Forget Diane, Langelier Marie-France, Thérien Cynthia, Trinh Vincent, Coulombe Benoit
Laboratory of Gene Transcription, Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada H2W 1R7.
Mol Cell Biol. 2004 Feb;24(3):1122-31. doi: 10.1128/MCB.24.3.1122-1131.2004.
The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIE alpha closely approaches promoter DNA at nucleotide -10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIE alpha is obtained when the DNA template is artificially premelted in the -11/-1 region, suggesting that TFIIE alpha facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.
利用凝胶纯化复合物的位点特异性蛋白质-DNA光交联技术，分析了TATA结合蛋白-TFIIB-TFIIF-RNA聚合酶II（RNAP II）-TFIIE-启动子复合物的拓扑结构。RNAP II亚基的交联结果用于确定启动子DNA相对于酶结构的路径。结果表明，启动子DNA环绕着RNAP II的移动钳。TFIIF和TFIIE在TATA元件上游和转录起始位点下游的交联表明，这两个因子都与RNAP II移动钳相关联。TFIIEα在核苷酸-10处紧密靠近启动子DNA，该位置在开放复合物中转录泡的紧邻上游。当DNA模板在-11/-1区域人工预熔解时，TFIIEα对转录起始的刺激增加，这表明TFIIEα可能通过与部分开放的转录泡的上游末端相互作用来促进开放复合物的形成。这些结果支持了RNAP II的移动钳和TFIIE在转录起始中的核心作用。
