利用含二苯甲酮的基因编码氨基酸进行蛋白质 - DNA 光交联
Protein-DNA photo-crosslinking with a genetically encoded benzophenone-containing amino acid.
作者信息
Lee Hyun Soo, Dimla Romerson D, Schultz Peter G
机构信息
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
出版信息
Bioorg Med Chem Lett. 2009 Sep 1;19(17):5222-4. doi: 10.1016/j.bmcl.2009.07.011. Epub 2009 Jul 9.
Abstract
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.
摘要
利用一对正交的tRNA/氨酰-tRNA合成酶，将光交联氨基酸对苯甲酰-L-苯丙氨酸（pBpa）响应琥珀色无义密码子，在细菌中基因编码到大肠杆菌分解代谢物激活蛋白（CAP）中。含有pBpa的突变型CAP（CAP-K26Bpa）在紫外线照射下与含有共有操纵序列的DNA片段形成共价复合物。由于这种氨基酸能够在对蛋白质结构产生最小干扰的情况下，基因编码到大肠杆菌、酵母或哺乳动物细胞中的任何DNA结合蛋白中，因此该方法对于研究DNA-蛋白质相互作用应具有普遍的实用性。
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