Effects of N- and C-terminal truncations in Brf on the TFIIIC-independent assembly of B′ (Brf plus TBP)-DNA and TFIIIB-DNA complexes. (a) The 75-bp TA-30-B6 probe for EMSA for TFIIIC-independent assembly of TBP, Brf, and B". (b) EMSA for TFIIIB-DNA complex formation. Ten femtomoles of the TA-30-B6 probe was incubated with 10 fmol of TBP, 50 fmol of full-length Brf or the indicated truncated Brf, and B" (1× = 50 fmol), as indicated above each lane. Reaction conditions were otherwise as specified in Materials and Methods, and electrophoresis was done in Mg²⁺-containing native gels. Control reactions (not shown) demonstrated that Brf-DNA complexes did not form in the absence of TBP. TBP-DNA, B′-DNA, and TFIIIB-DNA complexes are identified at the right.

Transcription activities and interactions of Brf fragments. Formation of TFIIIB-DNA and B′ (TBP-Brf)-DNA complexes with the TA-30-B6 DNA probe (Fig. 1a) was analyzed by gel retardation. Transcription was measured with the SNR6 gene-related pU6_LboxB construct (Fig. 3a). The locations of landmark features of Brf—the TFIIB-homologous putative Zn-binding domain, the two TFIIB-related sequence repeats, and the three segments of conserved sequence among fungal Brfs—are indicated at the top, and the N- and C-terminal Brf deletion mutants are designated at the left. The four columns at the right summarize the ability of each truncated Brf to allow formation of B′-DNA and TFIIIB-DNA complexes (nd, not determined; ∗, unstable to gel electrophoresis but detected by DNA-protein photochemical cross-linking) and to allow TFIIIC-independent transcription directed by the SNR6 (U6) and δ TATA boxes present on plasmid pU6_LboxB (+++, 100%; ++, 30 to 80%; +, 5 to 20%; ±, <1 to 4%; −, not reliably detected).

TFIIIC-independent transcription. (a) The pU6_LboxB construct. The start sites and termination sites of leftward and rightward transcription directed by the SNR6 (U6) TATA box and by the δ TATA box are indicated. Rightward transcription initiating to the right of the SNR6 TATA box can be directed by TFIIIC binding to boxes B and A. (b) TFIIIC-independent transcription of pU6_LboxB with Brf fragments individually and in combination. Transcripts are identified at the side [R.M., recovery marker; ∗, downstream-shifted initiation with Brf(284-596)], and Brf fragments are indicated above the lanes. Transcription was performed with 100 fmol of pU6_LboxB DNA, 16 fmol of pol III, 100 fmol of TBP, 100 fmol of B", and 300 fmol of Brf fragment or intact Brf (lanes a to h) or as specified above lanes i to s. The weaker complementation of Brf(1-320) with Brf(284-596) was analyzed in lanes q to s with additional TBP (200 fmol) and B" (300 fmol).

Mapping the disposition of Brf fragments in transcription factor complexes by photochemical cross-linking: Brf split at amino acid 283. (a) DNA probes. The sequence of the SNR6 gene probe with a TGT mutant TATA box is shown. Locations at which dTMP was replaced by the photoactive ABdUMP are circled. The primers used to generate these probes are indicated in capital letters above and below the sequence. These primers were extended by incorporating ABdUMP(u) and the indicated radioactive nucleotide (lowercase letters, underlined) and subsequently extended to full length. Probes are designated by the location of ABdUMP and by the photoactive strand (N, nontranscribed; T, transcribed). (b) Analysis of cross-linking to bp −39/−38, −33, and −28 in the nontranscribed strand. The proteins present with DNA are indicated above the lanes, and cross-linked proteins are identified at the right. The use of TBPm₃ in conjunction with the TGT-mutated TATA box allows unidirectional and TFIIIC-independent assembly of TFIIIB on this DNA. Samples contained 400 fmol of TBPm₃, 700 fmol of intact Brf (designated W), 150 or 300 fmol Brf(1-282) (designated n or N, respectively), 150 fmol Brf(284-596) (designated C), and 100 fmol B"(138-594), as indicated. (c to e) Quantitative analysis of cross-linking efficiencies along the DNA-binding site. The eight probes indicated between panels c and d were analyzed two, three, or four times, as indicated in brackets. Cross-linking was quantified, normalized to the level of the reference protein indicated on the ordinate of each panel, and averaged. Error bars indicate the average deviation (for experiments repeated twice) or the standard error of the mean (for experiments repeated three or four times). Because the normalization contributed substantially to these variations, rank orders that were consistently observed are also designated (• between columns) below the abscissas of panels c and d (for experiments repeated three or four times only and when not otherwise obvious). (c) Cross-linking efficiencies of Brf fragments separately and in combination relative to those of intact Brf in TFIIIB-DNA complexes; (d) cross-linking efficiencies of B" in TFIIIB-DNA complexes assembled with Brf fragments separately or in combination, normalized to that of B" in the TFIIIB-DNA complex with intact Brf; (e) cross-linking efficiencies of Brf fragments relative to that of intact Brf in heparin-challenged TFIIIB-DNA complexes reconstituted with Brf(1-282) and Brf(284-596).

Analysis of the assembly of DNA complexes with Brf(1-282) and Brf(435-596) by photochemical cross-linking. The cross-linking efficiency of Brf(435-596) in the B′-DNA complex is shown relative to that of intact Brf in the TFIIIB-DNA complex. Recruitment of B" to DNA by Brf(435-596) and Brf(284-596) is compared and normalized to the cross-linking efficiencies of B" in TFIIIB-DNA complexes formed with intact Brf.

The N- and C-proximal halves of Brf interact with opposite ends of TBP: analysis by photochemical cross-linking. TFIIIB-DNA complexes were formed with 200 fmol of hTBPm₃ (WT [wild type]), of the Brf interaction-defective hTBPm₃(R231E+R235E+R239S) (Brf⁻), or of the TFIIB interaction-defective hTBPm₃(E284R+E286R+L287E) (IIB⁻), 300 fmol each of Brf(1-282) and Brf(435-545) as indicated above each lane, 200 fmol of B"(137-594), and 8 fmol of −28^N DNA probe. Cross-linked proteins are indicated at the right. The asterisk marks a fragment of Brf(435-545) that appears to be competent to assemble TFIIIB-DNA complexes.

TFIIIC-dependent transcription of the SUP4 gene with Brf split at amino acids 282 and 435. Components (400 fmol of intact Brf or the indicated fragments, 100 fmol of TBP, 300 fmol of B", 76 fmol of TFIIIC, as indicated, 16 fmol of pol III, and 100 fmol of the SUP4 gene-containing plasmid pTZ1) are specified above each lane. SUP4 transcripts and the recovery marker (R.M.) are identified at the sides.

TFIIIC interaction. (a) MPE-Fe(II) footprints of TFIIIC-B-DNA complexes assembled on TA-30 DNA with intact Brf and with Brf split at amino acid 283. Reaction mixtures were assembled as described below and subjected to reaction with MPE-Fe(II) as specified in Materials and Methods. The resulting material was separated electrophoretically, and the gel was exposed without drying to a phosphorimaging screen as shown in panel b. Gel slices corresponding to the appropriate bands were excised; DNA was extracted, processed, separated on a sequencing gel as specified in Materials and Methods, and analyzed on a phosphorimaging detector. The density traces shown have been normalized at 5′ unprotected sequence. Green trace, TFIIIC-DNA complex; red trace, TFIIIC-TFIIIB-DNA complex assembled with intact Brf; blue trace, TFIIIC-TFIIIB-DNA complex assembled with Brf(1-282) and Brf(284-596). (b) EMSA for assembly and stability of TFIIIC-B′-DNA and TFIIIC-TFIIIB-DNA complexes with Brf(1-282) and Brf(284-596). Nine femtomoles of TA-30 DNA probe was incubated with 31 fmol of TFIIIC, 200 fmol of TBP, 300 fmol of B", and 200 fmol of intact Brf (as marked above lanes a to d) or with 31 fmol of TFIIIC, 500 fmol of TBP, 500 fmol of Brf fragment, and 300 fmol of B" (as indicated above lanes e to h). Electrophoresis was in Mg²⁺-containing gel. The result shown was generated in a footprinting experiment: DNA-protein mixtures were reacted with MPE-Fe(II) before electrophoresis, and the exposure shown was made without drying the gel.

Model of the disposition of Brf in the TFIIIB-DNA complex. Two views of the TBP-DNA complex are shown (derived from reference 35). The existing DNA sequence was changed to place T at −33^N, −32^T, −23^T, and −22^T, and DNA was also extended at each end with undistorted B helix (corresponding to SNR6 DNA sequence). DNA strands are in dark (transcribed) and light (nontranscribed) blue; core TBP (Arabidopsis thaliana residues 12 to 198) is in red. The photoactive side chains of ABdUMP at bp −39, −38, −33, −28, −13, and −12 are shown, fully extended and projecting out of the major groove, in green, with their nitrene nitrogens in yellow. The TBP residues corresponding to the surface required for stable Brf binding (40) are shown in black. The corresponding TBP epitope required for cross-linking of Brf(1-282) and for TFIIB binding is shown in gray. Gray, dashed, and black arrows point to sites of proximity and interaction of Brf fragments 1-282, 284-434, and 435-545, respectively. At the right is shown the TFIIB-TBP-DNA complex (35). The structure comprises amino acids 113 to 316 of TFIIB (in green), homologous to amino acids 79 to 289 of yeast Brf.