The C-terminal HEPN domains of TtCsm6 form a composite endoribonuclease active site. (A) Overall view of the HEPN domain interface in the TtCsm6 dimer. The inset shows a close-up view of the HEPN domain active site. Conserved active site residues are shown in stick format. The Ni2+ ion present in the TtCsm6 crystal structure is shown as a green sphere. (B) Comparison of the HEPN ribonuclease active sites of yeast Ire1 (left), TtCsm6 (middle), and SmCsm6 (right). Active site residues are shown in stick format. The structures were aligned by least-squares superposition of the active site Asn, Arg, and His residues and are shown in identical orientations. (C) Nuclease activity assays of active site mutants of TtCsm6. The assays were performed using a 24-nt ssRNA substrate labeled with Cy5 at the 3′-end. Reactions were resolved on a 16% denaturing polyacrylamide gel and analyzed on a fluorescence gel scanner. For conciseness, the panel depicts a cropped region of the denaturing gel; a full-size image of the gel is shown in Supplemental Figure 7. (D) Ribonuclease activities of Csm6 orthologs. Recombinant T. thermophilus, S. epidermidis (SeCsm6), and P. horikoshii (PhCsm6) Csm6 proteins were incubated with a 24-nt Cy5-labeled ssRNA substrate at 37°C. Reactions were sampled at indicated time points and analyzed as for Figure 1C. For conciseness, the panel depicts a cropped region of the denaturing gel; a full-size image of the gel is shown in Supplemental Figure 8.
