MUTAGENESIS OF THE HAIRPIN RIBOZYME

Extensive in vitro mutagenesis studies have been performed on the hair pin ribozyme and substrate in an effort to refine the overall secondar y structure of the molecule and provide further insight into what elem ents are essential for activity. A secondary structure consisting of f our helices and five loop regions remains the basic model as originall y proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our re sults suggest that helices 3 and 4 are smaller than previously propose d, consisting of four base pairs and three base pairs respectively. He lix 4 can be extended without loss of activity and loop 3 at the close d end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 con sisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made bet ween the new data and previously published mutagenesis and phylogeneti c data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage () occurring in a preferred 5'(g/c/u)n*guc3' s equence of the substrate.