A noncanonical RNA polymerase assembly pathway in B. 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: , , , and .In Escherichia coli, RNAP assembly follows a wellestablished sequential pathway: + ().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: + () ().We provide in vivo evidence for both and intermediates in B. subtilis, whereas E. coli supports only the intermediate.These findings uncover a previously unrecognized plasticity in bacterial RNAP assembly, attributable to distinct - and - 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.