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.

打开细胞 RNA 聚合酶 (RNAP) 的 DNA 结合裂缝是转录起始所必需的，但潜在的分子机制尚不清楚。在这里，我们报告了来自古生菌Thermococcus kodakarensis的RNAP 、RNAP-TFEα 二元和 RNAP-TFEα-启动子 DNA 三元复合物的低温电子显微镜结构). 这些结构表明 TFEα 桥接 RNAP 钳位和茎结构域以打开 DNA 结合裂缝。将启动子 DNA 定位到裂缝中会关闭它，同时保持 TFEα 与 RNAP 移动模块的相互作用。结构和光交联结果还表明，TFEα 的扩展翼螺旋结构域中的保守芳香族残基与启动子 DNA 相互作用，以稳定转录气泡。本研究为 TFEα 的功能提供了结构基础，并阐明了在含茎 RNAP（包括古细菌和真核 RNAP）的转录起始过程中 DNA 结合裂隙打开的机制。