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. 2014 Sep 17;289(45):31503–31512. doi: 10.1074/jbc.M114.575472

FIGURE 5.

FIGURE 5.

Binding affinity assays of PPR10 containing cysteine to serine mutations with RNA element. A and D, EMSAs are performed on wild type PPR10, PPR10 variants containing each single cysteine to serine mutation, and PPR10 with quadruple cysteine-to-serine mutations in the presence of psaJ or atpH RNA. The RNA sequences of psaJ and atpH are indicated in Fig. 1B. Binding reactions contain ∼40 pm radiolabeled RNAs and PPR10 at the indicated concentrations. B and E, the PPR10 fractions of RNA bound and concentrations in each lane of Fig. 4, A and D, are plotted and fitted to the calculate equilibrium Kd values. C and F, the summary table of equilibrium Kd values for reactions of different PPR10 mutants with psaJ or atpH RNA elements. In the presence of psaJ RNA, the quadruple mutant displays >10-fold higher Kd value than others, indicative of much weaker binding affinity of quadruple mutant for psaJ RNA. In the presence of atpH RNA, the wild type, four single mutants, and the quadruple mutants exhibit uniform EMSA profiles and similar Kd values.