Structures of the RNA polymerase-σ⁵⁴ reveal new and conserved regulatory strategies

Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ⁵⁴) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate-dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ⁵⁴ holoenzyme at 3.8 angstroms reveals molecular details of σ⁵⁴ and its interactions with RNAP. The structure explains how σ⁵⁴ targets different regions in RNAP to exert its inhibitory function. Although σ⁵⁴ and the major s factor, σ⁷⁰, have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.