Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.