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 DEMONSTRATIONOF THE IRON SWITCH

The translation of ferritin and erythroid 5-aminolevulinate synthase m RNAs 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 pr otein, the iron-regulatory factor. Iron-regulatory factor was expresse d in vaccinia-virus-infected HeLa cells (hIRF(vac)) and in Escherichia coli (hIRF(eco)). An N-terminal histidine tag allowed a rapid one-ste p purification of large quantities of soluble recombinant protein. Bot h hIRF(vac) and hIRF(eco) bound specifically to iron-responsive elemen ts and were immunoprecipitated by iron-regulatory-factor anti-bodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bea ring an iron-responsive element in the 5' untranslated region, specifi c 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. Tr eatment of hIRF(vac) and hIRF(eco) with FeSO4 resulted in a drastic re duction 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 p rotein; after purification, it binds to iron-responsive elements and r epresses translation in vitro. Following iron treatment, iron-responsi ve-element binding is lost and aconitase activity is gained. No eukary otic co-factor seems to be required for the conversion of the iron-res ponsive-element binding to the aconitase form of the protein.