abstract
- Transcription by RNA polymerase II (Pol II) requires general transcription factors that bind with Pol II at the promoters of protein-coding genes to form preinitiation complexes (PICs). Among these is TFIIE, which recruits TFIIH to the PIC and stimulates the kinase and translocase activities of TFIIH, thereby regulating the fate of formed PICs. In this study, we used a purified reconstituted human Pol II transcription system and single molecule total internal reflection fluorescence microscopy to monitor TFIIE binding dynamics in PICs under different conditions in real time. We observed dynamic interactions of the two subunits of TFIIE (TFIIEα and TFIIEβ) with PICs, consistent with rapid on/off binding as a heterodimer. TFIIH exclusion increased the rates of association and dissociation for both subunits, and enabled TFIIEα to associate with PICs more often than TFIIEβ; hence, TFIIEα and TFIIEβ behave asynchronously in the absence of TFIIH. Additionally, two disease-related TFIIEβ point mutations destabilized TFIIEβ and altered its kinetic behaviors within PICs. Our results contribute to an emerging model that PICs are not static assemblies and highlight important connections between the structural arrangement and kinetic behaviors of GTFs in PICs.