Using Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during σ(70) Binding

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