Holstege F C, van der Vliet P C, Timmers H T
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
EMBO J. 1996 Apr 1;15(7):1666-77.
We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that creating a region of heteroduplex DNA around the start site of the adenovirus major late (AdML) promoter circumvents the requirement for TFIIE and TFIIH in transcription. The critical size and position of the heteroduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from -4 to +2. Promoter opening was investigated directly with potassium permanganate (KMnO4), a chemical probe specific for single-stranded thymidines. We found that KMnO4-detectable opening of the AdML promoter requires the presence of the complete pre-initiation complex, DBpolFEH, and that opening occurs in two discrete steps. First, dependent on ATP but prior to initiation, the -9 to +1 region becomes single-stranded. Second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. Our results lead to a model in which the critical function of the TFIIH-associated DNA helicases is to create a single-stranded region. This gives RNA polymerase II access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond.
我们利用纯化的RNA聚合酶II和基础转录因子在体外重建实验中研究了启动子开放。我们发现，在腺病毒主要晚期（AdML）启动子起始位点周围创建一个异源双链DNA区域，可规避转录过程中对TFIIE和TFIIH的需求。减轻对TFIIE和TFIIH需求的异源双链区域的关键大小和位置是从-4到+2的六个核苷酸。我们使用高锰酸钾（KMnO4）直接研究了启动子开放，KMnO4是一种对单链胸腺嘧啶具有特异性的化学探针。我们发现，KMnO4可检测到的AdML启动子开放需要完整的预起始复合物DBpolFEH的存在，并且开放过程分两个离散步骤发生。首先，在依赖ATP但在起始之前，-9到+1区域变为单链。其次，第一个磷酸二酯键的形成导致开放区域扩展到+8位置。我们的结果得出了一个模型，其中与TFIIH相关的DNA解旋酶的关键功能是创建一个单链区域。这使RNA聚合酶II能够接触模板链的核苷酸，并在第一个磷酸二酯键形成时允许开放区域扩展。
