We describe a technique to determine sites on proteins involved in protein-DNA interactions. DNA was synthesized via polymerase chain reaction (PCR) to produce four polynucleotide products with phosphorothioate nucleotides at the A, T, G, or C residues. Limited conjugation with the chemical protease FeBABE results in the surface of DNA being randomly labeled at the phosphorothioate sites with this protein-cleaving reagent. After formation of a protein-DNA complex, the proteolytic DNA can be activated to cleave the protein backbone at sites near the DNA. This technique was used to study the bacterial RNA polymerase/lacUV5 DNA open promoter complex, about which significant structural information is available. Cleavage sites on the two largest subunits of RNA polymerase, β and β′, agree well with a recent model based on the crystal structure of the core enzyme α2ββ′ [Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. H. (2000) Cell 101,601-611]. The cleavage site present on α supports previous studies regarding DNA binding regions of the α subunit. Cleavage sites identified throughout the σ70 subunit help to orient it with respect to the open promoter complex.
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