SARP interacts with σ^HrdB R4, β FTH, β′ ZBD.

(A) The SARP protomers interact with σHrdB, β, and β′. (B) The upstream SARP protomer contacts σHrdB R4 by its ODB domain. Salt bridges are shown as red dashed lines. (C) The H499 of σHrdBR4 is enfolded in an amphiphilic pocket of the BTA domain. The K496 of σHrdBR4 makes a salt bridge with the E246 of the BTA domain. (D) The downstream SARP protomer make extensive interactions with the β FTH, the preceding loop (TPL) and the following loop (TFL) of the β flap. SARP is colored orange and β flap is colored blue. Hydrogen bonds, salt-bridges, and van der Waals interactions are shown as yellow, red, and gray dashed lines, respectively. (E) Interactions between the β′ ZBD and the ODB of downstream SARP. The positively charged R67 and R69 of β′ ZBD contact the negatively charged E76 and E77 of the HTH loop of the ODB domain. (F) Mutating interfacial residues of SARP impaired transcription activation. The data underlying this figure can be found in S1 Data; error bars, SEM; n = 3; *P P Streptomyces strains, highlighting the residues interacting with β (blue), σ R4 (cyan), and αCTD (purple). The black boxes highlight the positions conserved. BTA, bacterial transcriptional activation; FTH, flap tip helix; ODB, OmpR-type DNA-binding; SARP, Streptomyces antibiotic regulatory protein; ZBD, zinc-binding domain.</p

Scheme model summarizing the ATP-dependent and phosphorylation-dependent regulation of AfsR.

Unmodified full-length AfsR is autoinhibited by its C-terminal domains. AfsR is activated by ATP binding and significantly stimulates the production of transcripts. The phosphorylation of AfsR results in increased ATPase activity, potentially counteracting the effects of ATP binding. Phosphorylated AfsR forms more oligomers and shows slightly higher transcriptional activation compared to the unmodified form. (TIF)</p

Phosphorylation modulates AfsR transcriptional activation.

(A) LC-MS/MS analysis showing that S22, T337, S391, T506, and S953 are phosphorylated in AfsR by AfsKΔC. The lowercase letter in the peptide sequence indicates phosphorylated residue. “b” and “y” denote peptide fragment ions retaining charges at the N and C terminus, respectively. The subscript numbers indicate their positions in the identified peptide. (B) Sequence alignment of Streptomyces AfsR family members highlighting the consensuses sequences neighboring phosphorylation sites S22, T337, S391, T506, and S953. Orange boxes represent phosphorylation sites. (C) Transcription assays with increasing concentrations of phosphorylated AfsR by AfsKΔC (phos-AfsR) and untreated AfsR. (D) Transcription assays of 500 nM AfsR mutants mimicking dephosphorylation (T337A) and phosphorylation (S22E, T337E, T506E, S953E, and S391E/T337E/T506E/S953E (4E)). Data are presented as mean ± SEM from 3 independent assays. n.s. means no significance; *P P T337A and AfsR4E. (F) Fluorescence polarization assay of AfsR4E and dephosphorylated AfsR (dephos-AfsR) with afs box. The concentration of afs box was 10 nM. Error bars represent mean ± SEM of n = 3 experiments. The data underlying A, C, D, E, and F are provided in S1 Data. (TIF)</p

Purification and assembly of protein complexes.

(A) Assembly of S. coelicolor RNAP-σHrdB. The peak of RNAP-σHrdB was analyzed by SDS-PAGE. (B) Purification of AfsR. (C) Purification of ΔTPR. (D) Purification of SARP. (E) Purification of RbpA. (F) Purification of CarD. (G) Purification of AfsKΔC. (H) Assembly of AfsRT337A-TIC in the presence of 1 mM ATPγS. The protein compositions in the dotted line boxed fractions are shown in the SDS-PAGE. The original gel images can be found in S1 Raw Images. (TIF)</p

Comparison of SARP with EmbR (PDB ID: 2FEZ) comprising an additional C-terminal FHA domain.

Comparison of SARP with EmbR (PDB ID: 2FEZ) comprising an additional C-terminal FHA domain.</p

List of primer sequences used in this study.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the −35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β′ zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.</div

RbpA and CarD do not influence the transcriptional activation capability of SARP.

In vitro transcription assays with or without RbpA and CarD on the afsS promoter (left) and transcription assays with or without 500 nM SARP in the presence of RbpA and CarD (right). The data underlying this figure can be found in S1 Data; error bars, SEM; n = 3. (TIF)</p

In vitro assays of the <i>Streptomyces coelicolor</i> AfsR SARP.

(A) Core promoter sequences used for in vitro assays. The mutations introduced into the afs box are highlighted. The mutated target sites contained mutations in upstream repeat (M1), the downstream repeat (M2), or both repeats (M1M2). The actII-4 promoter was used as a control. (B) Fluorescence polarization assays of the SARP with mutant afs box (M1, M2, and M1M2). Error bars represent mean ± SEM of n = 3 experiments. (C) In vitro MangoIII-based transcription assays with or without 500 nM SARP in the absence of RbpA and CarD. Error bars represent mean ± SEM of n = 3 experiments. The data underlying B and C can be found in S1 Data. (TIF)</p

Numerical values used to generate graphs.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the −35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β′ zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.</div

ATPγS activates phosphorylated AfsR and AfsR_4E.

Transcription assays involving 500 nM phosphorylated AfsR (phos-AfsR) or AfsR4E, with and without preincubation with 1 mM ATP or ATPγS. CK represents the control group without the addition of AfsR. The data underlying this figure can be found in S1 Data; error bars, SEM; n = 3. (TIF)</p