ESSENTIAL NUCLEOTIDE-SEQUENCES AND SECONDARY STRUCTURE ELEMENTS OF THE HAIRPIN RIBOZYME

In vitro selection experiments have been used to isolate active varian ts of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme -substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, an d by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essentia l nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the cat alytic core of the ribozyme. Activity and substrate binding assays sho w that point mutations at these eight sites eliminate cleavage activit y but do not significantly decrease substrate binding, demonstrating t hat these bases contribute to catalytic function. The mutation U39C ha s been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutat ion in the wild-type ribozyme and in a variety of mutant backgrounds s how that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleot ides and a secondary structure model of four short helices, encompassi ng 18 bp for the ribozyme-substrate complex are derived.