Abstract

Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σ^A controls housekeeping functions, S. aureus σ^B regulates virulence, biofilm formation, persistence, cell internalization, membrane transport, and antimicrobial resistance. Besides the sequence difference, the spacers between the −35 element and −10 element of σ^B regulated promoters are shorter than those of σ^A regulated promoters. Therefore, how σ^B recognizes and initiates transcription from target promoters can not be inferred from that of the well studied σ. Here, we report the cryo-EM structures of S. aureus RNAP-promoter open complexes comprising σ^A and σ^B, respectively. Structural analyses, in combination with biochemical experiments, reveal the structural basis for the promoter specificity of S. aureus transcription. Although the −10 element of σ^A regulated promoters is recognized by domain σ^A₂ as single-stranded DNA, the −10 element of σ^B regulated promoters is co-recognized by domains σ^B₂ and σ^B₃ as double-stranded DNA, accounting for the short spacers of σ^B regulated promoters. S. aureus RNAP is a validated target of antibiotics, and our structures pave the way for rational drug design targeting S. aureus RNAP.