REPLACEMENT OF THE CONSERVED G-CENTER-DOT-U WITH A G-C PAIR AT THE CLEAVAGE SITE OF THE TETRAHYMENA RIBOZYME DECREASES BINDING, REACTIVITY,AND FIDELITY

There is a phylogenetically conserved G.U pair at the 5'-splice site o f group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. We have used a ribozyme derived fro m the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G.U or a G-C pair at this posi tion. Ribozyme binding of oligonucleotides at 42 degrees C was measure d by native gel electrophoresis and equilibrium dialysis. Binding of G GCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G.U pair at the cl eavage site. This is surprising since a terminal G-C enhances the bind ing between oligonucleotides by 20-fold relative to a terminal G.U. Th ermal denaturation studies indicate that C(-1)P and several analogs wi th deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGG AAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds th e ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. T he substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly th an GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleav ed at the wrong nucleotide position. Taken together, the results sugge st that a G-C pair at the ribozyme cleavage site makes docking of the guide-sequence substrate helix into the catalytic site less favorable than a G.U pair. The resulting consequences of weaker binding, slower reaction, and reduced cleavage fidelity provide a rationale for the ph ylogenetic conservation of the G.U.