Structural insights into transcription activation of the Streptomyces antibiotic regulatory protein, AfsR.

The Streptomyces antibiotic regulatory proteins (SARPs) are ubiquitously distributed transcription activators in Streptomyces and control antibiotics biosynthesis and morphological differentiation. However, the molecular mechanism behind SARP-dependent transcription initiation remains elusive. We here solve the cryo-EM structure of an AfsR-loading RNA polymerase (RNAP)-promoter intermediate complex (AfsR-RPi) including the Streptomyces coelicolor RNAP, a large SARP member AfsR, and its target promoter DNA that retains the upstream portion straight. The structure reveals that one dimeric N-terminal AfsR-SARP domain (AfsR-SARP) specifically engages with the same face of the AfsR-binding sites by the conserved DNA-binding domains (DBDs), replacing σHrdBR4 to bind the suboptimal -35 element, and shortens the spacer between the -10 and -35 elements. Notably, the AfsR-SARPs also recruit RNAP through extensively interacting with its conserved domains (ß flap, σHrdBR4, and αCTD). Thus, these macromolecular snapshots support a general model and provide valuable clues for SARP-dependent transcription activation in Streptomyces.

Structural basis of promoter recognition by Staphylococcus aureus RNA polymerase.

Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σA controls housekeeping functions, S. aureus σB 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 σB regulated promoters are shorter than those of σA regulated promoters. Therefore, how σB 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 σA and σB, 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 σA regulated promoters is recognized by domain σA2 as single-stranded DNA, the -10 element of σB regulated promoters is co-recognized by domains σB2 and σB3 as double-stranded DNA, accounting for the short spacers of σB 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.