FIGURE 2 - available via license: Creative Commons Attribution 4.0 International
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SyCsx1 shows cA4-dependent ribonuclease activity. (A) Multiple sequence alignment of SyCsx1 and homologous Csx1 proteins showing the conserved DxTHG motif from the CARF domain and the RxxxxH active site of the HEPN domain. An asterisk marks residues that were shown to impact protein function. (B) His-tagged SyCsx1 (48.7 kDa) was isolated from E. coli by affinity chromatography and further purified by sizeexclusion chromatography on a Superdex 200 10/300 column (upper panel). Elution fractions were analyzed by SDS-PAGE (lower panel, see Supplementary Figure S1). (C) 400 ng of Synechocystis total RNA were incubated in the presence or absence of SyCsx1 and 300 nM cOAs for 1 h and subsequently separated on a 12% denaturing PAA gel. The dotted line indicates a non-contiguous sample that was omitted from the gel. (D) A fluorogenic ribonuclease activity assay measuring the cleavage of 40 nM RNaseAlert substrate by SyCsx1 depending on the addition of 300 nM cA4 (excitation 490 nm; emission 520 nm). The cleavage of the RNaseAlert substrate separates a fluorophore/quencher pair, generating a fluorescent signal. (E) Fluorogenic ribonuclease activity assay of SyCsx1 variants with point mutations in the ligand-binding site of the CARF domain or the active site of the HEPN domain. All fluorogenic assays were performed in triplicates and mean values of relative fluorescence normalized to the maximum substrate turnover are shown.
Source publication
Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate...
Citations
CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1–5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6–8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2′-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.
