RECONSTITUTION OF HAIRPIN RIBOZYME ACTIVITY FOLLOWING SEPARATION OF FUNCTIONAL DOMAINS

The hairpin ribozyme is a 50-nucleotide RNA enzyme of unknown three-di mensional structure. Here, we demonstrate that interdomain interaction s are required for catalytic function by reconstitution of activity fo llowing separation of an essential, independently folding domain (loop B) from the substrate binding strand at a helical junction. The resul ting construct relies on long range tertiary contacts for catalysis. F or this work, we used an optimized ribozyme and substrate, which inclu ded sequence changes to minimize the formation of nonproductive confor mational isomers. Kinetic analysis was carried out using both single a nd multiple turnover methods and shows that the catalytic efficiency ( h(cat)/K-m) of the reconstituted ribozyme is 10(4)-fold lower than tha t of the intact ribozyme. The decrease in k(cat)/K-m results entirely from a 10(4)-fold increase in the apparent K-m, whereas the k(cat) par ameter is essentially unchanged. Therefore, cleavage chemistry appears to be unimpaired, but the reaction is limited by the productive assem bly of the two domains, Our results strongly support a previously prop osed model in which the catalytic topology of the ribozyme contains a bend at a helical junction.