Using disultide bond engineering to study conformational changes in the β′260-309 coiled-coil region of Escherichia coli RNA polymerase during σ70 binding

RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holloenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the beta' subunit (beta'260-109, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with (sigma(70) both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to sigma-(70) In this work, we engineered a disulfide bond within beta'240-309 that locks the beta' coiled-coil region in the coiled-coil conformation, and we show that this "locked" peptide is able to bind to sigma(70). We also show that the locked coiled-coil is capable of inducing a conformational change within sigma(70) that allows recognition of the -10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding sigma-(70) or upon recognition of the -10 nontemplate strand of DNA.