Mutational analysis of loops 1 and 5 of the hairpin ribozyme.

A comprehensive analysis of base preferences for all positions in loops 1 and 5 of the hairpin ribozyme-substrate complex was carried out using a cis-ribozyme tethered to substrate by a pentapyrimidine loop. Ribozyme-substrate molecules were mutated to contain each of the three non-native base variations at each of the eight positions within these loops. Catalytic activity was measured for each mutant and compared to the activity of the original native sequence. This was the first time all base positions in these loops have been mutated to all variants and kinetically characterized. Various effects were found, ranging from invariant base positions to those with nearly complete tolerance of any base change. Two positions resulted in cleavage rates below the lower limit of accurate quantification for all non-wild-type base substitutions. These positions are G8 in the ribozyme and Gs6 in the substrate. When A10 was substituted with a pyrimidine, self-cleavage activity fell below the lower limit of detection while the remaining positions showed varying base preferences. The information reported here on loops 1 and 5 combined with previous mutagenesis data on loops 2 and 4 [Siwkowski, A., Shippy, R., and Hampel, A. (1997) Biochemistry 36, 3930-3940] completed a comprehensive mutational/kinetic analysis of every base position located within all the required loops of the hairpin ribozyme-substrate complex and allowed for the development of a mechanism for catalysis which is proposed.