Author: Phelan Marie Banks Ryan J. Conn Graeme
Publisher: Oxford University Press
ISSN: 1362-4962
Source: Nucleic Acids Research, Vol.32, Iss.16, 2004-01, pp. : 4715-4724
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Abstract
The structure and Mg2+ binding properties of a conserved 75mer RNA motif of the internal ribosome entry site (IRES) element of encephalomyocarditis virus picornavirus have been investigated by 1H-NMR and UV melting experiments. The assignment of the imino proton resonances with characteristic chemical shift dispersion for canonical and non-canonical base pairs confirmed the predicted secondary structure of the 75mer and its fragments. Addition of Mg2+ resulted in a dramatic increase in apparent melting temperature, with the 75mer RNA registering the biggest increase, from 63 to 80°C, thus providing evidence for enhanced stability arising from Mg2+ binding. Similarly, addition of Mg2+ induced selective changes to the chemical shifts of the imino protons of a GCGA tetraloop in the 75mer, that is essential for IRES activity, thereby highlighting a possible structural role for Mg2+ in the folding of the 75mer. Significantly, the same protons show retarded exchange to water solvent, even at elevated temperature, which suggest that Mg2+ induces a conformational rearrangement of the 75mer. Thus, we propose that Mg2+ serves two important roles: (i) enhancing thermodynamic stability of the 75mer RNA (and its submotifs) via non-specific interactions with the phosphate backbone and (ii) promoting the folding of the 75mer RNA by binding to the GCGA tetraloop.
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