A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.
We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151.
Neupert
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B
Swiss Institute for Experimental Cancer Research, Genetics Unit, Epalinges.
Menotti
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E
Kühn
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LC
D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D035925:Iron-Regulatory Proteins; D016297:Mutagenesis, Site-Directed; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D035925:Iron-Regulatory Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA