A simple crosslinking method, CLAMP, to map the sites of RNA-contacting domains within a protein.
A large number of proteins contain multiple RNA recognition motifs (RRMs). How multiple RRMs contribute to RNA recognition in solution is, however, poorly understood. Here, we describe a simple biochemical approach called CLAMP (crosslinking and mapping of protein domain) to identify an RRM that is crosslinked to a specific nucleotide in RNA. It involves site-specific incorporation of a chromophore, photochemical RNA-protein crosslinking, and site-specific chemical cleavage of the protein. This technique is suitable for numerous other RNA binding proteins that have multiple RNA binding domains.
Banerjee
Hiren
H
Department of Molecular, University of Colorado at Boulder, Boulder, Colorado, USA.
Singh
Ravinder
R
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D014498:Uracil / Q000031:analogs & derivatives / Q000737:chemistry
D003432:Cross-Linking Reagents; D011506:Proteins; D013388:Succinimides; C015752:N-chlorosuccinimide; D014498:Uracil; D012313:RNA; C048747:5-iodouracil