Phylogenetic conservation among > 100 group I introns and previous in vitro studies have implicated a G.U pair as defining the 5'-splice site for exon ligation. The U residue defines the 3' end of the 5' exon, and the complementary G residue is part of the internal guide sequence (IGS) that base pairs to the 5' exon. We now quantitate the effect of this pair on individual reaction steps using the L-21ScaI ribozyme, which is derived from the group I intron of Tetrahymena thermophila pre-rRNA. The following results indicate that interactions with this G.U pair contribute to the binding of the 5'-exon, the positioning of the 5'-splice site with respect to the catalytic site, and the chemical step. The oligonucleotide, CCCUCU, binds to the ribozyme approximately 20-fold stronger than CCCUCC despite the fact that the U-containing oligonucleotide forms an approximately 5-fold less stable duplex with an oligonucleotide analog of the IGS, GGAGGG. This and two independent experimental observations indicate that the G.U pair contributes approximately 100-fold (3 kcal/mol, 50 degrees C) to tertiary interactions that allow the P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core. The approximately 50-80-fold increase in miscleavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCCUCU with C or upon removal of the 3'-terminal U suggests that the tertiary interactions with the G.U pair not only contribute to docking but also ensure correct positioning of the 5'-splice site with respect to the catalytic site, thereby minimizing the selection of incorrect splice sites. Comparison of the rates of the chemical cleavage step with G.U vs G.C suggests that the G.U pair contributes approximately 10-fold to the chemical step. It was previously suggested that the 2'-hydroxyl of this U residue helps stabilize the 3'-oxyanion leaving group in the chemical transition state via an intramolecular hydrogen bond. Relative reactivities of oligonucleotide substrates with ribose and deoxyribose U and C are consistent with a model based on a recent X-ray crystallographic structure in which the exocyclic amino group of G helps orient the 2'-hydroxyl of U via a bridging water molecule, thereby strengthening the hydrogen bond donated from the 2'-hydroxyl group to the neighboring incipient 3'-oxyanion. Finally, kinetic and thermodynamic evidence for the formation of a G.C+ wobble pair is presented.(ABSTRACT TRUNCATED AT 400 WORDS)