MAPPING THE SIGMA(70) SUBUNIT CONTACT SITES ON ESCHERICHIA-COLI RNA-POLYMERASE WITH A SIGMA(70)-CONJUGATED CHEMICAL PROTEASE

The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by bindin g one of several a subunits. To characterize the proximity between sig ma(70), the major a for transcription of the growth-related genes, and the core enzyme subunits (alpha(2) beta beta'), we analyzed the prote in-cutting patterns produced by a set of covalently tethered FeEDTA pr obes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of sigma(70) by using seven mu tants, each carrying a single cysteine residue at position 132, 376, 3 96, 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 cleav age [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 differ ent probe-modified sigma(70) proteins are clustered in distinct region s of the subunits. On the beta subunit, cleavage is observed in two re gions, 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 formin g the catalytic center of RNA polymerase. On the beta' subunit, the cl eavage was identified within the sequence 228-461, including beta' con served regions C and D (which comprise part of the catalytic center).