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. 2022 Feb 19;298(4):101752. doi: 10.1016/j.jbc.2022.101752

Figure 1.

Figure 1

RbpA E17 and R10 synergize to promote Fdx activity against M. tuberculosis RNAP-σAin vitro.A, schematic of RbpA’s four domain structure including the location of the substituted residues, R10, E17, R79, R88, and the two M. tuberculosis truncation mutants, RbpA 26–111 lacking the NTT, and RbpA 72–111 lacking the NTT and CD. B, representative gels showing Fdx (0 μM, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM) inhibition of M. tuberculosis RNAP-σA production of three nucleotide transcripts alone or in complex with RbpAMtbWT, RbpAMtbR10A, RbpAMtbE17A, RbpAMtbR10A/E17A, RbpAMtbR79A, RbpAMtbR88A, RbpAMtb26–111, or RbpAMtb72–111 from a linear dsDNA template containing positions −80 to +70 of M. tuberculosis rrnAP3 (relative to the +1 transcription start site). C, dose–response curves of the experiments shown in (A). The curves are generated from at least four replicates from at least two different experiments. Percent inhibition at each Fdx concentration included in the plots compared to no drug is depicted as the mean ± SD. The IC50 for each replicate was calculated by nonlinear regression analysis with four-parameter (EC50, Hill Slope, top and bottom curve plateaus) fitting of log transformed Fdx concentration versus normalized response, with the mean IC50 and 95% confidence interval listed in the table. D, structural modeling of Fdx binding pocket on the RbpA-bound M. tuberculosis RNAP-σA from PDB structure 6BZO. Fdx and RNAP residues involved in the RNAP-Fdx binding interface are shown with PyMol stick representation while the rest of the structure is shown with PyMol cartoon representation. Polar interactions are indicated by red dashed lines, and potential van der Waals interactions are shown as gray lines. E, structural modeling of RbpA-bound M. tuberculosis RNAP-σA from PDB structure 6C04. RbpA R10 and RNAP σA D441 are shown with PyMol stick representation while the rest of the structure is shown with PyMol cartoon representation. The polar interaction between RbpA R10 and RNAP σA D441 is indicated by the red dashed line. CD, core domain; Fdx, fidaxomicin; NTT, N-terminal tail; RbpA, RNA polymerase binding protein A; RNAP, RNA polymerase.