Recombinant iron‐regulatory factor functions as an iron‐responsive‐element‐binding protein, a translational repressor and an aconitase
Open Access
- 1 December 1993
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 218 (2), 657-667
- https://doi.org/10.1111/j.1432-1033.1993.tb18420.x
Abstract
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.Keywords
This publication has 51 references indexed in Scilit:
- Translational regulation by mRNA/protein interactions in eukaryotic cells: Ferritin and beyondBioEssays, 1993
- Sequence-Specific Binding of Transfer RNA by Glyceraldehyde-3-Phosphate DehydrogenaseScience, 1993
- Regulating the fate of mRNA: The control of cellular iron metabolismCell, 1993
- Coordmation of cellular iron metabolism by post-transcriptional gene regulationJournal of Inorganic Biochemistry, 1992
- Enhanced Degradation of the Ferritin Repressor Protein During Induction of Ferritin Messenger RNA TranslationScience, 1992
- Structural relationship between an iron-regulated RNA-binding protein (IRE-BP) and aconitase: Functional implicationsCell, 1991
- Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B" and their cognate RNAsNature, 1990
- Derepression of Ferritin Mmessenger RNA Translation by Hemin in VitroScience, 1990
- A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNACell, 1989
- Binding of a Cytosolic Protein to the Iron-Responsive Element of Human Ferritin Messenger RNAScience, 1988