Mapping protein-protein interactions with a library of tethered cutting reagents: The binding site of sigma(70) on Escherichia coli RNA polymerase

Surface-exposed lysine amino groups and other reactive nucleophiles of the sigma(70) protein were conjugated with the cutting reagent iron (S)-1-[p-(b romoacetamido)benzyl]ethylenediaminetetraacetate (FeBABE) via 2-iminothiola ne (2IT) with low efficiency. The result is a library of sigma(70) conjugat es, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein. Model calcul ations indicate that the conjugates in this library should be capable of cu tting nearby sites on the backbone of almost any protein or nucleic acid to which sigma(70) binds. Since cutting occurs only when the protein is bound , the cleaved sites indicate proximity; since only proximal sites are cleav ed, interpretation of the results is straightforward. We used this library to map the periphery of the binding site on the core enzyme (alpha(2)beta b eta') of Escherichia coli RNA polymerase. The beta subunit was cut primaril y within its conserved regions C, D, Rif I, and G; additional sites were al so cut between A and B and near conserved regions E and H. The cut sites wi thin the beta' subunit were intensely clustered between residues 250-450, w hich include its conserved regions C and D, along with two additional cut s ites in conserved regions A and G. No cut sites on the a subunit were obser ved. These results recapitulate and extend those obtained using FeBABE conj ugates of seven strategically placed single-Cys sigma(70) mutants [Owens, J . T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., an d Meares, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6021-6026]. This t echnique provides a straightforward, general approach to mapping protein in teractions without mutagenesis.