Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.

Abstract

An approach is presented for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. This approach was tested by examining the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, .lambda. cI gene, and to the uninduced and isopropyl .beta.-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. The distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant and promoter mutant strains was also examined. The in vivo data are in complete agreement with the predictions of the attenuation model of regulation. The resolution, sensitivity and generality of these methods are discussed.