Photochemical cross-linking of lac repressor to nonoperator 5-bromouracil-substituted DNA

Ultraviolet irradiation of lac repressor bound to 5-bromouracil-substituted nonoperator DNA 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 X 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 interaction. It has been possible to cross-link two DNA molecules to one repressor molecule, thus showing the existence of at least two binding sites for nonoperator DNA on the repressor. The attached peptides were analyzed after extensive proteolytic cleavage, and the most abundant peptide found was peptide 23-33. Histidine-29 seems to be the photo-cross-linked amino acid. Analysis of the results required a computation method discussed in the Appendix.