A noncanonical RNA polymerase assembly pathway in Bacillus subtilis: α Dimer associates with either β or β′ before forming the core enzyme

The bacterial RNA polymerase (RNAP) core enzyme, responsible for transcription, is composed of five conserved subunits: α 2 , β, β′, and ω. In Escherichia coli , RNAP assembly follows a well-established sequential pathway: α + α → α 2 → α 2 β → α 2 ββ′(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 → α 2 → α 2 β′(ω) → α 2 ββ′(ω). We provide in vivo evidence for both α 2 β and α 2 β′ intermediates in B. subtilis , whereas E. coli supports only the α 2 β 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.