Structure of the transcription initiation complex mediated by a distinct ECF sigma factor from E. coli

Transcription initiation is a critical regulatory step in bacterial gene expression that requires the RNA polymerase (RNAP) core enzyme to associate with σ factors. Although the molecular mechanisms of most σ⁷⁰-family factors in Escherichia coli have been well characterized, the smallest extracytoplasmic function (ECF) sigma factor, σ^FecI, has remained structurally and functionally uncharacterized. Here, we determined the cryo-EM structure of the σ^FecI-dependent RNAP promoter open complex (σ^FecI-RPo) at 3.0 Å resolution through in vitro reconstitution. Structural and biochemical analyses revealed an unconventional mechanism of σ^FecI-mediated transcription initiation. Notably, the σ₂/σ₄ linker region of σ^FecI adopts an atypical conformation relative to the RNAP active-center cleft, distinct from that observed in canonical ECF sigma factors. Furthermore, we identified previously unreported hydrophobic interactions between the σ⁴ region of σ^FecI and the N-terminal helix (NTH) of the RNAP β' subunit, which are essential for RNAP recruitment, open-complex stability, and transcription activation. Together, these findings expand our understanding of σ factor-RNAP interactions and reveal a previously unrecognized mode of ECF sigma factor-dependent transcription initiation. Our work provides a structural framework for understanding σ^FecI function and offers a basis for the rational regulation of gene expression in E. coli.