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

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Abstract
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.