title: Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB.
abstract: Transcription factor (TF) IIIB, the central transcription initiation factor of RNA polymerase III (pol III), is composed of three subunits, Bdp1, Brf1 and TATA-binding protein (TBP), all essential for normal function in vivo and in vitro. Brf1 is a modular protein: Its N-proximal half is related to TFIIB and binds similarly to the C-terminal stirrup of TBP; its C-proximal one-third provides most of the affinity for TBP by binding along the entire length of the convex surface and N-terminal lateral face of TBP. A structure-informed triple fusion protein, with TBP core placed between the N- and C-proximal domains of Brf1, has been constructed. The Brf1-TBP triple fusion protein effectively replaces both Brf1 and TBP in TFIIIC-dependent and -independent transcription in vitro, and forms extremely stable TFIIIB-DNA complexes that are indistinguishable from wild-type TFIIIB-DNA complexes by chemical nuclease footprinting. Unlike Brf1 and TBP, the triple fusion protein is able to recruit pol III for TATA box-directed transcription of linear and supercoiled DNA in the absence of Bdp1. The Brf1-TBP triple fusion protein also effectively replaces Brf1 function in vivo as the intact protein, creating a TBP paralogue in yeast that is privatized for pol III transcription.
authors.affiliation: Division of Biological Sciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA. gak@biomail.ucsd.edu
authors.forename: George A
authors.initials: GA
authors.lastname: Kassavetis
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Soragni
authors.forename: Robert
authors.initials: R
authors.lastname: Driscoll
authors.forename: E Peter
authors.initials: EP
authors.lastname: Geiduschek
mesh_terms: D004247:DNA / Q000378:metabolism; D012320:RNA Polymerase III / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000737:chemistry*; D035361:Transcription Factor TFIIIB / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic
chemical_list: C090956:BRF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D004247:DNA; D012320:RNA Polymerase III