Fig. 6. Craspase proteolytically cleaves Csx30 in an RNA-dependent manner.

(A) Genetic context for RpoE, Csx31 and Csx30 co-expression with Craspase wild-type (WT) or mutant (MT; H585A C627A) and a target RNA in E. coli BL21-AI. (B) Protein gel showing the eluted protein content from Streptavidin purifications of Tag-RpoE, Tag-Csx31 and Tag-Csx30 after co-expression with either Craspase WT or Craspase MT (H585A C627A). Colored arrows indicate the expected size for full length protein. (C) Protein gels after Craspase WT or Craspase MT (H585A C627A) incubation with Csx30 in the presence of target RNA or non-target RNA. Protein cleavage products are indicated with a red asterisk. (D) Protein gel after Craspase WT incubation with target RNA containing either a non-matching PFS (NPFS) or matching PFS (PFS). (E) Left: protein gel after incubation of Tag-Csx30 with target RNA and Craspase WT or Craspase D698A R294A, with or without prior incubation with MgCl₂. Right: protein gel after incubation of Tag-Csx30 with target ssDNA and Craspase D698A R294A. (F) Model for Craspase functionality. Once unbound Craspase has bound a target RNA, the peptidase activity is activated. This results in proteolytic cleavage of Csx30 between L407 and D408. Upon target RNA cleavage by Craspase, the peptidase activity is shut off.