Fig. 6: Analysis of CvkR binding motifs.

a DNase I footprinting of the FAM-labeled P_cas12k and P_tnsB promoter sequences in the absence (red graphs) and presence of 0.1 µg CvkR (blue graphs). The protected region is boxed. Dideoxynucleotide sequencing traces are displayed (green, A; blue, C; black, G; red, T) and the DNA sequences protected by CvkR are indicated below (for P_cas12k) or above (for P_tnsB) the corresponding regions in capitalized bold letters. The sequence overlap is marked by vertical black lines. A dashed line indicates a single different nucleotide. The area adjacent to the CvkR-protected regions is displayed in lowercase letters. The TSS (red), −10 (green) and −35 elements (blue) are highlighted. b Sequence logo of the CvkR motif based on sequence alignments of cas12k and tnsB promoters of 15 CAST systems with cvkR genes (for sequence details see Supplementary Fig. 6). In the sequence logos, the −35 and −10 promoter regions are shaded in blue and green, and the TSSs in brown. The protected areas in the DNase I footprinting assays are boxed in blue and the palindromic CvkR binding motif in orange. c Wild-type and mutated promoter fragments used for EMSA assays. The probes P_cas12kcenter and P_tnsBcenter indicate the CvkR-protected region of cas12k and tnsB promoters in the DNase I footprinting assay, respectively. TSS, −10, and −35 elements in P_cas12kcenter and P_tnsBcenter are indicated as in a. CvkR-binding sites are pink-boxed. The inverted repeat is displayed in bold letters and highlighted by the pink arrows, while pink letters indicate the mutated sites. d Interaction between CvkR and P_cas12k variants analyzed by EMSA. The data are presentative from two or three independent experiments. The free probe and complexes of protein-DNA complexes are marked as “Free” and “Bound”, respectively. Source data are provided as a Source Data file.