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Lukas C. Kühn - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of the 4fe 4s cluster converting Iron Regulatory Factor from its rna binding form to cytoplasmic aconitase
The EMBO Journal, 1994Co-Authors: Harald Hirling, Beric R Henderson, Lukas C. KühnAbstract:The control of cellular Iron homeostasis involves the coordinate post-transcriptional regulation of ferritin mRNA translation and transferring receptor mRNA stability. These Regulatory events are mediated by a soluble cytoplasmic protein, Iron Regulatory Factor (IRF), which binds specifically to mRNA hairpin structures, termed Iron-responsive elements (IREs), in the respective mRNAs. IRF is modulated by variations of cellular Iron levels and exists as either an apo-protein or a [4Fe-4S]-cluster protein. The two conformations show distinct, mutually exclusive functions. High-affinity IRE binding is observed with the apo-form induced by Iron deprivation, but is lost under high Iron conditions when IRF is converted to the [4Fe-4S]-cluster form which shows cytoplasmic aconitase activity. Moreover, IRE binding is inactivated by the sulfhydryl-oxidizing agent diamide and fully activated in vitro by 2% 2-mercapto-ethanol, whereas alkylation of IRF inhibits IRE binding. In the present study, we analyzed each of the above features using site-directed mutants of recombinant human IRF. The results support the bifunctional nature of IRF. We conclude that cysteines 437, 503 and 506 anchor the [4Fe-4S]-cluster, and are essential to the aconitase activity. Mutagenesis changing any of the cysteines to serine leads to constitutive RNA binding in 0.02% 2-mercaptoethanol. Cysteine 437 is particularly critical to the RNA-protein interaction. The spontaneous or diamide-induced formation of disulfide bonds between cysteines 437 and 503 or 437 and 506, in apo-IRF, as well as its alkylation by N-ethylmaleimide, inhibit binding to the IRE.(ABSTRACT TRUNCATED AT 250 WORDS)
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characterization of a second rna binding protein in rodents with specificity for Iron responsive elements
Journal of Biological Chemistry, 1993Co-Authors: Beric R Henderson, Christian Seiser, Lukas C. KühnAbstract:Iron Regulatory Factor (IRF) is a cytoplasmic RNA-binding protein involved in regulating Iron homeostasis. IRF controls expression of ferritin and transferrin receptor post-transcriptionally via specific binding to stem-loop Iron-responsive elements (IREs) located in the untranslated regions of the respective mRNAs. We have confirmed by RNA band-shift analysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulated by intracellular Iron levels. This faster migrating complex appears to represent a specific interaction between the ferritin IRE and an Iron-regulated protein that is distinct from IRF, as concluded from the following lines of evidence. First, UV cross-linking and partial digestion with different proteases revealed different peptide patterns for the two IRE-protein complexes. Second, antiserum raised against IRF peptides immunoprecipitated only authentic IRF and not the protein of the faster migrating complex, as determined by band-shift analysis. Following separation of the two IRE-binding proteins by ion-exchange chromatography, only the IRF-containing fraction reacted with the antibodies on Western blots. The second protein binds IREs with an affinity similar to that of IRF as demonstrated by competition with a ferritin IRE and related stem-loop RNAs. UV cross-linking experiments indicate that this second protein, tentatively named IRFB, has a molecular mass of approximately 105 kDa. Analysis of mouse tissues revealed differences in the distribution of IRF and IRFB. Whereas IRF protein and IRE binding activity were predominant in liver, intestine, and kidney, the IRFB protein(s) revealed highest binding activity in intestine and brain. Our data support the existence of two distinct Iron-regulated IRE-binding proteins in rodents.
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recombinant Iron Regulatory Factor functions as an Iron responsive element binding protein a translational repressor and an aconitase a functional assay for translational repression and direct demonstration of the Iron switch
FEBS Journal, 1993Co-Authors: Nicola K Gray, Lukas C. Kühn, Sabine Quick, B Goossen, Anne Constable, Harald Hirling, Matthias W. HentzeAbstract:The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an Iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the Iron-Regulatory Factor. Iron-Regulatory Factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to Iron-responsive elements and were immunoprecipitated by Iron-Regulatory-Factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an Iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in Iron-responsive-element-binding of Iron-Regulatory Factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant Iron-Regulatory Factor is a bifunctional protein; after purification, it binds to Iron-responsive elements and represses translation in vitro. Following Iron treatment, Iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-Factor seems to be required for the conversion of the Iron-responsive-element binding to the aconitase form of the protein.
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interleukin 2 dependent transcriptional and post transcriptional regulation of transferrin receptor mrna
Journal of Biological Chemistry, 1993Co-Authors: Christian Seiser, Santuza M R Teixeira, Lukas C. KühnAbstract:Interleukin-2 (IL-2) controls the proliferation of the murine T cell line B6.1 and induces transferrin receptor (TfR) mRNA steady-state levels 50-fold when added to arrested, IL-2-deprived cells. In addition, TfR mRNA is post-transcriptionally regulated by intracellular Iron. Low Iron levels activate a cytoplasmic RNA-binding protein, called Iron Regulatory Factor (IRF) or Iron-responsive element-binding protein, which coordinately stabilizes TfR mRNA and inhibits ferritin mRNA translation. Since ferritin expression is known to be modulated by cytokines, we decided to investigate the mechanism by which IL-2 activates TfR gene expression in B6.1 cells. Induction by IL-2 of both nuclear and cytoplasmic TfR RNA was compared with run-on transcription rates in isolated nuclei. The results revealed a 3-fold increase in TfR gene transcription and a 6-fold rise in nuclear TfR RNA reaching its steady-state level within 2 h. The main accumulation of mature mRNA in the cytoplasm occurred after 6 h in parallel with the activation of IRF. However, stimulation of IRF binding activity by the Iron chelator desferrioxamine, in the absence of IL-2, failed to induce TfR mRNA. Moreover, deprivation of growing B6.1 cells of IL-2 resulted in cell arrest and a rapid decay of TfR mRNA, which was not prevented by the activation of IRF with desferrioxamine. TfR mRNA stabilization appears, therefore, to depend on IL-2. We conclude that TfR mRNA expression is controlled by at least three steps at the onset of cell proliferation: (i) the growth Factor-dependent activation of transcription; (ii) mRNA stabilization by IRF in the cytoplasm; and (iii) an additional IL-2-dependent activity which prevents TfR mRNA degradation. Our results indicate that expression of TfR, like ferritin, is controlled by both Iron and cytokines.
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The putative Iron-responsive element in the human erythroid 5-aminolevulinate synthase mRNA mediates translational control.
The Journal of biological chemistry, 1993Co-Authors: C R Bhasker, G Burgiel, B. Neupert, A. Emery-goodman, Lukas C. Kühn, B. K. MayAbstract:The 52-nucleotide 5'-untranslated region of the human erythroid 5-aminolevulinate synthase mRNA contains a 28-nucleotide Iron-responsive element-like stem-loop motif. We fused the 5'-untranslated region upstream to the coding sequence of the human growth hormone cDNA. A chimeric construct containing a mutated variant of the presumptive Iron-responsive element was similarly synthesized. Translation of the wild type chimeric transcript was markedly repressed (approximately 95%) in rabbit reticulocyte lysates as opposed to the mutant. Both transcripts translated with comparable efficiency in wheat germ extracts. Purified placental Iron Regulatory Factor selectively and markedly inhibited translation of the wild type chimeric transcript (> 90%) when tested in wheat germ extracts. By contrast, translations of either the mutant chimeric transcript or other control mRNA species were unaffected. The proximal position of the Iron-responsive element relative to the cap site was shown to be important for translational control, in vitro. Our studies suggest that interaction of the Iron Regulatory Factor with the Iron-responsive element sterically hinders formation of the preinitiation complex, resulting in translational repression. Thus inactivation of the repressor protein by critical levels of Iron or heme would trigger translation of this mRNA in erythroid cells. Consequently, protoporphyrin and heme synthesis would be subtly coordinated with Iron supply.
Matthias W. Hentze - One of the best experts on this subject based on the ideXlab platform.
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recombinant Iron Regulatory Factor functions as an Iron responsive element binding protein a translational repressor and an aconitase a functional assay for translational repression and direct demonstration of the Iron switch
FEBS Journal, 1993Co-Authors: Nicola K Gray, Lukas C. Kühn, Sabine Quick, B Goossen, Anne Constable, Harald Hirling, Matthias W. HentzeAbstract:The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an Iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the Iron-Regulatory Factor. Iron-Regulatory Factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to Iron-responsive elements and were immunoprecipitated by Iron-Regulatory-Factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an Iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in Iron-responsive-element-binding of Iron-Regulatory Factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant Iron-Regulatory Factor is a bifunctional protein; after purification, it binds to Iron-responsive elements and represses translation in vitro. Following Iron treatment, Iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-Factor seems to be required for the conversion of the Iron-responsive-element binding to the aconitase form of the protein.
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translational regulation via Iron responsive elements by the nitric oxide no synthase pathway
The EMBO Journal, 1993Co-Authors: Gunter Weiss, B Goossen, Wolfgang Doppler, Dietmar Fuchs, Kostas Pantopoulos, Gabriele Wernerfelmayer, H Wachter, Matthias W. HentzeAbstract:Abstract Nitric oxide (NO) produced from L-arginine by NO synthases (NOS) is a transmitter known to be involved in diverse biological processes, including immunomodulation, neurotransmission and blood vessel dilatation. We describe a novel role of NO as a signaling molecule in post-transcriptional gene regulation. We demonstrate that induction of NOS in macrophage and non-macrophage cell lines activates RNA binding by Iron Regulatory Factor (IRFs), the central trans regulator of mRNAs involved in cellular Iron metabolism. NO-induced binding of IRF to Iron-responsive elements (IRE) specifically represses the translation of transfected IRE-containing indicator mRNAs as well as the biosynthesis of the cellular Iron storage protein ferritin. These findings define a new biological function of NO and identify a Regulatory connection between the NO/NOS pathway and cellular Iron metabolism.
Harald Hirling - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of the 4fe 4s cluster converting Iron Regulatory Factor from its rna binding form to cytoplasmic aconitase
The EMBO Journal, 1994Co-Authors: Harald Hirling, Beric R Henderson, Lukas C. KühnAbstract:The control of cellular Iron homeostasis involves the coordinate post-transcriptional regulation of ferritin mRNA translation and transferring receptor mRNA stability. These Regulatory events are mediated by a soluble cytoplasmic protein, Iron Regulatory Factor (IRF), which binds specifically to mRNA hairpin structures, termed Iron-responsive elements (IREs), in the respective mRNAs. IRF is modulated by variations of cellular Iron levels and exists as either an apo-protein or a [4Fe-4S]-cluster protein. The two conformations show distinct, mutually exclusive functions. High-affinity IRE binding is observed with the apo-form induced by Iron deprivation, but is lost under high Iron conditions when IRF is converted to the [4Fe-4S]-cluster form which shows cytoplasmic aconitase activity. Moreover, IRE binding is inactivated by the sulfhydryl-oxidizing agent diamide and fully activated in vitro by 2% 2-mercapto-ethanol, whereas alkylation of IRF inhibits IRE binding. In the present study, we analyzed each of the above features using site-directed mutants of recombinant human IRF. The results support the bifunctional nature of IRF. We conclude that cysteines 437, 503 and 506 anchor the [4Fe-4S]-cluster, and are essential to the aconitase activity. Mutagenesis changing any of the cysteines to serine leads to constitutive RNA binding in 0.02% 2-mercaptoethanol. Cysteine 437 is particularly critical to the RNA-protein interaction. The spontaneous or diamide-induced formation of disulfide bonds between cysteines 437 and 503 or 437 and 506, in apo-IRF, as well as its alkylation by N-ethylmaleimide, inhibit binding to the IRE.(ABSTRACT TRUNCATED AT 250 WORDS)
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recombinant Iron Regulatory Factor functions as an Iron responsive element binding protein a translational repressor and an aconitase a functional assay for translational repression and direct demonstration of the Iron switch
FEBS Journal, 1993Co-Authors: Nicola K Gray, Lukas C. Kühn, Sabine Quick, B Goossen, Anne Constable, Harald Hirling, Matthias W. HentzeAbstract:The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an Iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the Iron-Regulatory Factor. Iron-Regulatory Factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to Iron-responsive elements and were immunoprecipitated by Iron-Regulatory-Factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an Iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in Iron-responsive-element-binding of Iron-Regulatory Factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant Iron-Regulatory Factor is a bifunctional protein; after purification, it binds to Iron-responsive elements and represses translation in vitro. Following Iron treatment, Iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-Factor seems to be required for the conversion of the Iron-responsive-element binding to the aconitase form of the protein.
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Iron Regulatory Factor expressed from recombinant baculovirus conversion between the rna binding apoprotein and fe s cluster containing aconitase
Nucleic Acids Research, 1993Co-Authors: Alice Emerygoodman, Harald Hirling, Leo Scarpellino, Beric R Henderson, Lukas C. KühnAbstract:Iron Regulatory Factor (IRF) is a cytoplasmic mRNA-binding protein that coordinates post-transcriptionally the expression of several important proteins in Iron metabolism. Binding of IRF to Iron-responsive elements (IRE) in the 5' untranslated region (UTR) of ferritin and erythroid 5-aminolevulinic acid-synthase mRNAs inhibits their translation, whereas binding to IREs in the 3' UTR of transferrin receptor (TfR) mRNA prevents the degradation of this mRNA. IRF binds RNA strongly after Iron deprivation, but is inactive, yet present, under conditions of high cellular Iron supply. Recently, IRF was also shown to have aconitase activity indicating the existence of an Fe-S cluster in the protein. In the current study we expressed human IRF in insect cells from recombinant baculovirus and analysed IRE-binding and aconitase activities under various culture conditions. Newly made apoprotein, synthesized in the absence of Iron, was fully active in IRE-binding, but showed no aconitase activity. In contrast, IRF made by cells grown in high Iron medium bound RNA poorly, but exhibited high aconitase activity with a Km of 9.2 microM for cis-aconitate. Apo-IRF was converted in vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost its RNA-binding capacity. These results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster.
L C Kuhn - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of nitric oxide activates Iron Regulatory Factor in macrophages
The EMBO Journal, 1993Co-Authors: Jeanclaude Drapier, H Hirling, J Wietzerbin, P Kaldy, L C KuhnAbstract:Biosynthesis of nitric oxide (NO) from L-arginine modulates activity of Iron-dependent enzymes, including mitochondrial acontiase, an [Fe-S] protein. We examined the effect of NO on the activity of Iron Regulatory Factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called Iron responsive elements (IREs). Murine macrophages were activated with interferon-gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG-substituted analogues of L-arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria-free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN-gamma and/or LPS stimulation. Authentic NO gas as well as the NO-generating compound 3-morpholinosydnonimine (SIN-1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post-transcriptional regulation of genes involved in Iron homeostasis and support the hypothesis that the [Fe-S] cluster of IRF mediates Iron-dependent regulation.
Beric R Henderson - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of the 4fe 4s cluster converting Iron Regulatory Factor from its rna binding form to cytoplasmic aconitase
The EMBO Journal, 1994Co-Authors: Harald Hirling, Beric R Henderson, Lukas C. KühnAbstract:The control of cellular Iron homeostasis involves the coordinate post-transcriptional regulation of ferritin mRNA translation and transferring receptor mRNA stability. These Regulatory events are mediated by a soluble cytoplasmic protein, Iron Regulatory Factor (IRF), which binds specifically to mRNA hairpin structures, termed Iron-responsive elements (IREs), in the respective mRNAs. IRF is modulated by variations of cellular Iron levels and exists as either an apo-protein or a [4Fe-4S]-cluster protein. The two conformations show distinct, mutually exclusive functions. High-affinity IRE binding is observed with the apo-form induced by Iron deprivation, but is lost under high Iron conditions when IRF is converted to the [4Fe-4S]-cluster form which shows cytoplasmic aconitase activity. Moreover, IRE binding is inactivated by the sulfhydryl-oxidizing agent diamide and fully activated in vitro by 2% 2-mercapto-ethanol, whereas alkylation of IRF inhibits IRE binding. In the present study, we analyzed each of the above features using site-directed mutants of recombinant human IRF. The results support the bifunctional nature of IRF. We conclude that cysteines 437, 503 and 506 anchor the [4Fe-4S]-cluster, and are essential to the aconitase activity. Mutagenesis changing any of the cysteines to serine leads to constitutive RNA binding in 0.02% 2-mercaptoethanol. Cysteine 437 is particularly critical to the RNA-protein interaction. The spontaneous or diamide-induced formation of disulfide bonds between cysteines 437 and 503 or 437 and 506, in apo-IRF, as well as its alkylation by N-ethylmaleimide, inhibit binding to the IRE.(ABSTRACT TRUNCATED AT 250 WORDS)
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characterization of a second rna binding protein in rodents with specificity for Iron responsive elements
Journal of Biological Chemistry, 1993Co-Authors: Beric R Henderson, Christian Seiser, Lukas C. KühnAbstract:Iron Regulatory Factor (IRF) is a cytoplasmic RNA-binding protein involved in regulating Iron homeostasis. IRF controls expression of ferritin and transferrin receptor post-transcriptionally via specific binding to stem-loop Iron-responsive elements (IREs) located in the untranslated regions of the respective mRNAs. We have confirmed by RNA band-shift analysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulated by intracellular Iron levels. This faster migrating complex appears to represent a specific interaction between the ferritin IRE and an Iron-regulated protein that is distinct from IRF, as concluded from the following lines of evidence. First, UV cross-linking and partial digestion with different proteases revealed different peptide patterns for the two IRE-protein complexes. Second, antiserum raised against IRF peptides immunoprecipitated only authentic IRF and not the protein of the faster migrating complex, as determined by band-shift analysis. Following separation of the two IRE-binding proteins by ion-exchange chromatography, only the IRF-containing fraction reacted with the antibodies on Western blots. The second protein binds IREs with an affinity similar to that of IRF as demonstrated by competition with a ferritin IRE and related stem-loop RNAs. UV cross-linking experiments indicate that this second protein, tentatively named IRFB, has a molecular mass of approximately 105 kDa. Analysis of mouse tissues revealed differences in the distribution of IRF and IRFB. Whereas IRF protein and IRE binding activity were predominant in liver, intestine, and kidney, the IRFB protein(s) revealed highest binding activity in intestine and brain. Our data support the existence of two distinct Iron-regulated IRE-binding proteins in rodents.
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Iron Regulatory Factor expressed from recombinant baculovirus conversion between the rna binding apoprotein and fe s cluster containing aconitase
Nucleic Acids Research, 1993Co-Authors: Alice Emerygoodman, Harald Hirling, Leo Scarpellino, Beric R Henderson, Lukas C. KühnAbstract:Iron Regulatory Factor (IRF) is a cytoplasmic mRNA-binding protein that coordinates post-transcriptionally the expression of several important proteins in Iron metabolism. Binding of IRF to Iron-responsive elements (IRE) in the 5' untranslated region (UTR) of ferritin and erythroid 5-aminolevulinic acid-synthase mRNAs inhibits their translation, whereas binding to IREs in the 3' UTR of transferrin receptor (TfR) mRNA prevents the degradation of this mRNA. IRF binds RNA strongly after Iron deprivation, but is inactive, yet present, under conditions of high cellular Iron supply. Recently, IRF was also shown to have aconitase activity indicating the existence of an Fe-S cluster in the protein. In the current study we expressed human IRF in insect cells from recombinant baculovirus and analysed IRE-binding and aconitase activities under various culture conditions. Newly made apoprotein, synthesized in the absence of Iron, was fully active in IRE-binding, but showed no aconitase activity. In contrast, IRF made by cells grown in high Iron medium bound RNA poorly, but exhibited high aconitase activity with a Km of 9.2 microM for cis-aconitate. Apo-IRF was converted in vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost its RNA-binding capacity. These results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster.
