MODELING STUDY ON THE CLEAVAGE STEP OF THE SELF-SPLICING REACTION IN GROUP-I INTRONS

A three-dimensional model of the Tetrahymena thermophila group I intro n is used to further explore the catalytic mechanism of the transphosp horylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., W esthof, E.J MoL Biol. 216,585-610 (1990)), we first converted their li gation step model into a model of the cleavage step by the substitutio n of several bases and the removal of helix P9. Next, an attempt to pl ace a trigonal bipyramidal transition state model in the active site r evealed that this modified model for the cleavage step could not accom modate the transition state due to insufficient space. A lowering of P 1 helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to m odel the molecular contacts proposed by Pyle et al. (Pyle, A. M., Murp hy, F. L., Cech, T. R. Nature 358,123-128.(1992)), based on mutational studies involving the J8/7 segment Two hydrated Mg2+ complexes were p laced in the active site of the ribozyme model, using the crystal stru cture of the functionally similar Klenow fragment (Beese, L.S., Steitz , T.A. EMBO J. 10,25-33(1991)) as a guide. The presence of two metal i ons in the active site of the intron differs from previous models, whi ch incorporate one metal ion in the catalytic site to fulfill the post ulated roles of Mg2+ in catalysis. The reaction profile is simulated b ased on a trigonal bipyramidal transition state, and the role of the h ydrated Mg2+ complexes in catalysis is further explored using molecula r orbital calculations.