MODELING OF A POSSIBLE CONFORMATIONAL CHANGE ASSOCIATED WITH THE CATALYTIC MECHANISM IN THE HAMMERHEAD RIBOZYME

Here we describe a possible model of the cleavage mechanism in the ham merhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine rum placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G 16.2 residue. The observed conformational change in the uridine rum su ggests an explanation for the conservation of G5. In the crystal struc ture of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3 Angstrom away from G16.2. However, the uridine turn is sufficiently f lexible to allow this conformational change with relatively modest cha nges in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions an alogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (199 1)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F Setlik et al., J. Biomol. Str: Dyn. 10, 945- 972 (1993)) and other phosphotransferases. Comparison of this model wi th one in which the uridine rum conformation was not changed showed th at although the changes in the C17 sugar pucker could be modeled, insu fficient space existed for the magnesium ions in the active site.