Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB.
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.
Kassavetis
George A
GA
Division of Biological Sciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA. gak@biomail.ucsd.edu
Soragni
Elisabetta
E
Driscoll
Robert
R
Geiduschek
E Peter
EP
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
C090956:BRF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D004247:DNA; D012320:RNA Polymerase III