NMR studies of the structure and Mg2+ binding properties of a conserved RNA motif of EMCV picornavirus IRES element.
The structure and Mg(2+) binding properties of a conserved 75mer RNA motif of the internal ribosome entry site (IRES) element of encephalomyocarditis virus picornavirus have been investigated by (1)H-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 Mg(2+) resulted in a dramatic increase in apparent melting temperature, with the 75mer RNA registering the biggest increase, from 63 to 80 degrees C, thus providing evidence for enhanced stability arising from Mg(2+) binding. Similarly, addition of Mg(2+) 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 Mg(2+) in the folding of the 75mer. Significantly, the same protons show retarded exchange to water solvent, even at elevated temperature, which suggest that Mg(2+) induces a conformational rearrangement of the 75mer. Thus, we propose that Mg(2+) 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.
Phelan
Marie
M
Department of Chemistry, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 1QD, UK.
Banks
Ryan J
RJ
Conn
Graeme
G
Ramesh
Vasudevan
V
D001665:Binding Sites; D017124:Conserved Sequence; D004686:Encephalomyocarditis virus / Q000235:genetics*; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D013056:Spectrophotometry, Ultraviolet
D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D008274:Magnesium