The cleavage reaction of rRNA by M5 occurs through the nucleophilic attack of a water molecule to the scissile phosphate, followed by departure of the 3 ′ leaving group (see Figure ), similarly to other RNases. However, it is a priori unclear whether the attacking water molecule is deprotonated before the attack-and in that case, where the proton is transfered, either to Glu96 or a backbone phosphate-or whether its proton is shuttled to the leaving group through the scissile phosphate oxygen atoms (see Figure ). In addition, the two proton transfers can happen in a more or less concerted fashion with the nucleophilic attack and departure of the 3 ′ leaving group. Only few computational studies of RNases are reported in the literature, almost exclusively on RNase H, where these points are still debated, including the identity of a possible general acid to reprotonate the leaving group.