Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3′-terminal G414 residue of the intron 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 investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3′-terminal G414 docked into the G-binding site. This docking is destabilized by ∼100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K int ) 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5′ exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.Self-splicing of the Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps ( Figure 1A; Cech, 1990). In the first step, an exogenous guanosine (G) 1 acting as a nucleophile attacks the 5′ splice site, leaving the 5′ exon with a 3′-hydroxyl terminus. In the second step, the 3′-terminal residue of the intron, G414, binds to the active site, and its 3′ phosphoryl group is attacked by the 3′-hydroxyl of the 5′ exon. This gives ligated exons and the free intron.The first step of self-splicing has been studied by using a shortened version of the intron. This version, referred to as the L-21ScaI ribozyme ( Figure 2B), has the first 21 and last 5 nucleotides removed and mimics the first step of the selfsplicing reaction ( Figure 1B). Extensive studies of the L-21ScaI ribozyme have provided insights into the mechanism of RNA catalysis and the first step of self-splicing (Cech et al., 1992).The second step of self-splicing, however, has not been characterized in as much detail. Several components have been shown to be involved in this step (Figure 2A). Guanosine is universally conserved a...