Published online 7 September 2004
Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved
NMR studies of the structure and Mg2+ binding properties of a conserved RNA motif of EMCV picornavirus IRES element
Department of Chemistry and 1 Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 1QD, UK
* To whom correspondence should be addressed. Tel: +44 161 200 4539; Fax: +44 161 200 4559; Email: vasudevan.ramesh{at}umist.ac.uk
Received June 15, 2004; Revised and Accepted August 17, 2004
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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