FIGURE 5.

Immunopurified Cas6 cleaves CRISPR repeat RNA and associates with substrate and product RNAs. (A) Cleavage activity. Uniformly ³²P-labeled CRISPR repeat RNA was incubated in the absence (RNA) or presence of recombinant Cas6 (rCas6), whole cell extract (WCE), or samples from immunoprecipitation reactions using Cas6 antibodies; (Pre) preimmune; (Imm) immune; (S) supernatant; (P) pellet. The RNAs were separated on a 15% denaturing, 7 M urea-containing polyacrylamide gel, along with 5′-end-labeled RNA markers (M). (B) Northern blot analysis of Cas6 immunoprecipitation. RNAs extracted from WCE or from IPs using preimmune (Pre) or immune (Imm) Cas6 or Cmr2 antibodies were separated on 15% denaturing, 7 M urea-containing polyacrylamide gels, along with 5′-end-labeled RNA markers (M). A 5′-end-labeled DNA oligonucleotide antisense to psiRNA 6.01-specific sequences, or to the repeat sequence of P. furiosus CRISPRs 1, 5, and 6 were used as probes, as indicated. The positions of the 2× intermediate (2×), 1× intermediate (1×), and mature psiRNAs (arrows) are indicated.