FIG 1.

Acetylation of K193 site affects the DNA-binding ability of CRP. (a) DNA-binding abilities of CRP^WT and CRP mutants. EMSA was used to evaluate the DNA-binding abilities of CRP^WT and its derivatives at the indicated concentrations (0, 5, and 15 μM) to biotin-labeled target DNA 2D3 following a 20-min incubation. Lane 1 represents the labeled DNA alone. (b) DNA-binding abilities of CRP^WT were evaluated with different concentrations of cAMP. (c) Effect of cAMP on DNA-binding abilities of CRP^K193R, CRP^K193Q, and CRP^K193A evaluated by EMSA. (d) Western blot analysis of purified CRP^WT and CRP^K193Ace using a specific antibody against the acetylated K193 residue of CRP_Mtb, in which K193 was successfully acetylated (CRP^K193Ace). Antibodies specific for CRP (anti-CRP; 1:2,000) and acetylated K193 peptides (anti-K193^Ace; 1:2,000) were used. (e) DNA-binding abilities of CRP^K193Ace and CRP^WT. The binding of CRP^WT to the target DNA 2D3 could be completed by a 500-fold excess of unlabeled 2D3 DNA as shown in lane 5 of panel a and lane 8 of panel e. The concentrations of cAMP and proteins used in the EMSA are indicated, and each EMSA result is representative of three independent replicates. K193Q, substitution of K193 with glutamine to mimic acetylation; K193R, substitution of K193 with arginine to mimic deacetylation; K193A, substitution of K193 with glycine.