Chen Hung-Ta, Warfield Linda, Hahn Steven
Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Mailstop A1-162, Seattle, Washington 98109, USA.
Nat Struct Mol Biol. 2007 Aug;14(8):696-703. doi: 10.1038/nsmb1272. Epub 2007 Jul 15.
We incorporated the non-natural photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa) into the RNA polymerase II (Pol II) surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photo-cross-linking of preinitiation complexes (PICs) with these Pol II derivatives and hydroxyl-radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9, while TFIIE cross-links to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains alter both Pol II-TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9 and TFIIB. Together with previous biochemical and structural studies, these findings illuminate the structural organization of the PIC and the network of protein-protein interactions involved in transcription start site selection.
我们将非天然光反应性氨基酸对苯甲酰-L-苯丙氨酸(Bpa)引入到由Rpb1和Rpb2亚基形成的中央裂隙周围的RNA聚合酶II(Pol II)表面。预起始复合物(PIC)与这些Pol II衍生物的光交联以及羟基自由基切割试验表明,TFIIF二聚化结构域与Rpb2叶和与Rpb9相邻的突出结构域相互作用,而TFIIE与Pol II中央裂隙另一侧的Rpb1钳结构域发生交联。Rpb2叶和突出结构域中的突变会改变Pol II-TFIIF结合以及转录起始位点,这一表型与TFIIF、Rpb9和TFIIB中的突变相关。与之前的生化和结构研究一起,这些发现阐明了PIC的结构组织以及转录起始位点选择中涉及的蛋白质-蛋白质相互作用网络。