Figure 5. σ³² is partially membrane associated.

The extent of association of σ³² and the β′ subunit of RNA polymerase with the membrane fraction was determined by quantitative immunoblotting of the soluble and nonsoluble fractions. Membrane association of σ³² and β′ was assessed in several relevant strain backgrounds. In addition to endogenous σ³², all strains contained a plasmid-encoded variant of σ³² lacking its 21 C-terminal amino acids (σ³²Δ21aa). Ectopically expressed σ³²Δ21aa or I54Nσ³²Δ21aa were present at levels comparable to native σ³² and were distinguished from endogenous σ³² on a 12% SDS-PAGE gel. All fractionation experiments were performed ≥8 times, and % fractionation was calculated from experiments where probed cytoplasmic (RuvB) and membrane (RseA) proteins separated properly.