Purification and analytical characterization of NudC, NudC Mutants and NudE

E. coli One Shot BL21 Star (DE3) containing the respective plasmid were induced at OD₆₀₀=0.8 with 1 mM IPTG for 3 h. Purification was performed as described⁴. All proteins yielded a single band on SDS page (Coomassie staining, Supplementary Fig. 6a). For preparation of RNA-free pure NudC, 1 M urea was added to His-Trap buffer A and His-Trap buffer B. To ensure dimer formation, all NudC variants were analyzed by size exclusion chromatography on a Superdex 200 10/300 column (50 mM Tris-HCl pH 7.5, 150 mM NaCl, Supplementary Fig. 6b–f). Folding of all mutants was confirmed by CD spectroscopy (J-810 Jasco spectropolarimeter, 0.2 mg/mL protein in 5 mM Tris-HCl pH 7.5, 5 mM MgSO₄, 0.5 mM EDTA and 0.5 mM DTT, 1 nm bandwidth, 0.5 nm data pitch, 20 nm min⁻¹ scanning speed, 1 s response time, Supplementary Fig. 7).