FAST FOLDING MUTANTS OF THE TETRAHYMENA GROUP-I RIBOZYME REVEAL A RUGGED FOLDING ENERGY LANDSCAPE

A model for the kinetic folding pathway of the Tetrahymena ribozyme ha s been proposed where the;two main structural domains, P4-P6 and P3-P7 , form in a hierarchical manner with P4-P6 forming first and P3-P7 fol ding on the minute timescale. Recent studies in our laboratory identif ied a set of mutations that accelerate P3-P7 formation, and all of the se mutations appear to destabilize a native-like kinetic trap. To bett er understand the microscopic details of this slow step in the Tetrahy mena ribozyme folding pathway, we have used a previously developed kin etic oligonucleotide hybridization assay to characterize the folding o f several fast folding mutants. A comparison of the temperature depend ence of P3-P7 folding between the mutant and wild-type ribozymes demon strates that a majority of the mutations act by decreasing the activat ion enthalpy required to reach the transition state and supports the e xistence of the native-like kinetic trap. Ln several mutant ribozymes, P3-P7 folds with biphasic kinetics, indicating that only a subpopulat ion of molecules can evade the kinetic barrier. The rate of folding of the wild-type increases in the presence of urea, while for the mutant s urea merely shifts the distribution between the two folding populati ons. Small structural changes or changes in solvent can accelerate fol ding, but these changes lead to complex folding behavior, and do not g ive rise to rapid two-state folding transitions. These results support the recent view of folding as an ensemble of molecules traversing a r ugged energy landscape to reach the lowest energy state. (C) 1998 Acad emic Press.