A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator

Figures

• 

  Fig. 1. Type III CRISPR locus of T. thermophilus HB8 (TTHB) and model structure of TTHB144 with cA4 bound. (a) Gene neighborhood of TTHB144 encoded on plasmid pTT27. Three genes encoding CARF domain-containing proteins (shown in purple) are present in the type III CRISPR locus of TTHB. TTHB152 is a Csm6 family protein, while TTHB144 and TTHB155 are hypothetical proteins of unknown function. (b) TTHB144 structure modeled using Phyre2. Each subunit of the predicted homodimer is shown by a different color (blue or cream). The highly conserved residues Thr-11, Lys-94 and His-368 are shown. (c) cA4 modeled into the CARF domain of TTHB144. Lys-94 is situated centrally beneath the cA4 molecule, and the side-chain of Thr-11 is suitably positioned to interact with cA4.

• 

  Fig. 2. RNA degradation and cA4 cleavage occur in separate domains of TTHB144. (a) Phosphorimage of denaturing PAGE visualizing the degradation of 50 nM radiolabeled A1 RNA, as previously described [21], by TTHB144 (0.5 μM dimer), its CARF domain variants K94A and T10A/T11A and the HEPN domain variant H368A. The reaction was incubated at 70 °C for 60 min in pH 8.0 buffer containing 20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA and 3 units SUPERase•In™ inhibitor, before quenching by phenol–chloroform extraction. RNA was cleaved by wild-type (wt) protein and the T10A/T11A variant in the presence of 1 μM cA4, but not by the K94A or H368A variants. (b) Phosphorimage of denaturing PAGE visualizing degradation of 50 nM radiolabeled RNA by TTHB144 (1 μM dimer) when incubated with 20 μM cA4 at 70 °C. Control reactions incubating RNA with buffer (c1) or RNA with protein in the absence of cA4 (c2) are shown. All of the substrate RNA was degraded within 15 s (lane 3). (c) Plot of fluorescence emitted when RNaseAlert™ substrate (1.5 μM; Integrated DNA Technologies) was degraded by 125 nM dimer TTHB144 in the absence or presence of 500 nM cA4 at 50 °C. Fluorimetry was carried out in a Cary Eclipse…

• 

  Fig. 3. cA4 binding and cleavage by wild-type and variant TTHB144 enzymes. (a) Panels are phosphorimages of denaturing PAGE visualizing degradation of 200 nM radiolabeled cA4 by TTHB144 and variants (8 μM dimer) at 70 °C. cA4 is degraded to A2 > P. Time in minutes is indicated. Protein and radiolabeled cA4 were incubated in pH 8.0 buffer containing 20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA and 3 units SUPERase•In™ inhibitor, and reactions were quenched at the indicated time-points by phenol–chloroform extraction. A cA4 degradation assay was also carried out in the presence of 1 μM A1 RNA in order to evaluate the effect of RNA binding and cleavage at the HEPN active site on cA4 degradation at the CARF domain. (b) Plot of the fraction of cA4 cut versus time, generated by quantifying the densiometric signals from the phosphorimages depicted in panel a. All data points are the average of at least three technical replicates and are fitted to an exponential rise equation to derive the rate of cA4 degradation, as described previously [21]. Data points for TTHB144 are the average of six replicates encompassing two biological replicates with three technical replicates for each. Error bars show the…