Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs. This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells. To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system. We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE. Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon. The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE-IRP-1 complex. In contrast, cap-distal IRE-IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts. Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5 untranslated region of an mRNA can regulate translation. In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE-IRP-1 complexes positioned within the open reading frame. These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.The regulation of iron metabolism by the iron-responsive element (IRE)-iron regulatory protein (IRP) system represents an intensively studied example of translational control in higher eukaryotes. Several mRNAs encoding proteins involved in cellular iron metabolism harbor an IRE at a cap-proximal position of their 5Ј untranslated regions (UTRs). The IRE is specifically recognized by IRP-1 and IRP-2, which bind to and repress the translation of IRE-containing mRNAs both in vivo and in vitro (17,39). Translational control by specific mRNAprotein interactions is commonly enacted at the level of translation initiation (e.g., caudal [6, 38], 15-lipoxygenase [35, 36], and oskar [22,43]). IRP binding to the IRE of ferritin mRNAs affects an early step of translation initiation: it prevents the recruitment of the 43S translation preinitiation complex (which includes the small ribosomal subunit) (13, 33). Transfection studies using mammalian tissue culture cells revealed a characteristic feature of this IRE-IRP regulatory mechanism: for IRP binding to efficiently block translation, the IRE must be located within Ͻ60 nucleotides from the m 7 GpppN-cap structure of the mRNA (9, 10). An IRE placed Ͼ60 nucleotides downstream from the cap structure mediates only partial translational inhibition by IRP binding. In keeping with this position effect, the cap-...
Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.Intracellular iron homeostasis in mammals and many other eukaryotes is controlled at the post-transcriptional level by two related cytoplasmic proteins, the iron regulatory proteins (IRP-1 and IRP-2) 1 (for reviews see Refs. 1 and 2). These two proteins bind to a conserved RNA stem-loop structure, the so-called iron-responsive element. An increasing number of mRNAs have been shown to carry one IRE motif in the 5Ј-untranslated regions of mRNAs encoding ferritin (L-and Hchains), erythroid 5-aminolevulinate synthase, mitochondrial aconitase, and succinate dehydrogenase, as well as in the 3Ј-untranslated region of transferrin receptor (TfR) and Nramp2 mRNAs, thus modulating their expression according to iron availability (3-12). For instance, the interaction of IRP with a single IRE in the 5Ј-untranslated region of ferritin mRNA inhibits translation by preventing ribosome binding (13-15). On the other hand, IRP binding to the five IREs present in the 3Ј-untranslated region of TfR mRNA stabilizes the message against degradation (10, 16). Therefore, the specific interaction between IRP and mRNA ensures a coordinated regulation of the expression of proteins involved in the uptake (TfR), storage (ferritin), and utilization (erythroid 5-aminolevulinate synthase) of iron (2). Because the IRPs appear to regulate two enzymes of the citric acid cycle, they may also play an important role in mediating iron regulation of mitochondrial energy production (9, 10).IRP-1 and IRP-2 are affected by iron in different manners. Whereas iron increases the rate of degradation of IRP-2 (17, 18), i...
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