In order to understand the catalysis mechanism of the hairpin ribozyme, mutant ribozymes were constructed. The distance between the loop A domain and the loop B domain was extended by inserting various lengths of nucleotide linkers at the hinge region in cis mutants, or the domains were separated physically in a trans mutant. All the mutant ribozymes, including the trans mutant, could cleave substrate RNA at the predicted site. A cis mutant with a single nucleotide insertion exhibited cleavage activity about twice as high as that of the wild-type (wt) ribozyme. The insertion of 2-5 nucleotides (nt) gradually reduced the activity to the level of the wt ribozyme, Insertion of a longer linker, up to 11 nt, resulted in the reduction of activity to one half of that of the wt ribozyme. The ribozyme with a single nucleotide insertion at the hinge region seems to form a more suitable conformation for catalysis by three-dimensional fold-back of the loop B to loop A containing the cleavage site. The trans mutant, in which the A and B domains were physically separated, maintained a significant level of activity, suggesting that both domains are necessary for catalysis, but separable. These results demonstrate that interaction between the A and B domains results in catalysis.
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