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. 1995 Jul 25;23(14):2579–2583. doi: 10.1093/nar/23.14.2579

A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

B Neupert 1, E Menotti 1, L C Kühn 1
PMCID: PMC307077  PMID: 7544459

Abstract

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.

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Selected References

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