NMR studies of the structure and Mg2+ binding properties of a conserved RNA motif of EMCV picornavirus IRES element

Abstract

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.

NMR and Molecular Modelling Studies of the Binding of Amicetin Antibiotic to Conserved Secondary Structural Motifs of 23S Ribosomal RNAs

“…1c, d), has a low free energy (Hh Ϫ16.0 kcal mol Ϫ1 and E. coli Ϫ18.0 kcal mol Ϫ1 ) and hence is predicted to be endowed with good stability. These values correlate well with the data noted for an analogous 38mer RNA from EMCV IRES, endowed with a GNRA tetraloop, an internal 7nt asymmetrical bulge and a single C-U mismatch, which showed a predicted free energy of Ϫ14.0 kcal mol Ϫ1 and demonstrated to produce good NMR spectra [15]. CD spectroscopy is a useful and rapid method to identify experimental conditions before commencing an NMR structural investigation.…”

“…We provide direct evidence for the interaction between the RNA motifs and amicetin. There is increasing evidence that smaller synthetic oligonucleotide analogues of the well conserved rRNA secondary structural motifs can adopt the relevant three dimensional fold and function autonomously [15]. Determining the structures of such motifs will be an essential step in understanding the specificity of RNA-drug or RNA-protein interactions at the molecular level [16].…”

New insights into internal ribosome entry site elements relevant for viral gene expression

“…Subsequently, nuclear magnetic resonance analysis confirmed the presence of a GNRA tetraloop in EMCV and PV IRES elements (Phelan et al, 2004;Du et al, 2004). Site-directed substitutions in the FMDV GNRA motif led to a local reorganization of the apical region that allowed the identification of a second motif (Fernandez-Miragall et al, 2006), presumably encompassing the GNRA receptor.…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…Interestingly, the GNRA motif and the C-rich loop are conserved in IRES elements classified as type I or II (Fernandez et al, 2011a;Yu et al, 2011b). The GNRA motif, which is essential for IRES activity in both FMDV and EMCV (Lopez de Quinto and Martinez-Salas, 1997; Robertson et al, 1999), adopts a tetraloop conformation (Fernandez-Miragall and Martinez-Salas, 2003;Phelan et al, 2004), and determines long-range interactions (Fernandez-Miragall et al, 2006;Ramos and Martinez-Salas, 1999). Domain 4 consists of a Yshape RNA structure (corresponding to subdomains J and K) and provides the binding-site for eIF4G (Bassili et al, 2004;Kolupaeva et al, 1998;Lopez de Quinto and Martinez-Salas, 2000;Lozano et al, 2014).…”