Insights into RNA tertiary folding: Examination of structure formation between the P1 helix and the pre-folded Tetrahymena group I ribozyme

To function, many RNAs must fold into complex tertiary structures. To reveal the basic properties of RNA folding, the formation of tertiary structure between an isolated duplex, termed P1, and the pre-folded core of the Tetrahymena ribozyme was examined. The P1 duplex is composed of the oligonucleotide substrate (S) and a single-stranded region of the ribozyme. Docking of P1 positions S for cleavage within the active site of the ribozyme, so cleavage can be used to monitor docking. Studies with substrates of various lengths revealed the properties of different steps of the ribozyme reaction. S binding was found to be independent of temperature and pH, consistent with the model that the P1 duplex is similar to a free model duplex. The temperature dependence of the rate constant of docking suggested that the transition state for docking was unrelated to the docked state. In addition, a single-molecule fluorescence resonance energy transfer (smFRET) assay was developed to follow docking. Examination of single molecules allows the properties of states that do not accumulate to be measured. Control experiments indicated that the smFRET assay faithfully reports on the cleavage activity, overall folding, and P1 docking of the ribozyme. SmFRET studies facilitated a thorough characterization of the transition state for docking. Modification of the eight functional groups of P1 known to form tertiary interactions gave no effects on the rate constant for docking, suggesting that the transition state for docking is early in the docking process. The time scale for the docking of seconds is slow compared to other RNA folding events with many more degrees of freedom. This and experimental observations suggest that docking is slow due to escape from a kinetic trap. However, urea does not accelerate the docking rate, suggesting that there is not an increase in accessible surface area between the undocked state and the transition state. Breaking and forming structure in the process of folding may explain the failure of urea to accelerate this and other slow folding events in RNA. Overall, the results suggest that specifying a single native structure may generally be a challenge for RNA.