Binding of the σ⁷⁰ protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (α₂ββ′) and the holoenzyme (core + σ⁷⁰) shows that absence of the σ⁷⁰ subunit is associated with the appearance of several cleavage sites on the subunits β (within 10 residues of sequence positions 745, 764, 795, and 812) and β′ (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near β residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when σ⁷⁰ binds. No differences are observed for the α subunit.