Effects of complementary loop composition in truncated R3C ligase ribozymes on kiss switch activation

摘要

The formation of a kissing-loop through the introduction of complementary 7-membered loops is known to dramatically increase the activity of truncated R3C ligase ribozymes that otherwise display reduced activity. Restoration of activity is thought to result from kissing complex formation-induced rearrangement of two molecules with complementary loops. By combining two types of R3C ligase ribozyme mutants, A and hairpin -Delta U , the influence of loop composition on ligation activity was investigated. Substrate ligation occurred in hairpin-Delta U , but not in A , despite the absence of a substrate-binding site in hairpin -Delta U . Loop-loop interactions of A - and hairpin -Delta U -variants with complementary 6-membered loops also resulted in proper kissing-complex formation-induced substrate ligation. However, heterogeneous combinations of 7- and 6-membered loops, and/or of 6- and 5-membered loops had distinct results that depended upon the sequence and bulged nucleotides of the loop regions. These differences suggest that both thermodynamic and kinetic controls act upon the kissing-loop interaction-mediated rearrangement of the shortened trans-R3C ribozymes.