Initially, HSPC117/RtcB proteins were assumed to catalyze the direct nucleophilic attack of the 2 0 ,3 0 -cyclic phosphate by a 5 0 -hydroxyl group, which seemed likely for two main reasons. First, 2 0 ,3 0 -cyclic phosphates are energyrich substrates with a favorable leaving group [108] and second, HSPC117/RtcB-catalyzed ligation reactions did not seem to be strictly dependent on the addition of nucleotide triphosphate cofactors [102] [103] [104] 108] . However, only enzyme preparations that were rigorously purified in the presence of chelating agents proved to yield RtcB preparations sufficiently pure to demonstrate the real cofactor requirements of RtcB-catalyzed 3 0 -5 0 RNA ligation [108] . Thus, 3 0 -5 0 RNA ligation by RtcB proteins occurs as a sequential reaction involving the stoichiometric hydrolysis of nucleotide triphosphates rather than direct nucleophilic attack of the 2 0 ,3 0 -cyclic phosphate by a 5 0 -hydroxyl group [108] . One advantage of this somewhat counterintuitive strategy might lie in the suppression of the backward reaction-the cleavage of the newly formed phosphodiester-as it is catalyzed by bacterial 2 0 -5 0 -ligases (see below) [59] .