Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGU CC, binds to an RNA substrate, GGACC down arrow GAGCCAG, cleaving the RNA s ubstrate at one site. We have investigated the effect of the substrate sequ ence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadz yme acted as a catalyst for single site cleavage of a C5 deletion mutant su bstrate, GGAC down arrow GAGCCAG, as well as the wild-type substrate. Howev er, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved, Kinetic studies by surface plasmon resonance i ndicated that the difference between active and inactive structures reflect ed the slow association and dissociation rate constants of complex formatio n induced by Pb2+ rather than differences in complex stability, CD spectra showed that the active form of the substrate-leadzyme complex was rearrange d by Pb2+ binding, The G8 of the wild-type substrate, which was absent in t he inactive complex, is not near the cleavage site, Thus, these results sho w that the active substrate-leadzyme complex has a Pb2+ binding site at the junction between the unpaired region (asymmetric internal loop) and the st em region, which is distal to the cleavage site. Pb2+ may play a role in re arranging the bases in the asymmetric internal loop to the correct position for catalysis.