Specific pre-crRNA binding and cleavage by Cas5d. (A) Secondary structure of T. thermophilus Dvulg repeat RNA. (B) Gel mobility-shift assays for RNA binding by Cas5d with increasing concentrations of protein (2, 30, 40, 500, 1000 nM). (Left to right) A 37-nucleiotide model of pre-crRNA as shown in A, reverse complement of A RNA, 4- and 8-nt truncations of pre-crRNA. Cleaved RNA is indicated (*). (C) Time course for gel mobility-shift assay of pre-crRNA binding by 100 nM of Cas5d. (Left to right) 1, 2, 5, 10, 15, 30, 45, 60 min. Cleaved RNA is indicated (*). (D) Gel mobility-shift assays for binding of pre-crRNA substituted with dG at position 26 by Cas5d with increasing concentrations of protein (2, 30, 40, 500, 1000 nM).
