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Nucleic Acids Research, 1995, Vol. 23, No. 14 2579-2583
© 1995

MOLECULAR BIOLOGY

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

Barbara Neupert, Eric Menotti and Lukas C. Kühn*

Swiss Institute for Experimental Cancer Research, Genetics Unit Chemin des Boveresses, CH-1066 Epalinges, Switzerland

*To whom correspondence should be addressed

Received May 15, 1995. Accepted May 31, 1995.

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 peptlde 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-proteln 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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