In vitro enzymatic assays

The in vitro adenylylation assay using purified unmodified HepT and MntA was modified based on the in vitro assay of Truttmann et al. (26). The unmodified HepT and MntA proteins are very similar in size (133 aa and 139 aa, respectively), making it difficult to distinguish the reaction products. Therefore, 24 extra aa (including the 6× His tag) were fused to the N terminus of MntA, a strategy we previously used that has potentially minimal effect on the activity of MntA (27). Purified MntA (10 μg) was added to 40 μl of reaction buffer (containing 20 mM Tris–HCl, 10 mM MgCl₂ and 5 mM DTT) supplemented with 0 to 12 μM ATP and incubated at room temperature for 30 min. Unmodified HepT (20 μg) was then added to the reaction and incubated at 37°C for another 60 min. The reaction was stopped by adding SDS-PAGE loading buffer and the samples were analyzed by tricine-SDS-PAGE. To capture the intermediate products, a reaction with 3 μM ATP serving as the substrate was stopped by adding EDTA, and the products were analyzed by HPLC/Q-TOF-MS as described above.

For the in vitro RNase cleavage assay, ompA mRNA was synthetized, and the assay was performed as previously described using various of concentrations of HepT (0–160 nM) (17). The reaction mixture was incubated at 37°C for 30 min. To obtain the polyadenylylated HepT protein, the lysate from cells coexpressing hepT and mntA was shaken vigorously after incubating with Ni-NTA agarose, and polyadenylylated HepT with purity >90%, was obtained after a second elution (Supplementary Figure S1).