Structural basis of σ⁵⁴ displacement and promoter escape in bacterial transcription

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ⁷⁰ class, represented by the σ⁷⁰ that regulates housekeeping genes. σ⁵⁴ forms a class on its own and regulates stress response genes. Extensive studies on σ⁷⁰ have revealed the molecular mechanisms of the σ⁷⁰ dependent process while how σ⁵⁴ transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ⁵⁴ initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ⁵⁴ and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ⁵⁴ and upstream DNA, enabling the transition to elongation.