Direct binding of TFEα opens DNA binding cleft of RNA polymerase

• Published in: Nature communications 2020-11, Vol.11 (1), p.6123-6123, Article 6123
• Main Authors: Jun, Sung-Hoon, Hyun, Jaekyung, Cha, Jeong Seok, Kim, Hoyoung, Bartlett, Michael S., Cho, Hyun-Soo, Murakami, Katsuhiko S.
• Format: Article
• Language: English
• Subjects: 101/28; 631/337/572; 631/45/173; 631/45/500; 631/45/535/1258/1259; 631/45/535/1266; Amino Acid Sequence; Archaea; Binding; Conformation; Crosslinking; Cryoelectron Microscopy; Crystallography; Crystallography, X-Ray; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Domains; Electron microscopy; Humanities and Social Sciences; Models, Molecular; multidisciplinary; Polytetrafluoroethylene; Promoter Regions, Genetic; Protein Binding; Protein Conformation; Ribonucleic acid; RNA; RNA polymerase; Science; Science (multidisciplinary); Sequence Analysis, Protein; Thermococcus - enzymology; Thermococcus - genetics; Thermococcus kodakarensis; Transcription, Genetic; X-ray crystallography
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-19998-x

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. How clamp conformation is regulated in the transcription cycle of stalk-containing archaeal and eukaryotic RNA polymerase (RNAP) systems is still not well understood. Here, the authors combine cryo-EM, X-ray crystallography and photo-crosslinking assays to structurally characterise RNAP, the RNAP-TFEα binary and RNAP-TFEα-promoter DNA ternary complexes from the archaea Thermococcus kodakarensis and enables them to describe the dynamic conformational changes of the general transcription factor TFEα and RNAP during the early stage of transcription cycle.