In trans activation of wild-type Ire1 RNase by Ire1 with RNase with a double mutation. (a) Schematic representation of RNase dimers formed via interface IF1c . Due to a twofold symmetry, each RNase dimer can accommodate RNA stem-loop in two equivalent orientations (such as A and B). Both orientations are productive for wild-type Ire1. Neither orientation is productive for the H1061N mutant (C) and (D) due to disrupted catalysis. Only one orientation (E) is productive for the wild-type/H1061N chimera. Only one orientation (G) is also productive for the wild-type/H1061N+R1039A chimera, however this orientation is impaired by the R1039A mutation in the helix-loop element (HLE). (b) Titration of wild-type Ire1KR32 with Ire1KR32(H1061N) single mutant and Ire1KR32(H1061N, R1039A) double mutant. Reactions were conducted as in Figure 2d but contained 0.3 μM wild-type Ire1KR32 throughout the titrations to ensure subsaturating (k2/K1/2) regime for RNA cleavage.