T7 RNA polymerase-mediated incorporation of 8-N(3)AMP into RNA for studying protein-RNA interactions.
Ultraviolet (UV)-dependent photochemical crosslinking is a powerful approach that can be used for the identification of RNA-protein interactions. Although 8-azidoATP (8-N(3)ATP) 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-N(3)AMP 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.
Gaur
Rajesh K
RK
Beckman Research Institute of the City of Hope, Duarte, California, USA.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011506:Proteins; D014764:Viral Proteins; D000249:Adenosine Monophosphate; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases