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 (hIRF_vac) and in Escherichia coli (hIRF_eco). An N‐terminal histidine tag allowed a rapid one‐step purification of large quantities of soluble recombinant protein. Both hIRF_vac and hIRF_eco 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 hIRF_vac and hIRF_eco was demonstrated in wheat‐germ extract. In addition, hIRF_vac and hIRF_eco were shown to display aconitase activity. Treatment of hIRF_vac and hIRF_eco 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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