Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.
Bibliographical noteFunding Information:
We thank the staff at the Jinju Bio-industry Foundation for supporting Tko cell fermentation and Macromolecular X-ray science at the Cornell High Energy Synchrotron Source (MacCHESS) for supporting the crystallographic data collection. The cryo-EM experiment was performed at the Cryo-EM facility in Electron Microscopy Research Center, Korea Basic Science Institute (KBSI) and supported by KBSI grant C030221 and C021820. We thank Carmen V. Amoah-Kusi for assistance with cross-linking and transcription assays. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2015R1D1A1A01059097 to S.-H.J.) and by the Ministry of Science, ICT & Future Planning (NRF-2016R1A5A1010764 and NRF-2017M3A9F6029755 to H.-S.C. and NRF-2020R1F1A1072050 to S.-H.J.) and NIH grants (R01 GM087350 and R35 GM131860 to K.S.M. and R15 GM083306 to M.S.B.).
© 2020, The Author(s).
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Physics and Astronomy(all)