Mapping Protease Susceptibility Sites on the Escherichia coli Transcription Factor σ70

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Abstract
N-terminally and C-terminally histidine-tagged versions of Escherichia coli RNA polymerase initiation factor σ70 were subjected to limited proteolysis and electrophoretic separation. The protein fragments were transferred to nitrocellulose, and biotinylated nitrilotriacetic acid was used to detect the His-tagged ladder that resulted. Using size markers of known lengths derived from chemical cleavage of the same His-tagged σ70, we were able to map the sites of proteolysis for σ70 free in solution, bound to core RNA polymerase, and in the Mg2+-dependent open complex with λPR promoter DNA. Numerous sites of changed susceptibility were mapped. Most of these sites mapped near residues 100 and 500. In addition, the highly acidic region around residue 190 became susceptible to cleavage in the open promoter complex. These results suggest that σ70 undergoes significant conformational changes upon binding to core RNA polymerase and upon open promoter complex formation.