Structural Basis ofStreptomycesAntibiotic Regulatory Proteins Activating Transcription

Abstract

AbstractStreptomycetes are renowned antibiotic producers, withStreptomycesantibiotic 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 ofStreptomyces coelicolorAfsR (SAS) forms a side-by-side dimer contacting theafs boxcentered at −29.5 relative to the transcription start site. The upstream protomer binds to the direct repeat encompassing the −35 element while the σHrdBregion 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 α subunits, while specific regions of the RNAP β flap tip helix and β’ zinc-binding domain also engage with SAS. Key interfacial residues accounting for transcriptional activation were confirmed by mutational studies andin vitrotranscriptional assays. Overall, our results present a detailed molecular view of how SARPs serve to activate transcription.