title: Mapping the protein interaction network for TFIIB-related factor Brf1 in the RNA polymerase III preinitiation complex.
abstract: 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.
authors.affiliation: Molecular and Cell Biology, Taiwan International Graduate Program, Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Seok-Kooi
authors.initials: SK
authors.lastname: Khoo
authors.forename: Chih-Chien
authors.initials: CC
authors.lastname: Wu
authors.forename: Yu-Chun
authors.initials: YC
authors.lastname: Lin
authors.forename: Jin-Cheng
authors.initials: JC
authors.lastname: Lee
authors.forename: Hung-Ta
authors.initials: HT
authors.lastname: Chen
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D012320:RNA Polymerase III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism; D035581:Transcription Factor TFIIB / Q000235:genetics / Q000378:metabolism; D035361:Transcription Factor TFIIIB / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061785:Transcription Initiation, Genetic
chemical_list: C090956:BRF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D035361:Transcription Factor TFIIIB; D012320:RNA Polymerase III; C077488:RPC34 protein, S cerevisiae