Dumay-Odelot Hélène, Acker Joël, Arrebola Rosalia, Sentenac André, Marck Christian
Service de Biochimie et de Génétique Moléculaire, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France.
Mol Cell Biol. 2002 Jan;22(1):298-308. doi: 10.1128/MCB.22.1.298-308.2002.
Yeast transcription factor IIIC (TFIIIC) plays a key role in assembling the transcription initiation factor TFIIIB on class III genes after TFIIIC-DNA binding. The second largest subunit of TFIIIC, tau131, is thought to initiate TFIIIB assembly by interacting with Brf1/TFIIIB70. In this work, we have analyzed a TFIIIC mutant (tau131-DeltaTPR2) harboring a deletion in tau131 removing the second of its 11 tetratricopeptide repeats. Remarkably, this thermosensitive mutation was selectively suppressed in vivo by overexpression of B"/TFIIIB90, but not Brf1 or TATA-binding protein. In vitro, the mutant factor preincubated at restrictive temperature bound DNA efficiently but lost transcription factor activity. The in vitro transcription defect was abolished at high concentrations of B" but not Brf1. Copurification experiments of baculovirus-expressed proteins confirmed a direct physical interaction between tau131 and B". tau131, therefore, appears to be involved in the recruitment of both Brf1 and B".
酵母转录因子IIIC（TFIIIC）在TFIIIC与DNA结合后，于III类基因上组装转录起始因子TFIIIB的过程中发挥关键作用。TFIIIC的第二大亚基tau131，被认为是通过与Brf1/TFIIIB70相互作用来启动TFIIIB组装的。在这项研究中，我们分析了一个TFIIIC突变体（tau131-ΔTPR2），该突变体的tau131发生缺失，去除了其11个四肽重复序列中的第二个。值得注意的是，这种温度敏感型突变在体内被B"/TFIIIB90的过表达选择性抑制，而不是被Brf1或TATA结合蛋白抑制。在体外，在限制温度下预孵育的突变因子能有效结合DNA，但失去了转录因子活性。在高浓度的B"存在时，体外转录缺陷被消除，但Brf1不存在时则不能消除。杆状病毒表达蛋白的共纯化实验证实了tau131与B"之间存在直接的物理相互作用。因此，tau131似乎参与了Brf1和B"的募集过程。
