Abstract
Synchrotron hydroxyl radical (·OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes time-scales. The Mg 2+-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200 mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4–P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200 mM NaCl and folded by the addition of 10 mM MgCl 2, multiple kinetic phases are observed for ·OH protections throughout the ribozyme. The independently folding P4–P6 domain is the first to fold with its protections displaying 50–90% burst phase amplitudes. That the folding of P4–P6 within the ribozyme does not display the 100% burst phase of isolated P4–P6 at 200 mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, ·OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions.
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