The ferritin IRE, a highly conserved (96-99% in vertebrates) mRNA translation regulatory element in animal mRNA, was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Cobalt(III) hexammine was used to model hydrated Mg2+. IRE isoforms in other mRNAs regulate mRNA translation or stability; all IREs bind IRPs (iron regulatory proteins). A G.C base pair, conserved in ferritin IREs, spans an internal loop/bulge in the middle of an A-helix and, combined with a dynamic G.U base pair, formed a pocket suitable for Co(III) hexammine binding. On the basis of the effects of Co(III) hexammine on the 1H NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III) hexammine at the pocket in the major groove; Mg2+ may bind to the IRE at the same site on the basis of an analogy to Co(III) hexammine and on the Mg2+ inhibition of Cu-(phen)2 cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP cross-linking site suggests a role for the pocket in ferritin IRE/IRP interactions.

Most leaf phosphorus is remobilized to the seed during reproductive development in soybean. We determined, using (31)P-NMR, the effect phosphorus remobilization has on vacuolar inorganic phosphate pool size in soybean (Glycine max [L.] Merr.) leaves with respect to phosphorus nutrition and plant development. Phosphate compartmentation between cytoplasmic and vacuolar pools was observed and followed in intact tissue grown hydroponically, at the R2, R4, and R6 growth stages. As phosphorus in the nutrient solution decreased from 0.45 to 0.05 millimolar, the vacuolar phosphate peak became less prominent relative to cytoplasmic phosphate and hexose monophosphate peaks. At a nutrient phosphate concentration of 0.05 millimolar, the vacuolar phosphate peak was not detectable. At higher levels of nutrient phosphate, as plants progressed from the R2 to the R6 growth stage, the vacuolar phosphate peak was the first to disappear, suggesting that storage phosphate was remobilized to a greater extent than metabolic phosphate. Under suboptimal phosphate nutrition (

The role of modified nucleosides in tRNA structure and ion binding has been investigated with chemically synthesized RNAs corresponding to the yeast tRNA(Phe) anticodon stem and loop (tRNA(ACPhe). Incorporation of d(m5C) at position 14 of the stem of tRNA(ACPhe)-d(m5C14), CCAGACUGAAGAU-d(m5C14)-UGG, analogous to m5C40 in native tRNA(Phe), introduced a strong Mg2+ binding at a site distant from the m5C. A Mg(2+)-induced structural transition, detected by circular dichroism spectroscopy, was similar to that observed for the DNA analog of tRNA(ACPhe) (Guenther et al., 1992; Dao et al., 1992). In contrast, Mg2+ had little effect on unmodified tRNA(ACPhe)-rC14 or tRNA(ACPhe)-d(C14). Modified tRNA(ACPhe)-d(m5C14) bound two Mg2+ ions, and the binding was cooperative. The dissociation constant of the two Mg2+ ions from tRNA(ACPhe)-d(m5C14), 2.5 x 10(-9) M2, is the result of an RNA structure significantly stabilized by Mg2+ binding, delta G = -11.7 kcal/mol. The tRNA(ACPhe)-d(m5C14) structure, investigated by 1H NMR, had a double stranded stem of five base pairs and two additional base pairs across what was a seven membered loop in the unmodified tRNA(Phe)AC. Methylation of cytidine in the yeast tRNA(ACPhe) enables the molecule to form more than one conformation through a process regulated by Mg2+ concentration. Thus, the simplest of posttranscriptional modifications of tRNA, a methylation, is involved in a somewhat distant, internal-site Mg2+ binding and stabilization of tRNA structure, especially that of the anticodon stem and loop.

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