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Model of the RNA polymerase II preinitiation complex containing Pol II and general transcription factors TFIIB, TFIIF, and TFIIE and the function of conserved TFIIE structured domains in transcription initiation

Subject Area Biochemistry
Term from 2010 to 2013
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 193122574
 
Final Report Year 2013

Final Report Abstract

In eukaryotes, RNA polymerase (Pol) II is specialized for transcription of protein coding genes. Upon activation of transcription, Pol II and a set of Pol II-specific general transcription factors (GTFs: TFIIA, TBP, TFIIB, TFIIF, TFIIE, and TFIIH) are recruited to gene promoters where they form the transcription preinitiation complex (PIC), a key intermediate in transcription initiation. The Pol II general factors TFIIE and TFIIH function in transition of the PIC to the open complex (OC), a state containing unwound DNA in the Pol II active site. Detailed molecular information on the location of these factors in the PIC and how they function was lacking, leading to a major gap in understanding of the transcription initiation mechanism. In the course of the DFG Forschungsstipendium, the position of the three TFIIE winged helix (WH) domains within the PIC was determined using site-specific protein cleavage and crosslinking probes. My data shows that the WH domain on the large TFIIE subunit Tfa1 anchors TFIIE to the Pol II clamp and dimerizes with a tandem WH domain of the small TFIIE subunit Tfa2. This positions the Tfa2 tandem WH domain to span the Pol II cleft and encircle the region of promoter DNA that becomes single stranded in the open complex. Comparison of the TFIIE location in the Pol II PIC with models for Pol I and Pol III PICs revealed that all eukaryotic Pols utilize a tandem WH fold at or over the active site cleft, suggesting a general function for this motif in transcription initiation. Importantly, positioning of TFIIE enabled us to use TFIIE-FeBABE probes to map the location of the TFIIH subunit Ssl2/XPB within the PIC. Ssl2 lies adjacent to TFIIE, enclosing downstream DNA. Comparison of the PIC and OC models strongly suggests that Ssl2 promotes DNA opening by functioning as a double stranded DNA translocase, feeding 15 bp of DNA into the Pol II active site cleft by right-handedly threading of the promoter DNA into the Pol II active site. Combined with the fixed position of upstream promoter DNA, this leads to DNA unwinding and the OC. Mapping of Ssl2/XPB in the PIC, together with crosslinking of Ssl2 to TFIIB, suggests that TFIIB exists in two alternate conformations in the PIC: one similar to the X-ray structure, where the B- linker and -reader are buried inside the Pol II cleft and one in which both B-loops are flipped-out close to Ssl2. This finding suggests that even though positioning of the reader and linker in the cleft is required for initiation, the flipped-out state might not be poised for initiation and could probably represent the scanning state of the Pol enzyme. Thus, in yeast, alternate TFIIB conformations may be involved in switching between TSS scanning and effective initiation. The findings made in the course of the Forschungsstipendium have been highlighted in the News & Views section of NSMB.

Publications

  • Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nat Struct Mol Biol 19, 788-96 (2012)
    Grünberg, S., Warfield, L. & Hahn, S.
 
 

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