T7 RNA polymerase-mediated incorporation of 8-N3AMP into RNA for studying protein-RNA interactions

Abstract

Ultraviolet (UV)-dependent photochemical crosslinking is a powerful approach that can be used for the identification of RNA-protein interactions. Although 8-azidoATP (8-N3ATP) has been widely used to elucidate the ATP binding site of a variety of proteins, its inability to serve as an efficient substrate for bacteriophage RNA polymerases apparently restricted its actual potential as a photocrosslinking agent. In this chapter, in vitro transcription conditions that allow for template-dependent incorporation of 8-N3AMP into RNA are described. In addition, it is shown that a high-affinity MS2 coat protein binding sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2. This approach can be extended to identify almost any RNA binding protein. © 2008 Humana Press.