MECHANISTIC INVESTIGATIONS OF A RIBOZYME DERIVED FROM THE TETRAHYMENAGROUP-I INTRON - INSIGHTS INTO CATALYSIS AND THE 2ND STEP OF SELF-SPLICING

Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phos phoryl transesterification steps. One major difference between these s teps is that in the first an exogenous guanosine (G) binds to the acti ve site, while in the second the 3'-terminal G414 residue of the intro n binds. The first step has been extensively characterized in studies of the L-21Scal ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are inves tigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in th e second step and a nucleophile in its reverse. The rate constant of c hemical step is the same with exogenous G bound to the L-21Scal ribozy me and with the intramolecular guanosine residue of the L-21G414 riboz yme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and th e overall active site structure is the same in both steps of the splic ing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G41 4 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by similar to 100-fol d when G414 is attached to an electron-withdrawing pA group, The inter nal equilibrium with K-int = 0.7 for the ribozyme reaction indicates t hat bound substrate and product are thermodynamically matched and is c onsistent with a degree of symmetry within the active site. These obse rvations are consistent with the presence of a second Mg ion in the ac tive site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kineti c mechanism for ensuring efficient ligation of exons and raises new qu estions about the overall self-splicing reaction.