Modification of a free Fe-S cluster cysteine residue in the active iron- responsive element-binding protein prevents RNA binding

C. C. Philpott, D. Haile, T. A. Rouault, R. D. Klausner

Research output: Contribution to journalArticle

81 Scopus citations

Abstract

The iron-responsive element-binding protein (IRE-BP) binds to specific RNA stem-loop structures called iron-responsive elements (IREs), which mediate the post-transcriptional regulation of a variety of genes involved in iron metabolism. The IRE-BP is cytosolic aconitase, and a [4Fe-4S] cubane cluster is required for aconitase activity but is associated with loss of IRE binding affinity. Chemical modification of the IRE-BP can abrogate RNA binding and the 3 cysteines predicted to coordinate the Fe-S cluster in the IRE-BP could be targets for modification. We report the expression of recombinant IRE-BP in which the three putative cluster cysteines (Cys-437, Cys-503, and Cys- 506) have been mutated to serine residues. Replacement of any or all of these cysteine residues results in a complete loss of aconitase activity. While all of the mutants bind RNA, substitution of Cys-437 specifically renders the IRE-BP resistant to inactivation by low concentrations of N-ethylmaleimide or diamide. These results identify Cys-437 as the target of in vitro regulation of RNA binding in the IRE-BP and suggest that, in the RNA-binding form of the protein, Cys-437 is free and therefore available for modifications that inhibit RNA binding.

Original languageEnglish (US)
Pages (from-to)17655-17658
Number of pages4
JournalJournal of Biological Chemistry
Volume268
Issue number24
StatePublished - Jan 1 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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