DISSECTION OF THE ROLE OF THE CONSERVED G-CENTER-DOT-U PAIR IN GROUP-I RNA SELF-SPLICING

Phylogenetic conservation among >100 group I introns and previous in v itro 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, a nd 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, wh ich is derived from the group I intron of Tetrahymena thermophila pre- rRNA. The following results indicate that interactions with this G.U p air contribute to the binding of the 5'-exon, the positioning of the 5 '-splice site with respect to the catalytic site, and the chemical ste p. The oligonucleotide, CCCUCU, binds to the ribozyme similar to 20-fo ld stronger than CCCUCC despite the fact that the U-containing oligonu cleotide forms an similar to 5-fold less stable duplex with an oligonu cleotide analog of the IGS, GGAGGG. This and two independent experimen tal observations indicate that the G.U pair contributes similar to 100 -fold (3 kcal/mol, 50 degrees C) to tertiary interactions that allow t he P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core. The similar to 50-80-fold increase in miscl eavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCC UCU with C or upon removal of the 3'-terminal U suggests that the tert iary 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 spl ice sites. Comparison of the rates of the chemical cleavage step with G.U vs G.C suggests that the G.U pair contributes similar to 10-fold t o 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. Relati ve reactivities of oligonucleotide substrates with ribose and deoxyrib ose U and C are consistent with a model based on a recent X-ray crysta llographic structure in which the exocyclic amino group of G helps ori ent the 2'-hydroxyl of U via a bridging water molecule, thereby streng thening the hydrogen bond donated from the 2'-hydroxyl group to the ne ighboring incipient 3'-oxyanion. Finally, kinetic and thermodynamic ev idence for the formation of a G.C+ wobble pair is presented. The terti ary energy of recognition of the G.U wobble pair appears to be suffici ent to perturb the pK(a) of C to favor a G.C+ wobble pair instead of t he Watson-Crick pair, despite the loss of a hydrogen bond in the base pair. This provides an example of RNA tertiary structure determining s econdary structure.