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 Fluorescence Spectrophotometer (Agilent Technologies) with excitation and emission wavelengths set to 490 and 520 nm, respectively. (d) Phosphorimage of denaturing PAGE visualizing degradation of 400 nM radiolabeled cA4 generated using SsoCsm complex, as previously described [21], by TTHB144 (4 µM dimer) and variants at 70 °C for 120 min. cA4 was degraded to a slower migrating product. (e) High-resolution liquid chromatography mass spectrometry of cA4 produced using the SsoCsm complex and cleavage products generated on incubation with TTHB144 (40 µM dimer) at 70 °C. cA4 (~ 16 µM; top panel) was degraded to intermediate and product species (middle panel) with identical retention times to A4 > P and A2 > P, respectively (bottom panel). A4 > P and A2 > P standards were generated using the E. coli MazF toxin as previously described [8].

Fig 3: The dependences of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance.Curve 1 (solid line) represents the fluorescence intensity that was calculated according to Eq. 2; the circles on the curve represent the fluorescence intensity values recorded by the Cary Eclipse (Agilent Technologies, Australia) spectrofluorimeter. Curves 2 and 3 represent the fluorescence intensity recorded by a homemade spectrofluorimeter [20] and by a Fluorolog-3 (Horiba, Japan) spectrofluorimeter, respectively. The straight line F = 2.303AFLq is tangent to curves 1, 2 and 3 at AFL = 0. Here k' is chosen so that k'I0 = 1, and consequently numerically equals to q at . The inset represents the dependence of W on AFL calculated by Eq. 4 (curve 1) and determined experimentally for the homemade spectrofluorimeter (curve 2). For this plot, dyes with different fluorescence quantum yields were used: quinine sulfate (q = 0.52 [36], green circles) and NATA (q = 0.14 [21] red circles).

Fig 4: Mitochondrial EtBr efflux assays. Mitochondria were prepared as described [48]. The 2 ml reactions contained 0.24 mg ml-1 mitochondrial protein in buffer (250 mM sucrose, 30 mg ml-1 BSA, 5 mM MgCl2, 80 mM KC1, 10 mM MOPS, pH 7.2). After approximately 2 min, 10 µl of 1 mg ml-1 EtBr was added to each cuvette, followed by stepwise additions of 10 µl of 1 M succinate, 2 µl of 1 mM oligomycin and 10 µl of 2 mM CCCP. The assay was carried out at 25 °C, with continuous stirring, in an Agilent Cary Eclipse fluorescence spectrophotometer (excitation 520 nm and emission 590 nm; slit width 5 nm). The fluorescence was allowed to stabilize between each addition and fluorescence was measured every 15 s. WT, N. crassa wild-type (FGSC 9718); ?Por1, an isogenic porin deletion strain [50]. F5 is the buffer control and TBS (Tris-buffered saline) is a buffer control lacking BSA. Taken from [109], with permission.