New methods for the investigation of RNA refolding by NMR spectroscopy

A detailed characterization of the time scales and mechanisms by which RNA molecules change their folding structure upon binding of metabolites or during catalysis is important for the understanding of the different biological functions of RNA. The folding from an unfolded, denaturated state has been extensively studied, establishing hierarchical pathways, in which fast formation of secondary structural elements precedes the folding of tertiary elements. In contrast, only a few examples of time-resolved RNA refolding in the native state have been reported so far. Specifically, NMR spectroscopy has not yet been employed for the investigation of RNA refolding despite its intrinsic sensibility to dynamic structural changes. In the project presented here, new tools for the identification of RNA secondary structures and the quantification of RNA refolding processes by NMR spectroscopy have been developed and employed. Preparation of 15N-labeled phosphoramidites and their sequence-selective introduction into RNA sequences allowed a straightforward identification of base pairs and imino protons by HNN-COSY and 15N HSQC experiments, respectively. Synthesis and introduction of NPE-protected uridines and guanosines into bistable RNA sequences provided a powerful method for the photoinduced preparation of selected RNA folds far from equilibrium. When introduced into bistable RNA sequences they allow to disrupt selected base pairs, thereby destabilizing the associated secondary structures. As a consequence, one out of usually two coexisting fold could be prepared selectively in its caged form. Upon photolysis, the native sequence was released under physiological conditions with completely retained, preselected secondary structure arrangement. Refolding into the thermodynamic equilibrium was subsequently followed by real-time imino proton NMR spectroscopy, providing quantitative, time-resolved and structural information for the refolding processes of three bistable RNA sequences and a Mg2+-induced secondary and tertiary structure refolding. Depending on their topology, these RNA sequences refold either via a dissociative mechanism in which the disruption of existing base pairs precedes the formation of new ones or via an associative mechanism in which new base pairs are formed from initially unpaired sequence regions simultaneously to the detachement of existing base pairs. In the transition state approximately half of the base pairs are disrupted. The investigation of a bistable RNA sequence designed to undergo a topologically favored refolding processes was carried out by exchange sensitive NMR experiments. The introduction of sequence specific 15N-labeles into RNA sequences allowed the implementation of exchange sensitive 1D and 2D 1H/15N-heteronuclear NMR methods which were designed to map very slow exchange. We found that a 34mer RNA sequence exhibits two folds which exchange on the observable time scale (τobs = T1{15N} <5 s) and a third fold which is static on this time scale. A 1D version of the 15N exchange experiment allowed the measurement of the exchange rates between the two exchanging folds as a function of temperature and the determination of the corresponding activation energies Ea and frequency factors A. We found that the refolding rates are strongly affected by an entropically favorable preorientation of the replacing strand. The activation energies, however, are high and largely independent on the topology of the system. Interestingly, the activation energies determined for the secondary structure rearrangement of these quite small RNA sequences are similar to the values reported for secondary or tertiary structure rearrangements of much larger and more complex systems. The refolding rate constants are in the range of k = 0.002 - 90 min-1 at 25°C and therefore at least four orders of magnitude smaller than the rates observed for hairpin formation of similar systems from an unfolded state, which occur with k = 105-107 s-1. Thus, uncatalyzed secondary structure rearrangements of already folded RNAs happen at rates that are in the range of rate-limiting steps for biological reactions.

Pitsch, Stefan
Lausanne, EPFL
Other identifiers:
urn: urn:nbn:ch:bel-epfl-thesis3500-9

Note: The status of this file is: EPFL only

 Record created 2006-03-13, last modified 2018-01-27

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