Proteolytic DNA for Mapping Protein−DNA Interactions
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Abstract
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, beta and beta', agree well with a recent model based on the crystal structure of the core enzyme alpha(2)betabeta' [Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. H. (2000) Cell 101, 601-611]. The cleavage site present on alpha supports previous studies regarding DNA binding regions of the alpha subunit. Cleavage sites identified throughout the sigma(70) subunit help to orient it with respect to the open promoter complex.
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