Figure 5.

Dissection of cA₄ binding and catalysis by Csx1–Crn2. (A) Cartoon representation of Csx1–Crn2 showing sites targeted by mutagenesis. (B). Electrophoretic mobility shift assay (EMSA) showing binding of radiolabelled cA₄ (∼10 nM) by Csx1-Crn2 variants (0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.1, 1.0, 10.0 and 20.0 μM, respectively). On each gel is a control reaction (c) with cA₄ in the absence of protein. The ring nuclease defective variant H495A bound cA₄ with an apparent dissociation constant around 20 nM. The S452Y variant, which was designed to abolish cA₄ binding by the ring nuclease domain, had a severe binding defect, with cA₄ degradation apparent at high protein concentrations. The H129W variant, designed to abolish cA₄ binding in the CARF domain, showed cA₄ degradation starting from around 20 nM enzyme. The data are representative of three technical replicates. (C) TLC showing degradation of cA₄ (200 nM) by wild-type and variant enzymes (5 μM dimer) at 50°C. A time-course of 10 s, 1 min and 10 min was carried out and a control reaction (c) incubating buffer with radiolabeled cA₄ for 10 min is also shown. The H495A variant was inactive as shown previously. The H129W and H402A variants targeting the CARF and HEPN (Csx1) domains, respectively, had normal ring nuclease activity. Whereas the S452Y variant targeting the Crn2 domain had strongly reduced activity, consistent with weaker binding of cA₄ by the Crn2 domain. (D) Denaturing PAGE showing cleavage of radiolabelled substrate RNA (100 nM) by Csx1-Crn2 and variants (5 μM dimer) and 100 μM cA₄ at 50°C. RNA cleavage was examined at two time-points (5 & 30 min) and control reactions incubating radiolabelled RNA in buffer (c1) or RNA and protein in the absence of cA₄ activator (c2) for 30 min are also shown. The H129W and H402A variants were inactive as expected. The data are representative of three technical replicates.