The structure and catalytic mechanism of a pseudoknot-containing hammerhead ribozyme

We have determined the crystal structure of a pseudoknot (PK)-containing hammerhead ribozymethat closely resembles the pistol ribozyme, with essentially identical secondary structure andconnectivity. The activity is more sensitive to deletion of the of G8 2’OH than to the absence ofmagnesium ions, indicating that the catalytic mechanism is the same as the extended hammerhead,and distinct from the pistol ribozyme. Here we show that nucleophilic attack is almost perfectly in-line,and the G8 2’OH is well positioned to act as general acid, being directed towards the O5’ leavinggroup, and 2.9 Å away from it. Despite the similarity in overall structure to the pistol ribozyme, thelocal structure close to the cleavage site differs, and the PK hammerhead retains its uniquemechanistic identity and demonstrates enhanced activity over other hammerhead ribozymes understandard conditions.