Figure 3.

Identification of complex formation between σ⁷⁰ and Rsd. (A) Complex formation in vitro between Rsd and σ⁷⁰ was analyzed by the GST pull down assay. GST (lanes 1, 3, and 5) or GST–Rsd (lanes 2, 4, and 6) was mixed with equimolar amounts of Eσ⁷⁰ holoenzyme (lanes 1 and 2), core enzyme (lanes 3 and 4), or σ⁷⁰ subunit (lanes 5 and 6). Complexes formed were bound to glutathione–Sepharose beads. The bead-bound proteins were eluted with 50 mM glutathione and separated by SDS/PAGE on a 5–15% gradient gel. The gel was subjected to Western blot analysis by using a mixture of monospecific antibodies against RNA polymerase α, β, β′, and σ⁷⁰ subunits. The migration positions of RNA polymerase subunits can be identified in the holoenzyme lane. (B) Isolation of GST–σ⁷⁰-associated proteins in cell extracts. Cell lysates of a pGEXD transformant of E. coli W3110 were prepared at both exponential (lane, log) and stationary (lane, stationary) phases. GST–σ⁷⁰-bound proteins were isolated by the GST pull down assay, and the σ⁷⁰-bound proteins were separated by SDS/13.5% PAGE. The migration positions of core enzyme subunits, GST–σ⁷⁰, and ω proteins are indicated on the right. (C) Proteins isolated from stationary phase cells (see stationary lane in B) were subjected to heparin–Sepharose column chromatography. Proteins were eluted by a linear gradient of NaCl, and aliquots were analyzed by SDS/13.5% PAGE.