Figure 4.

Identification of the Rsd binding subunit and Rsd contact site. (A) GST (lane 1) or GST–Rsd (lane 2) was mixed with an equimolar mixture of σ⁷⁰, σ^54(N), σ^38(S), σ^32(H), σ^28(F), and σ^24(E). Complexes were isolated by the GST pull down method with glutathione–Sepharose beads. The bead-bound proteins were eluted with 50 mM glutathione and analyzed by SDS/13.5% PAGE. The gel was subjected to Western blot analysis by using a mixture of antibodies against all six σ subunits. The control lane contained all six σ subunits, which all reacted against the antibody mixture. (B) Trypsin-treated σ⁷⁰ (starting material, 1 nmol), shown in lane σ⁷⁰/trypsin, was mixed with two different concentrations of GST–Rsd (lane 1, 40 pmol; lane 2, 20 pmol), and the complexes formed were isolated by the GST pull down assay with glutathione–Sepharose beads. The bead-bound proteins were eluted with 50 mM glutathione and separated by 5–15% SDS/PAGE. The gel was analyzed by Western blotting by using monospecific polyclonal antibodies against σ⁷⁰. After N-terminal sequence analysis, R3–4 and R4 peptides were identified as C-terminal fragments downstream from 449 and 500, respectively.