Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.
We present an approach 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. To test this approach, we examine 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. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods.
D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects; D007763:Lac Operon; D007930:Leucine / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009876:Operon; D011506:Proteins / Q000378:metabolism*; D012475:Salmonella / Q000235:genetics*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D011506:Proteins; D012321:DNA-Directed RNA Polymerases; D007930:Leucine