Fig. 4.

Active site geometry and mutational study results. The scissile bond is between nucleotides 16 and 17. (A). Close-up view of the structure near the RNA cleavage site of the 24mer noncleavable complex (upper panel) and that of the 16mer product-like complex (lower panel). The scissile phosphate position is indicated by a red oval. Protein residues within 3.6 Å of any of the atoms of A16 and A17 are displayed and those within 3.2 Å are indicated by dashed lines to RNA. (B). RNA cleavage activity assay results indicate critical roles of four positively charged residues in catalysis. Trace amount of 5′-radiolabeled 24mer repeat RNA was incubated with 1 μM SsCas6 (WT) and various mutants (indicated by single letter amino acid codes) for 10 minutes at 45°C. The reaction products were separated on a denaturing polyacrylamide gel and visualized by phosphorimaging. The lane labeled with “-” contains RNA and the reaction buffer. (C) Progression curves of enzyme activities under a single turnover condition for the wild-type and four mutants of SsCas6. The single-turnover rates for the wild-type and the mutants are labeled near the respective curves.