Fig. 6. Target RNA-directed allosteric regulation of LdCsm involves activation and deactivation mechanisms.

a CTR activates the LdCsm DNase. b NTR mediates autoimmunity avoidance by deactivation. Reactions were set up with 5 nM S10–60 ssDNA, 100 nM of LdCsm, and 400 nM non-homologous RNA (non-h. RNA) or 500 nM target RNA. After addition of one of the target RNAs, the mixture was incubated at 37 °C for 3 min. Samples were then analyzed by non-denaturing PAGE (in this page) or denaturing PAGE (Supplementary Fig. S8). Red arrowheads indicate the LdCsm–ssDNA complex. Relative ssDNA binding and percentage ssDNA cleaved of LdCsm facilitated by each of these target RNAs were estimated by image quantification of bands on non-denaturing PAGE and denaturing PAGE, using the accessory analysis tool equipped with a Typhoon FLA 7000. For the quantification of the substrate binding, the amount of ssDNA-LdCsm-CTR⁺⁶ complex is arbitrarily defined as 1. Results of average of three independent assays are shown with bars representing the standard deviation (±SD).