Biofilms are multicellular aggregates of sessile bacteria encased by an extracellular matrix and are important medically as a source of drug-resistant microbes. In Bacillus subtilis, we found that an operon required for biofilm matrix biosynthesis also encoded an inhibitor of motility, EpsE. EpsE arrested flagellar rotation in a manner similar to that of a clutch, by disengaging motor force-generating elements in cells embedded in the biofilm matrix. The clutch is a simple, rapid, and potentially reversible form of motility control.

Highlights d Cryo-EM structures of 7 intermediates in promoter opening pathway from RPc to RPo d Intermediates populated by using an inhibitor and a promoter with unstable RPo d RNAP and DNA conformational changes in mobile regions mark the steps in the pathway d Transient interactions identified in intermediates are not found in RPc or RPo

In bacterial transcription initiation, RNA polymerase (RNAP) selects a transcription start site (TSS) at variable distances downstream of core promoter elements. Using next-generation sequencing and unnatural-amino-acid-mediated protein-DNA crosslinking, we have determined, for a library of 410 promoter sequences, the TSS, the RNAP leading-edge position, and the RNAP trailing-edge position. We find that a promoter element upstream of the TSS, the “discriminator,” participates in TSS selection, and that, as the TSS changes, the RNAP leading-edge position changes, but the RNAP trailing-edge position does not change. Changes in the RNAP leading-edge position but not the RNAP trailing-edge position are a defining hallmark of the “DNA scrunching” that occurs concurrent with RNA synthesis in initial transcription. We propose that TSS selection involves DNA scrunching prior to RNA synthesis.

Transcription by RNA polymerase in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ54) initially forms a transcriptionally silent complex requiring specialised ATP-dependent activators for initiation. Our crystal structure of the 450 kDa RNAP-σ54 holoenzyme at 3.8 Å 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 σ 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.