Specificity from steric restrictions in the guanosine binding pocket of a group I ribozyme

The 3' splice site of group I introns is defined, in part, by base pairs be tween the intron core and residues just upstream of the splice site, referr ed to as P9.0. We have studied the specificity imparted by P9.0 using the w ell-characterized L-21 Seal ribozyme from Tetrahymena by adding residues to the 5' end of the guanosine (G) that functions as a nucleophile in the oli gonucleotide cleavage reaction: CCCUCUA(5) (S)+ NNG half arrow right over h alf arrow left CCCUCU + NNGA(5). UCG, predicted to form two base pairs in P 9.0, reacts with a (k(cat)/K-M) value similar to 10-fold greater than G, co nsistent with previous results. Altering the bases that form P9.0 in both t he trinucleotide G analog and the ribozyme affects the specificity in the m anner predicted for base-pairing. Strikingly oligonucleotides incapable of forming P9.0 react similar to 10-fold more slowly than G, for which the mis paired residues are simply absent. The observed specificity is consistent w ith a model in which the P9.0 site is sterically restricted such that an en ergetic penalty, not present for G, must be overcome by G analogs with 5' e xtensions. Shortening S to include only one residue 3' of the cleavage site (CCCUCUA) eliminates this penalty and uniformly enhances the reactions of matched and mismatched oligonucleotides relative to guanosine. These result s suggest that the 3' portion of S occupies the P9.0 site, sterically inter fering with binding of G analogs with 5' extensions. Similar steric effects may more generally allow structured RNAs to avoid formation of incorrect c ontacts, thereby helping to avoid kinetic traps during folding and enhancin g cooperative formation of the correct structure.