Photochemical crosslinking of lac repressor to nonoperator 5-bromouracil-substituted DNA

Abstract

UV irradiation of lac repressor bound to 5-bromouracil-substituted nonoperator DNA [from Escherichia coli] leads to the formation of cross-links between the protein and the nucleic acid. The cross-links are formed between the DNA and the 1-51 N-terminal peptide of the repressor, the headpiece. The tetrameric core (4 .times. 60-360 amino acids) was never found to be cross-linked to the DNA. With isolated headpieces, which are able to bind DNA, no cross-link was detected. These results are discussed considering the fundamental role of the core in keeping the headpieces in an adequate geometry for the DNA-repressor intraction. It was possible to cross-link 2 DNA molecules to 1 repressor molecule, thus showing the existence of at least 2 binding sites for nonoperator DNA on the repressor. The attached peptides were analyzed after extensive proteolytic cleavage, and the most abundant was peptide 23-33. His-29 seems to be the photo-cross-linked amino acid. Analysis of the results required a special computation method.