Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease

The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several a subunits. To characterize the proximity between sigma(70), the major a for transcription of the growth-related genes, and the core enzyme subunits (alpha(2) beta beta'), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of sigma(70) by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated sigma(70) was bound to the core enzyme, which led to cleavage of nearby sites on the beta and beta' subunits (but not alpha). Unlike the results of random cleavage [Greiner, D. P., Hughes, K.A., Gunasekera, A. H. & Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71-75], the cut sites from different probe-modified sigma(70) proteins are clustered in distinct regions of the subunits. On the beta subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the beta' subunit, the cleavage was identified within the sequence 228-461, including beta' conserved regions C and D (which comprise part of the catalytic center).