Fig. 7: The CcpA HTH domain is required for efficient binding to RNA in vitro. | Nature Communications

Fig. 7: The CcpA HTH domain is required for efficient binding to RNA in vitro.

From: The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins

Fig. 7

a SDS-PAGE of recombinant CcpA WT and T18DT33D phosphomimetic mutant used for EMSA. The original picture of one representative gel was provided in the Source data file. b, c dsDNA and dsRNA substrates used for the EMSA. d, e EMSA performed with increasing amounts of recombinant CcpA proteins (0, 1.5, 3, 6, 9 15, 21, 30 and 40 µM) and IRD800-labelled dsDNA and dsRNA substrates (0.1 µM) as well as a 50-fold excess of a non-specific competitor (poly(dI-dC)). Complexes were subsequently resolved on 1% TBE-agarose gels. Two replicate experiments were performed. Source data are provided in the Source data file. f, g Quantification of the EMSA results. Band intensities were quantified by ImageQauntTL v8.2.0.0. The binding curve and Kd value were obtained from Graphpad 9.3.1, assuming CcpA binds DNA and RNA as dimer. Two independent replicate experiments were used for quantification. Data are plotted as mean ± SD. The binding affinity (Kd) of CcpA WT to dsDNA substrate is 7.97 ± 0.54 µM (95% confidence interval), while to dsRNA is 9.72 ± 0.92 (95% CI).

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