Structural visualization of key steps in human transcription initiation
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.
[…] Data collection and image processing were conducted using the Leginon data collection software and the Appion electron microscopy processing environment, respectively. Three-dimensional maps were calculated using libraries from the EMAN2 and SPARX software packages,. Volume segmentation, automatic rigid body docking, generation were performed using UCSF Chimera. […]
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