Regulatory landscapes and structural choreography of transcription initiation in spirochetes

Spirochaetota is a deeply rooted bacterial phylum of evolutionary and medical importance. We show that the spirochetal RNA polymerase (RNAP) has a diminished ability to melt promoters compared to its Escherichia coli counterpart. This deficiency is compensated by CarD and is not linked to the phylums natural resistance to the antibiotic rifampicin. CryoEM analysis revealed that the spirochetal RNAP disengages the -35 element during promoter unwinding, which contrasts with other bacterial RNAPs that release this element later during the initial transcription. Our research also details the unusually tight, non-sequence-specific binding of the spirochetal RNAP holoenzyme to DNA, which may be linked to the highly extended structure of the spirochetal nucleoid. We support these observations with a structural analysis of the dimeric holoenzyme non-specifically bound to DNA. Our results highlight the functional diversity of bacterial transcription systems and lay the foundation for understanding transcription regulation in pathogenic spirochetes.