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.
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.
Gray
N K
NK
European Molecular Biology Laboratory, Heidelberg, Germany.
Quick
S
S
Goossen
B
B
Constable
A
A
Hirling
H
H
Kühn
L C
LC
Hentze
M W
MW
D000154:Aconitate Hydratase / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002846:Chromatography, Affinity; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D035925:Iron-Regulatory Proteins; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014616:Vaccinia virus / Q000235:genetics
D018076:DNA, Complementary; D035925:Iron-Regulatory Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D005293:Ferritins; D015500:Chloramphenicol O-Acetyltransferase; D000154:Aconitate Hydratase