The TIC of the SARP, RNAP, and <i>afsS</i> promoter DNA.

Abstract

(A) Domain structures of SARPs. The N-terminal ODB together with the following BTA is referred as an SARP domain. S. coelicolor AfsR is a large SARP with additional NOD and TPR domains. (B) Hypothetical scheme for the regulation of S. coelicolor AfsR. (C) Fluorescence polarization assays of the SARP with afs box. Error bars represent mean ± SEM of n = 3 experiments. (D) Transcription assays with increasing concentrations (62.5 nM, 125 nM, 250 nM, 500 nM, 750 nM, 1,000 nM) of the SARP. CK represents the control group without the addition of the SARP. Data are presented as mean ± SEM from 3 independent assays. (E) Assembly of the SARP-TIC. The protein compositions in the dotted line boxed fractions are shown in the SDS-PAGE. The original gel image can be found in S1 Raw Images. (F) The afsS promoter fragment used for the SARP-TIC assembly. The −35 element, −10 element, the TSS, and the 6-bp noncomplementary bubble are denoted. The afs box is colored orange and contains DRup (the upstream direct repeat) and DRdown (the downstream direct repeat). The top (non-template, NT) strand and bottom (template, T) strand are colored light green and dark green, respectively. (G) Two views of cryo-EM map. The map was generated by merging the consensus map of the full SARP-TIC and the focused map of the SARP region in Chimera X. (H) Cartoon representation of the SARP-TIC structure. The subunits are colored as in the color scheme. The data underlying C, D, and E are provided in S1 Data. BTA, bacterial transcriptional activation; cryo-EM, cryo-electron microscopy; NOD, nucleotide-binding oligomerization domain; ODB, OmpR-type DNA-binding; RNAP, RNA polymerase; SARP, Streptomyces antibiotic regulatory protein; TIC, transcription initiation complex; TPR, tetratricopeptide repeat; TSS, transcription start site.</p