Protein-DNA photo-crosslinking with a genetically encoded benzophenone-containing amino acid.
The photo-crosslinking amino acid, p-benzoyl-L-phenylalanine (pBpa), was genetically incorporated into Escherichia coli catabolite activator protein (CAP) in bacteria in response to an amber nonsense codon using an orthogonal tRNA/aminoacyl-tRNA synthetase pair. The mutant CAP (CAP-K26Bpa) containing pBpa formed a covalent complex with a DNA fragment containing the consensus operator sequence upon UV irradiation. Because this amino acid can be genetically incorporated into any DNA-binding protein in E. coli, yeast or mammalian cells with minimal perturbation of protein structure, this method should be generally useful for investigating DNA-protein interactions.
Lee
Hyun Soo
HS
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Dimla
Romerson D
RD
Schultz
Peter G
PG
D000596:Amino Acids / Q000737:chemistry*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001483:Base Sequence; D001577:Benzophenones / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D014466:Ultraviolet Rays
D000596:Amino Acids; D001577:Benzophenones; D003432:Cross-Linking Reagents; D002373:Cyclic AMP Receptor Protein; C047723:benzophenone; D004247:DNA; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases