Elucidating the catalytic mechanism of the hairpin ribozyme

Abstract

The information reported here completes a mutational analysis of every base position located within the required loops of the hairpin ribozyme-substrate complex. In all, 24 base positions were analyzed using a point-mutational approach. In addition, two base pairs were studied using a compensatory mutational approach, in which two base positions are mutated simultaneously. The combination of these experiments were conducted in an effort to better understand the base requirements for catalysis as well as base preferences which may shed light on potential tertiary contacts. In this thesis I propose there are only six essential bases within the hairpin ribozyme-substrate complex: five in the ribozyme (G[sub 8], A[sub 22], A[sub 23], C[sub 25], A[sub 38]) and one in the substrate (sG[sub 6]). These results also support a purine requirement at the A[sub 10] position within loop 1 of the ribozyme. In addition, compensatory mutagenesis results support the identification of an A[sub 26]:G[sub 36] base pair as well as a purine bias at the G[sub 11] position. The information reported on loops 1 and 5 combined with previous mutagenesis data on loops 2 and 4, as well as new compensatory mutagenesis data presented here provide insight into potential base interactions as well as required base moeties for catalysis. Overall, this study represents a comprehensive mutational analysis of every base located within all the required loops of the hairpin ribozyme-substrate complex and allowed for a refinement on a unique mechanism for RNA catalysis.