RNAs must fold into unique three-dimensional structures to function in the cell, but how each polynucleotide finds its native structure is not understood. To investigate whether the stability of the tertiary structure determines the speed and accuracy of RNA folding, docking of a tetraloop with its receptor in a bacterial group I ribozyme was perturbed by site-directed mutagenesis. Disruption of the tetraloop or its receptor destabilizes tertiary interactions throughout the ribozyme by 2-3 kcal/ mol, demonstrating that tertiary interactions form cooperatively in the transition from a native-like intermediate to the native state. Nondenaturing PAGE and RNase T1 digestion showed that base pairs form less homogeneously in the mutant RNAs during the transition from the unfolded state to the intermediate. Thus, tertiary interactions between helices bias the ensemble of secondary structures toward native-like conformations. Time-resolved hydroxyl radical footprinting showed that the wild type ribozyme folds completely within 5-20 ms. By contrast, only 40-60% of a tetraloop mutant ribozyme folds in 30-40 ms, with the remainder folding in 30 -200 s via non-native intermediates. Therefore, destabilization of tetraloop-receptor docking introduces an alternate folding pathway in the otherwise smooth energy landscape of the wild type ribozyme. Our results show that stable tertiary structure increases the flux through folding pathways that lead directly and rapidly to the native structure.

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Positively charged counterions drive RNA molecules into compact configurations that lead to their biologically active structures. To understand how the valence and size of the cations influences the collapse transition in RNA, small angle X-ray scattering was used to follow the decrease in the radius of gyration (Rg) of the Azoarcus and Tetrahymena ribozymes in different cations. Small, multivalent cations induced the collapse of both ribozymes more efficiently than monovalent ions. Thus, the cooperativity of the collapse transition depends on the counterion charge density. Singular value decomposition of the scattering curves showed that folding of the smaller and more thermostable Azoarcus ribozyme is well described by two components, whereas collapse of the larger Tetrahymena ribozyme involves at least one intermediate. The ion-dependent persistence length, extracted from the distance distribution of the scattering vectors, shows that the Azoarcus ribozyme is less flexible at the midpoint of transition in low charge density ions than in high charge density ions. We conclude that the formation of sequence-specific tertiary interactions in the Azoarcus ribozyme overlaps with neutralization of the phosphate charge, while tertiary folding of the Tetrahymena ribozyme requires additional counterions. Thus, the stability of the RNA structure determines its sensitivity to the valence and size of the counterions.

Summary Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg2+, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s-1 at 37 °C. From comparison with time-resolved Fe(II)-EDTA footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the IC intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of non-native conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.

-upon-Tyne, NE2 4HH 1 In the kidney, a number of transport proteins involved in the secretion of permanently charged organic cations have recently been cloned. To evaluate the possible similarities between intestine and kidney in the handling of organic cations we investigated the transport of 1-methyl-4-phenylpyridinium (MPP + ) across monolayers of intestinal Caco-2 cells. MPP + is a prototypic substrate of the cloned organic cation transporters hOCT1 and hOCT2. 2 In Caco-2 cell monolayers, the basolateral to apical¯ux of MPP + was signi®cantly greater than the apical to basolateral¯ux, consistent with net secretion of MPP + . 3 Net secretion of MPP + was abolished by addition of either 10 mM cyclosporin A or 100 mM verapamil to the apical membrane. In contrast, secretion of MPP + was una ected by addition of either TEA (2 mM) or decynium-22 (2 mM) to either apical or basolateral membranes. These results suggest that MPP + secretion is mediated primarily by P-glycoprotein located at the apical membrane. We found no evidence of a role for hOCT1 or hOCT2 in the secretion of MPP + . 4 In addition to net secretion of MPP + , we found evidence of a Na + -dependent MPP + uptake mechanism at the apical membrane of Caco-2 cells. 5 Na + -dependent MPP + uptake was sensitive to inhibition by the organic cations; decynium-22 (2 mM), TEA (2 mM) and cimetidine (5 mM) but not by carnitine, guanidine or proline. 6 These results suggest that net secretion of MPP + across the apical membrane of Caco-2 cells is a function of the relative contributions of MPP + secretion mediated by P-glycoprotein and MPP + absorption mediated by a novel Na + -dependent transport mechanism.