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Identification of RNA‐binding surfaces in iron regulatory protein‐1
Author(s) -
Kaldy Pierre,
Menotti Eric,
Moret Rémy,
Kühn Lukas C.
Publication year - 1999
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/18.21.6073
Subject(s) - aconitase , internal ribosome entry site , biology , rna , messenger rna , ribosome , rna binding protein , translation (biology) , untranslated region , cytoplasm , mutant , binding site , microbiology and biotechnology , biochemistry , gene , mitochondrion
Post‐transcriptional regulation of mRNA translation and stability in iron metabolism involves the interaction between the trans ‐acting cytoplasmic iron regulatory proteins (IRP‐1 and IRP‐2) and cis‐ acting iron‐responsive elements (IREs) in mRNA 5′‐ or 3′‐untranslated regions. IRP‐1 can adopt two conformations: one with a [4Fe–4S]‐cluster, unable to bind IREs, which functions as a cytoplasmic aconitase; one lacking this cluster, which accumulates in iron‐deprived cells and binds mRNA firmly. We investigated which surfaces of IRP‐1 interact with IREs. Surface areas were predicted on the basis of the crystallized porcine mitochondrial aconitase structure. We selected nine sequences absent or different in mitochondrial and Escherichia coli aconitases, both being devoid of RNA‐binding properties. Mutations in two regions of domain 4 of IRP‐1 lowered the affinity for a wild‐type IRE up to 7‐fold in vitro , whereas the aconitase activity, a control for structural integrity, was not affected. Scatchard plot analysis with mutant IREs indicated that domain 4 is involved in the binding specificity. This conclusion was confirmed with hybrid proteins in which IRP‐1 surface loops were grafted into IRP‐2. The results indicate that arginines 728 and 732 contact the IRE bulge, whereas region 685–689 is necessary for recognition of the IRE loop.