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 σ subunits. To characterize the proximity between σ70, the major σ for transcription of the growth-related genes, and the core enzyme subunits (α2ββ′), 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 σ70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated σ70 was bound to the core enzyme, which led to cleavage of nearby sites on the β and β′ subunits (but not α). 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 σ70 proteins are clustered in distinct regions of the subunits. On the β 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 β′ subunit, the cleavage was identified within the sequence 228–461, including β′ conserved regions C and D (which comprise part of the catalytic center).

Protein-RNA interactions in the active center of transcription elongation complex.

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment.