (Table I), as though it formed an obstruction of the corresponding DNA-interacting segments of BЉ. Deleting BЉ amino acids 1-185 has the same effect on cross-linking (Table  I). A segment of BЉ extending approximately from amino acid 190 to amino acid 210 becomes more accessible to cleavage by hydroxyl radical upon entry into the TFIIIB-DNA complex (11). This is consistent with the notion that DNA access of BЉ might be blocked by its internal folding, and uncovered upon formation of the TFIIIB-DNA complex. The 137 N-terminal amino acids of BЉ are also removed in making 32 P-end-labeled BЉ. This may inadvertently facilitate the mapping of protein segments cross-linking to specific DNA sites upstream of the TATA box by generating higher efficiencies of cross-linking. However, if such an effect exists, it is quantitative rather than qualitative, since cross-linking of 32 P-labeled full-length BЉ to the SNR6 Ϫ39/Ϫ38 probe and the SUP4 Ϫ38/Ϫ37 probe likewise mapped to amino acids 299 -315 (data not shown).
The disposition of BЉ relative to DNA has been most clearly mapped in the SNR6 gene-TFIIIB complex, as detailed below.
3) Moving just 5 (or 6) bp toward the transcriptional start, to bp Ϫ33, shifts the site of the BЉ-DNA cross-link to the amino acid 435-485 segment. Thus, the intervening amino acids 316 -434 must be positioned away from DNA. This part of BЉ includes the amino acid 355-421 segment, which is required for transcription of linear DNA, and the amino acid 424 -449 segment, which is required for TFIIIC-dependent transcription; it extends into the amino acid 390 -470 "region I" segment, which is protected from hydroxyl radical cleavage upon forming the TFIIIB-DNA complex (11). Since the amino acid 316 -434 segment appears not to lie close to DNA, protection of region I from hydroxyl radical cleavage must be due to protein-protein interaction. A recently sequenced S. pombe open reading frame (NCBI accession CAA22645) considered to be a candidate homologue of S. cerevisiae BЉ has 31% identical (65% homologous) sequence in the region I segment.
4) Cross-linking to BЉ downstream of the SNR6 TATA box principally involves the C-proximal amino acid 371-594 segment (Fig. 3B); the cross-link appears to be located C-terminal to amino acid 425 (Fig. 6) and N-terminal to amino acid 487 (Table I), but more precise mapping has proved inconclusive (Fig. 7). This part of BЉ may not be uniquely positioned relative to DNA. 5) When BЉ enters into the TFIIIC-BЈ-DNA complex, it en-  Fig. 5A; gel-isolated free and DNA-linked [ 32 P]BЉ-(138 -594) were reacted with NTCB and cleaved as described under "Materials and Methods." The 32 P-labeled peptide fragments of the free and cross-linked [ 32 P]BЉ-(138 -594) were then resolved by SDS-PAGE and analyzed as described for Fig. 6. Phosphoimage density profiles of the NTCB cleavage products of free and DNA-linked [ 32 P]BЉ-(138 -594) (thin and thick lines, respectively), are normalized at the smallest 32 P-labeled polypeptide.
hances DNase I cleavage around the start site of transcription and increases DNase I protection between bp Ϫ14 and Ϫ30 attributable to the BЈ complex (11). Upon addition to a TFIIICindependent BЈ-DNA complex, BЉ generates markedly increased protection between bp Ϫ21 and Ϫ12 from MPE-Fe(II) cleavage (23) and de novo protection between bp Ϫ22 and-18 from hydroxyl radical cleavage (22). The weak cross-linking we have observed between BЉ and sites downstream of the TATA box appears to map to the C-terminal amino acid 426 -594 segment of BЉ. However, removal of most of this segment (amino acids 465-594) has no effect on the TFIIIB footprint (11). This could imply that, if BЉ directly generates the BЉ-dependent protection downstream of the TATA box, this interaction involves the amino acid 426 -464 segment, presumably through the minor groove, since cross-linking to the major groove-protruding photoreactive side chain of ABdUMP is weak (2). The decrease in cross-linking of BЉ⌬424 -438 with SNR6 probe Ϫ13/Ϫ12 might, accordingly, reflect the disposition of this seg-ment of BЉ 10 bp downstream of the TATA box. Alternatively, and more probably, the downstream protection generated by BЉ entry results from a change in the path of DNA that brings Brf into closer proximity with downstream DNA sequence. This may predominantly be the result of BЉ-Brf interactions that constrain the path of DNA within the TFIIIB-DNA complex.
Alternative Placements on the SUP4 Gene-The fact that TFIIIC-directed transcription of the SUP4 gene generates secondary start sites at ϩ4 and ϩ8 suggests that TFIIIB may not be placed at a unique location on this promoter by TFIIIC (20). A comparison of the BЉ mapping experiments in Figs. 6 and 8 is consistent with this interpretation. The amino acid 299 -314 segment principally cross-links to bp Ϫ38 and-37 of the SUP4 gene with a small contribution to cross-linking from amino acids 277-297. This is what is also seen at bp Ϫ39 and Ϫ38 of the SNR6 gene. However, the amino acid 299 -314 segment also cross-links to the SUP4 promoter at bp Ϫ33 and Ϫ32, and makes the major contribution to cross-linking at bp Ϫ30. The amino acid 435-512 segment makes no significant contribution to cross-linking at bp Ϫ33/Ϫ32, may make a minor contribution to cross-linking at bp Ϫ30, and only makes the major contribution further downstream at bp Ϫ26.
The BЉ-Brf Interface-The ability of DNA with more than one photoactive nucleotide to form multiple cross-links has been exploited in mapping the interface of BЉ and Brf. Detailed analysis (Fig. 4) shows that a high molecular weight DNAprotein adduct formed with the SUP4 Ϫ38/Ϫ37 probe contains both BЉ and Brf. High molecular weight bands were also generated at other sites by DNA probes with two neighboring ABdUMP residues. We conclude that BЉ and Brf share an extensive interface along the length of the TFIIIB-DNA complex. This is consistent with evidence that each half of Brf is able to recruit BЉ to a TFIIIB-DNA complex (7). The N-terminal half of Brf brings the amino acid 291-310 segment of BЉ into close proximity to DNA upstream of the TATA box, and the C-terminal half of Brf brings BЉ (presumably the amino acid 426 -487 segment) into less close DNA proximity downstream of the TATA box. It is the N-terminal half of Brf that dominates Brf cross-linking downstream of the TATA box in the TFIIIB-DNA complex, but this cross-linking requires both BЉ and the C-terminal half of Brf (7). This suggests that DNA protection (from DNase I) downstream of the TATA box is directly provided by the N-terminal half of Brf, held in place by BЉ and the C-terminal half of Brf.
This inquiry was partly motivated by the observation that BЉ-(263-594), BЉ⌬272-292, and BЉ⌬409 -421 generates start site-proximal aberrations in the TFIIIB-TFIIIC-DNA complex footprint, with BЉ⌬272-292 and BЉ⌬409 -421 generating identical effects (11). One interpretation of this observation is that regions I and II of BЉ are situated in close proximity to each other (11). The cross-linking of these two regions of BЉ to DNA sites that are separated by only 5 or 6 bp (Fig. 6) supports this contention. Additional cross-linking of the amino acid 426 -487 segment of BЉ to sites downstream of the TATA box suggests that TFIIIB bends DNA (23) so as to bring the two flanks of the TATA box closer together. Somewhat depressed cross-linking downstream of the TATA box is seen for BЉ263-594, BЉ⌬424 -438, and BЉ⌬409 -421 (Table I). This may reflect the proximity of these two parts of BЉ within the TFIIIB-DNA complex and subtle alterations in the path of DNA when these regions of BЉ are deleted.
Acknowledgments-We are grateful to A. Grove, S. Kolesky, M. Ouhammouch, C. Brent, and G. A. Letts for advice and technical help, and A. Grove for helpful comments on the manuscript.