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Mg2+  modulation of EMCV IRES key activity fragment equilibria and r(G·C) base-pair kinetics

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Abstract

NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino proton have been used to probe enhanced thermostability and conformational rearrangements induced by Mg2 +  in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus (EMCV) picornavirus internal ribosome entry site (IRES). We have measured some of their r(G·C) base-pair lifetimes and dissociation constants under different MgCl2 conditions, and we compare them with those of other short RNA duplexes. The RNA fragment r(CACCUGGCGACAGGUG) adopts two topologies, a palindromic duplex with two conformations and a hairpin, whose equilibrium can be monitored: the duplex form is destabilized by Mg2 +  and temperature, a delicate balance wherein the entropic contribution of the free energy helps populate the hairpin state. For both fragments, the opening rates of the r(G·C) pairs are lower in the presence of Mg2 +  and their dissociation constants are smaller or comparable. Analysis of the results suggests that Mg2 +  has a preferential and specific effect on the r(CACCUGGCGACAGGUG) hairpin in the region close to the r(G·C) closing pair of the GCGA tetraloop, and the ion moves diffusively around r(GGCCAAAAGCC), thereby differentiating the GNRA and RAAA hairpin motifs that are both involved in the biological regulation functions of the EMCV IRES.

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Acknowledgements

Julien A. Dupont is a studentship recipient of the F.R.I.A. (Fonds pour la formation à la Recherche dans l’Industrie et l’Agriculture). This work was supported by the Belgian National Foundation for the Scientific Research (F.N.R.S.) and the Catholic University of Louvain (Fonds Spéciaux de Recherche).

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  1. Free University of Brussels, 1050, Brussels, Belgium

    J. A. Dupont

  2. Catholic University of Louvain, 1348, Louvain-la-Neuve, Belgium

    K. Snoussi

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  1. J. A. Dupont
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  2. K. Snoussi
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Correspondence to K. Snoussi.

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Dupont, J.A., Snoussi, K. Mg2+  modulation of EMCV IRES key activity fragment equilibria and r(G·C) base-pair kinetics. J Biol Phys 35, 231–243 (2009). https://doi.org/10.1007/s10867-009-9151-2

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  • DOI: https://doi.org/10.1007/s10867-009-9151-2

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