b TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. Eukaryotic RNA polymerase (Pol) III transcribes precursor tRNAs, 5S rRNA, small nuclear RNAs such as U6 and 7SK RNAs, and a number of small nucleolar RNAs and microRNAs (1). In the yeast Saccharomyces cerevisiae, the Pol III transcription apparatus consists of 17-subunit Pol III and three other transcription factors: single-polypeptide TFIIIA, three-subunit TFIIIB, and six-subunit TFIIIC (2, 3). TFIIIA and TFIIIC function as the promoter recognition factors, and TFIIIB is recruited to the promoter through TFIIIC. TFIIIB is composed of TFIIB-related factor Brf1, TATA box binding protein TBP, and SANT domain-containing subunit Bdp1. Previous biochemical studies indicated that Brf1 and TBP cooperatively assemble onto DNA upstream of the transcription start site and Bdp1 binds to the Brf1-TBP-DNA complex mainly through its SANT domain (4-10). The TFIIIB-DNA assembly is required for subsequent Pol III recruitment and transcript initiation. Both Brf1 and Bdp1 have been found to interact with Pol III and function in promoter opening (4,(11)(12)(13)(14).The N-terminal domain of yeast Brf1 (Brf1n; amino acids [aa] 1 to 286) contains a zinc ribbon fold (aa 3 to 34) and a cyclin fold repeat subdomain (aa 83 to 282) (Fig. 1A), both of which are homologous to those in the general transcription factor TFIIB of the Pol II system. On the basis of biochemical and structural analyses, TFIIB ribbon and cyclin fold repeats are, respectively, positioned in the RNA exit tunnel and in the wall domain of Pol II (15)(16)(17)(18)(19)(20). In addition, the connecting region between the TFIIB ribbon and the cyclin repeat domain has been structurally resolved to contain B reader and B linker motifs that interact with the Pol active center. On the basis of sequence comparison, the connecting region in Brf1n, which we refer to as the N linker, contains low sequence homology ...

bThe RNA polymerase III (Pol III)-specific transcription factor Bdp1 is crucial to Pol III recruitment and promoter opening in transcription initiation, yet structural information is sparse. To examine its protein-binding targets within the preinitiation complex at the residue level, photoreactive amino acids were introduced into Saccharomyces cerevisiae Bdp1. Mutations within the highly conserved SANT domain cross-linked to the transcription factor IIB (TFIIB)-related transcription factor Brf1, consistent with the findings of previous studies. In addition, we identified an essential N-terminal region that cross-linked with the Pol III catalytic subunit C128 as well as Brf1. Closer examination revealed that this region interacted with the C128 N-terminal region, the N-terminal half of Brf1, and the C-terminal domain of the C37 subunit, together positioning this region within the active site cleft of the preinitiation complex. With our functional data, our analyses identified an essential region of Bdp1 that is positioned within the active site cleft of Pol III and necessary for transcription initiation. RNA polymerase III (Pol III) transcribes tRNAs and certain noncoding RNAs, such as 5S rRNA and U6 spliceosomal RNA (1). Pol III is a 17-subunit complex with a 12-subunit core structure homologous to Pol II and five other specific subunits (2). In the Pol III core structure, the two largest subunits, C160 and C128, form the active site cleft, and the other smaller subunits are localized in the periphery. The five Pol III-specific subunits form two separate subcomplexes, C53/C37 and C82/C34/C31, which are homologous to transcription factor IIF (TFIIF) and TFIIE, respectively, in the Pol II system. To initiate transcription, three transcription factors, TFIIIA, TFIIIB, and TFIIIC, cooperate to recruit Pol III to different types of gene promoters and form the preinitiation complex (PIC) (3, 4). For PIC formation at transfer DNA (tDNA) genes, TFIIIC recognizes the gene-internal box A and B elements, while TFIIIB localizes further upstream. TFIIIA is required only for the transcription of 5S rRNA, where it recognizes the gene-internal control element to further recruit TFIIIC and TFIIIB.TFIIIB is composed of three subunits: the TATA box-binding protein (TBP), TFIIB-related factor 1 (Brf1), and Bdp1. TBP and Brf1 bind tightly, allowing copurification, whereas Bdp1 mostly dissociates during purification (5). The copurified TBP/Brf1 fraction is termed B=, and the Bdp1 fraction is termed BЉ (B double prime). TBP is required for all three nuclear transcription systems, and Brf1 belongs to the class of TFIIB-related proteins that also include Rrn7 (in Saccharomyces cerevisiae) and TAF1B (in human) in the Pol I system (6, 7). The N-terminal half of Brf1 contains zinc ribbon and cyclin fold repeat domains related to TFIIB, while the structural features in the C-terminal half of Brf1 are Pol III specific. To date, no transcription factor homologous to Bdp1 has been found in the Pol I and Pol II systems.Biochemical analysis sugg...