PROBING THE HAMMERHEAD RIBOZYME STRUCTURE WITH RIBONUCLEASES

Susceptibility to RNase digestion has been used to probe the conformation of the hammerhead ribozyme structure prepared from chemically synthesised RNAs. Less than about 1.5% of the total sample was digested to obtain a profile of RNase digestion sites. The observed digestion profiles confirmed the predicted base-paired secondary structure for the hammerhead. Digestion profiles of both cis and trans hammerhead structures were nearly identical which indicated that the structural interactions leading to self-cleavage were similar for both systems. Furthermore, the presence or absence of Mg2+ did not affect the RNase digestion profiles, thus indicating that Mg2+ did not modify the hammerhead structure significantly to induce self-cleavage. The base-paired stems I and II in the hammerhead structure were stable whereas stem III, which was susceptible to digestion, appeared to be an unstable region. The single strand domains separating the stems were susceptible to digestion with the exception of sites adjacent to guanosines; G(L2.1) in the stem II loop and G(12) in the conserved GAAAC sequence, which separates stems II and III. The absence of digestion at G(L2.1) in the stem II hairpin loop of the hammerhead complex was maintained in uncomplexed ribozyme and in short oligonucleotides containing only the stem II hairpin region. In contrast, the G(12) site became susceptible when the ribozyme was not complexed with its substrate. Overall the results are consistent with the role of Mg2+ in the hammerhead self-cleavage reaction being catalytic and not structural.