Posttranscriptional regulation of genes of mammalian iron metabolism is mediated by the interaction of iron regulatory proteins (IRPs) with RNA stem-loop sequence elements known as iron-responsive elements (IREs)

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We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase. The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group ofmethionine and between the benzene ring of Tyr-15 and the 1& and f-CH2 groups of the substrate. An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine. Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase. Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme. At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP. Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme. Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme. However, enzymes R233Q, R233A, and D52A did not deacylate MettRNA. The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase.

Five furanocoumarins including a new one were isolated from the root of Angelica dahurica by repeated silica gel column chromatography. Their chemical structures were determined to be isoimperatorin (1), oxypeucedanin hydrate-3"-butyl ether (2), imperatorin (3), knidilin (4), and oxypeucedanin hydrate (5). This represents the first study in which the compound 2 has been isolated and identified. The long-range coupling (5J) in the 1H-NMR spectrum observed in the linear furanocoumarin skeleton was also investigated in detail.