RNA cleavage mechanism Previous computational studies of 2-ion RNases mostly focused on RNase H, and suggested different, possibly co-existing, mechanisms for the cleavage. A first study singled out two possible mechanisms: a first one where the proton of the attacking water molecule is shuttled to the leaving group through several water molecules (similar to our "Shuttling_2wat" mechanism), and a second where the reaction, starting with the attack of a hydroxide anion (deprotonated water), ends with stabilization of the deprotonated 3 ′ OH by the site B Mg 2+ ion. Another study 14 -starting from a slightly different active site conformation with an additional Glutamate Glu188 bound to site A Mg 2+ , which again echos our observation of a rich conformational landscape in such active sites-suggests that the water nucleophile donates its proton to the downstream phosphate (similar to our "A118_1wat" mechanism) and that the leaving group is reprotonated by an aspartate residue, initially protonated. It however could not exclude alternative reprotonation routes that were not explicitly considered.