Structural Basis of Streptomyces Antibiotic Regulatory Proteins Activating Transcription

Abstract

Streptomycetes are renowned antibiotic producers, with Streptomyces antibiotic regulatory proteins (SARPs) acting as activators for antibiotic biosynthesis. However, the precise mechanism underlying SARPs transcriptional activation remains elusive. Here, we used cryo-electron microscopy (cryo-EM) to unravel the interplay between SARP, DNA, and RNA polymerase (RNAP) during transcriptional activation. The SARP domain of Streptomyces coelicolor AfsR (SAS) forms a side-by-side dimer contacting the afs box centered at -29.5 relative to the transcription start site. The upstream protomer binds to the direct repeat encompassing the -35 element while the {sigma}HrdB region 4 (R4) is positioned on top of both protomers, causing the removal of R4 from the major groove of the -35 element. Both SAS protomers establish interactions with C-terminal domain of one RNAP subunits, while specific regions of the RNAP {beta} flap tip helix and {beta} zinc-binding domain also engage with SAS. Key interfacial residues accounting for transcriptional activation were confirmed by mutational studies and in vitro transcriptional assays. Overall, our results present a detailed molecular view of how SARPs serve to activate transcription.