Buratowski S, Hahn S, Guarente L, Sharp P A
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Cell. 1989 Feb 24;56(4):549-61. doi: 10.1016/0092-8674(89)90578-3.
A native gel electrophoresis DNA binding assay was used to resolve complexes formed on the adenovirus Major Late Promoter by general transcription factors and RNA polymerase II. Five sets of complexes containing distinct components were identified. These complexes were generated by sequential binding of TFIID, TFIIA, TFIIB, RNA polymerase II, and TFIIE. The relative positions of each of the factors in the complexes were determined by DNAase I footprint analysis. TFIIA, derived from yeast or mammalian cells, formed a complex with yeast TFIID and the TATA element. TFIIB bound to this complex and probably acts as a "bridge" to the polymerase and the initiation site. The addition of ATP or dATP, necessary for "activation" of transcription, resulted in an alteration of the footprint in the +20 to +30 region, the same area protected upon addition of TFIIE to the initiation complex. Addition of ribonucleotide triphosphates generated new complexes that contained accurately initiated transcripts associated with the transcription machinery and the template DNA. A model for the interactions of components in initiation of transcription by RNA polymerase II is proposed.
采用天然凝胶电泳DNA结合分析法来解析通用转录因子和RNA聚合酶II在腺病毒主要晚期启动子上形成的复合物。鉴定出了五组含有不同成分的复合物。这些复合物是通过TFIID、TFIIA、TFIIB、RNA聚合酶II和TFIIE的顺序结合产生的。通过DNA酶I足迹分析确定了复合物中各因子的相对位置。源自酵母或哺乳动物细胞的TFIIA与酵母TFIID和TATA元件形成复合物。TFIIB与该复合物结合，可能作为连接聚合酶和起始位点的“桥梁”。转录“激活”所需的ATP或dATP的添加导致+20至+30区域的足迹发生改变，该区域与向起始复合物中添加TFIIE时受到保护的区域相同。添加核糖核苷酸三磷酸产生了新的复合物，这些复合物包含与转录机制和模板DNA相关的准确起始转录本。提出了RNA聚合酶II转录起始中各成分相互作用的模型。
