Structural visualization of key steps in human transcription initiation.
Structural visualization of key steps in human transcription initiation.
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真核生物转录起始需要将一般转录因子组装成起始前复合物，以确保RNA聚合酶II在转录起始位点的准确装载。由于缺乏结构信息，该组装体的分子机制和功能仍然难以捉摸。我们利用体外重组系统，利用低温电子显微镜研究了人TBP、TFIIA、TFIIB、Pol II、TFIIF、TFIIE和TFIIH在启动子DNA上的逐步组装。我们的结构分析提供了转录起始各个阶段的伪原子模型，阐明了关键的分子相互作用，包括TFIIF如何结合Pol II和启动子DNA来稳定闭合PIC和开放启动子复合物并调节起始位点选择。开放和封闭起始前复合物的比较，结合TFIIH解旋酶XPD和XPB的定位，支持了XPB的DNA易位模型，并解释了它在启动子打开中的重要作用。
Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. We have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE, and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed PIC and the open-promoter complex and regulate start site selection. Comparison of open versus closed pre-initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, supports a DNA translocation model of XPB and explains its essential role in promoter opening.