A noncanonical RNA polymerase assembly pathway in Bacillus subtilis: α Dimer associates with either β or β' before...

The bacterial RNA polymerase (RNAP) core enzyme, responsible for transcription, is composed of five conserved subunits: α₂, β, β', and ω. In Escherichia coli, RNAP assembly follows a well-established sequential pathway: α + α → α₂ → α₂β → α₂ββ'(ω). This canonical scheme has long been considered universal. Here, we show that in Bacillus subtilis, RNAP assembly proceeds not only via the canonical route but also through an alternative pathway: α + α → α₂ → α₂β'(ω) → α₂ββ'(ω). We provide in vivo evidence for both α₂β and α₂β' intermediates in B. subtilis, whereas Escherichia coli supports only the α₂β intermediate. These findings uncover a previously unrecognized plasticity in bacterial RNAP assembly, attributable to distinct α-β and α-β' interfaces in different lineages. Our results highlight the evolutionary diversification of RNAP assembly and suggest new opportunities for developing species-specific antibiotics that target lineage-dependent assembly pathways.