PMID: 7544459Jul 25, 1995Paper

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

Nucleic Acids Research
B NeupertL C Kühn

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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Citations

Jan 16, 2003·Biochimica Et Biophysica Acta·Douglas M Templeton, Ying Liu
Aug 6, 1996·Proceedings of the National Academy of Sciences of the United States of America·M W Hentze, L C Kühn
Dec 23, 1998·Proceedings of the National Academy of Sciences of the United States of America·N M BrownR S Eisenstein
Oct 16, 1999·The American Journal of the Medical Sciences·D J Haile
May 1, 1996·Trends in Biochemical Sciences·T A Rouault, R D Klausner
Jan 7, 1998·Analytical Biochemistry·M M Ling, B H Robinson
Oct 8, 2011·PloS One·Inês DominguesSusana Constantino Rosa Santos
Oct 18, 2019·Biotechnology Progress·Christa Meingast, Caryn L Heldt
Feb 24, 2005·Molecular and Cellular Biochemistry·Zvezdana Popovic, Douglas M Templeton

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