Effect of Magnesium Ions on the Tertiary Structure of the Hepatitis C Virus IRES and Its Affinity for the Cyclic Peptide Antibiotic Viomycin

Abstract

A key ion-dependent folding unit within the hepatitis C IRES comprises the IIIef junction and pseudoknot. This region is also important in recruitment of the 40S ribosomal subunit. Here, circular dichroism is used to study the influence of metal ions on the structure and stability of this region. Comparison of the thermal stability of an IRES fragment encompassing subdomains IIIe/f and IV (named 3EF4) with that of a larger fragment also possessing subdomain IIId (3DEF4) indicates an additional stabilizing effect of Mg(2+) ions on the latter fragment. Magnesium and potassium ions stabilize both fragments through nonspecific counterion effects. The additional effect of magnesium on 3DEF4, observed in the absence or presence of 100 mM KCl, is attributed to a nonspecific but high-affinity site for metal ions created by a region of unusual high charge density. Subdomain IIId presumably participates in tertiary packing interactions that provide such a site. Viomycin binds to the full-length IRES and RNA fragments with K(d) values of 25-55 microM. Interestingly, viomycin binding to the two fragments is affected differently by Mg(2+); noncompetitive inhibition of binding to 3DEF4 is observed, whereas binding to 3EF4 is not impaired. Formation of a Mg(2+)-stabilized tertiary fold, involving subdomain IIId, may thereby hinder viomycin binding to 3DEF4 indirectly. Mutational and deletion studies locate viomycin binding within subdomains IIIe/f rather than within the pseudoknot. In pseudoknot mutants, Mg(2+) ions have different effects on viomycin binding and thermal stability, suggesting altered tertiary interactions involving subdomain IIId.

RNA-Binding Proteins HuR and PTB Promote the Translation of Hypoxia-Inducible Factor 1α

“…However, whether CoCl 2 regulates the function of PTB via any of the reported kinase pathways that control HIF-1␣ translation, dependently or independently of mTOR (3,22,47,49), remains to be investigated. It is also possible that cobalt itself directly elicits structural changes in the HIF-1␣ mRNA, as reported for other metal ions, such as iron and magnesium, which can affect mRNA tertiary structure (17,27,63). Thus, the possibility remains that cobalt directly alters the accessibility of RNA-binding proteins to the HIF-1␣ mRNA and thereby facilitates translation.…”

Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation

“…Samples contained 11–16 μM RNA in water or 1, 2 or 5 mM MgCl 2 were analyzed in a 1 mm CD quartz cuvette. RNA denaturation/renaturation experiments were performed as previously described (38). Wavelength scans were recorded from 200 to 300 nm at 50 nm/min at 25°C, using a 1 nm bandwidth, a 1 s response time.…”

“…Spectra were averaged over four to five accumulations. Results were expressed as mean residue ellipticity, in degrees cm 2 dmol –1 (38). …”

Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation

“…Experimental evidence for tertiary structure generated by RNA-RNA interactions is available for portions of IRES elements from FMDV (Ramos and Martí-nez-Salas 1999;Fernández-Miragall and Martínez-Salas 2003), HCV (Kieft et al 1999;Jubin et al 2000;Lyons et al 2001;Spahn et al 2001;Kieft et al 2002;Vos et al 2002;Lukavsky et al 2003;Rijnbrand et al 2004), and the intergenic region of insect picornavirus-like viruses (Jan and Sarnow 2002;Costantino and Kieft 2005). These elements appear to have a dynamic structure, suggesting that translation efficiency may be regulated at the level of RNA structure.…”