Figure 8
Sequence and structure influence the VapC-mt11 cleavage site. Cleavage assays of (A) tRNAPro14 mutant with a GG consensus sequence replacement by AA, (B) an open stem loop mutant, and a (C) GG −> AA and open stem loop mutant. Full length and cleavage product sizes shown on the left. The small 19 nucleotide cleavage product ran off the gel in (C). Secondary structures of the in vitro synthesized mutants of tRNAPro14 are depicted above cleavage reaction gels. Anticodon stem loop (ASL), anticodon sequence (shaded grey), base pairing represented as black dots (●), mutated bases (red), consensus sequence (blue shaded circles), weak cleavage at wild-type cleavage site (small yellow arrow), and alternate cleavage site (green arrow). Primer extension of these mutants shown below. G, A, T, and C correspond to DNA-sequencing ladders using the same oligonucleotide as in the primer extension reactions for each tRNA. Alternate cleavage sites are indicated by the green arrow head to the right of the primer extension gel images. RNA sequence corresponding to the alternate cleavage sites (green arrow) within the (A) ASL or (B,C) D-loop are shown below each primer extension gel image, mutations indicated by underlined red text. All uncropped images shown in Supplementary Information Fig. 8.
