A representative hatchet ribozyme requires divalent cations such as Mg2+ to promote RNA strand scission with a maximum rate constant of ~4/min.

Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal cations and that they have a complex active site which employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.

X-ray crystallographic structural studies on the hammerhead, hairpin, GlmS, hepatitis delta virus (HDV), Varkud satellite, and pistol ribozymes have defined the overall RNA fold, the catalytic pocket arrangement, the in-line alignment, and the key residues that contribute to the cleavage reaction.

[4] In addition, the removal of the nucleophilic hydroxyl renders the ribozyme inactive as it is not able to create the cleavage site.

The tertiary fold consists of four stem substructures which covalently stack upon each other forming the helical and loop structures, called P1, P2, P3, and P4, L1, L2 and L3 respectively (though not shown in the figure above).

P1 is composed of three or six base pairs roughly 40% and 60% of the time respectively in its natural state, suggesting that length corresponds to catalytic function.

The steep slope observed at lower Mg2+ concentrations suggests that more than one metal ion is necessary for each RNA to achieve maximal ribozyme activity.

It is possible that native unimolecular constructs, also carrying P0, might achieve saturation at concentrations of Mg2+ that are closer to normal physiological levels.

pH-dependent ribozyme activity increases linearly with a slope of 1 until reaching a kobs, of a Michaelis-Menten plot, plateau of ~4/min near a pH value of 7.5.

Both the pH dependency and the maximum rate constant have interesting implications for the possible catalytic strategies used by this ribozyme class.

[3] The Hatchet ribozyme construct remains completely inactive when incubated in the absence of Mg2+ in reactions containing only other monovalent cations at 1 M (Na+, K+, Rb+, Li+, Cs+), 2.5 M (Na+, K+), or 3 M (Li+).

[3] One standard application is to use flanking self-cleaving ribozymes to generate precisely cut out sequences of functional RNA molecules (i.e. shRNA, saiRNA, sgRNA).