FIGURE 2.

Detection of protein interactions using the E. coli two-hybrid assay. A, schematic shows the E. coli two-hybrid system used to detect interactions between the C-terminal region of σ⁷⁰ and other proteins. Positions of RNA polymerase subunits β, β′, and σ⁷⁰ and the α-σ⁷⁰ chimera are located upstream of the reporter gene (lacZ). The cI-bait fusion protein is indicated. The α-σ⁷⁰ chimera consists of the NTD of α fused to the C terminus of σ⁷⁰. B–D, relative β-galactosidase activity is shown for assays using α-σ⁷⁰ chimera with σ⁷⁰ mutations and cI-bait fusion proteins containing MotA^NTD (B), E. coli β-flap (C), or AsiA (D). Gray and red lines indicate normalized WT level and a 2-fold reduction, respectively. S.D. values (error bars) in B and C were determined from three or more independent experiments.