Reconfiguring the connectivity of a multiprotein complex: Fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB
Reconfiguring the connectivity of a multiprotein complex: Fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB
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DOI：
10.1073/pnas.0507653102
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发表时间：
2005-10-25
影响因子：
11.1
通讯作者：
Geiduschek, EP
中科院分区：
文献类型：
--
作者：
Kassavetis, GA;Soragni, E;Geiduschek, EP
转录因子（TF）Ⅲ B是RNA聚合酶Ⅲ（pol Ⅲ）的核心转录起始因子，由Bdp 1、Brf 1和TATA结合蛋白（TBP）三个亚基组成，它们在体内和体外都是维持正常功能所必需的。Brf 1是一种模块化蛋白：其N-近端的一半与TFIIB相关，并与TBP的C-末端结合类似;其C-近端的三分之一通过沿着TBP的凸面和N-末端侧面的整个长度结合而提供对TBP的大部分亲和力。一个结构知情的三重融合蛋白，与TBP核心之间的N-和C-近端结构域的Brf 1，已被构建。Brf 1-TBP三重融合蛋白在体外TFIIIC依赖性和非依赖性转录中有效地取代Brf 1和TBP，并形成非常稳定的TFIIIB-DNA复合物，其通过化学核酸酶足迹法与野生型TFIIIB-DNA复合物难以区分。与Brf 1和TBP不同，三重融合蛋白能够在Bdp 1不存在的情况下招募pol III用于TATA盒指导的线性和超螺旋DNA的转录。Brf 1-TBP三重融合蛋白也有效地取代Brf 1在体内的功能作为完整的蛋白质，在酵母中产生一个TBP蛋白，其被私有化用于pol III转录。
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.