Recombinant iron‐regulatory factor functions as an iron‐responsive‐element‐binding protein, a translational repressor and an aconitase

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 FeSO 4 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.