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

• Applicant: Dr. Sebastian Grünberg
• Subject Area: Biochemistry
• Term: from 2010 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 193122574

The formation of the preinitiation complex (PIC) is a key step in RNA polymerase (Pol) II transcription initiation, but most of the events leading to the formation of a transcription competent complex are unknown. Data from my preliminary FeBABE cleavage experiments suggest an unexpected location of the general transcription factor (GTF) TFIIE in PIC. I find that the conserved N-terminal winged helix domain (WH-D) of the TFIIE large subunit Tfa1 is located above the Pol II lobe between the Pol II interaction interfaces with the two structured domains of TFIIF. Interactions between TFIIE, TFIIF, and Pol II potentially anchor TFIIE in the PIC, modify TFIIF-Pol II interactions, and allow TFIIE to interact with upstream DNA. I also find that in the PIC, the conserved Tfa1 zinc binding domain (ZB-D) is positioned in the Pol II cleft, close to the active site and B-reader region of TFIIB, from where it may interact with flexible regions of Pol II, TFIIB, and the TFIIF Tfg2 linker domain to promote initiation and transcription start site selection. To answer how TFIIE functionally influences these elements, and directly and/or indirectly promotes transcription initiation, the functional contribution of the TFIIE Tfa1 ZB-D and WH-D in transcription initiation will be analyzed by a genetic assay and biochemical assays. I will further extend the preliminary mapping data of TFIIE interactions with Pol II and GTFs like TFIIF and TFIIB. To conclusively position TFIIE in the PIC, further FeBABE cleavage experiments with FeBABE linked to residues on the surface of other structured regions of TFIIE will also be required. Taken together, this project will enable us to create the most detailed model for the Pol II PIC so far, containing Pol II and the GTFs TFIIB, TFIIF, and TFIIE, thereby greatly contributing to the understanding of the complete structural composition of the PIC and the mechanistic steps leading to formation of the open complex and transcription initiation.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Dr. Steven Hahn