| RNA polymerase holoenzyme formation, where binding of the σ specificity factor to core RNA polymerase occurs, allows recognition of specific promoter sequences and confers the ability to regulate global gene expression. RNA polymerase core enzyme and σ70 undergo multiple conformational changes through the multiple steps of transcription initiation. However, the effects of these conformational changes on the functions of specific regions had not been well characterized. To address these issues, a disulfide bond recognition algorithm “Disulfide by Design” was used to strategically place disulfide bonds within regions of RNA polymerase core enzyme and σ 70, restricting the conformational flexibility of that region.; The coiled-coil of the β′ subunit of RNA polymerase is a major interaction domain for σ70 binding, but it was unknown if the β′ coiled-coil underwent a conformational change during transcription initiation. A disulfide bond was engineered within the β′ coiled-coil which “locks” this region in the coiled-coil conformation. These experiments suggested that the β ′ coiled-coil does not undergo a conformational change during σ 70 binding or recognition of the −10 nontemplate strand.; Conformational change within region 2 of σ70, which has been demonstrated to bind the β′ coiled-coil during holoenzyme formation, was also investigated through disulfide bond engineering. When holoenzymes containing disulfide-locked σ70 (one which locks regions 2.1–2.2, and the other 2.2–2.3) were characterized for gross defects in multiple-round transcription, we found that insertion of either disulfide bond yielded active enzyme. However, both disulfide-containing holoenzymes exhibited defects in formation and stability of the open complex. These experiments suggested that a major conformational change within region 2 of σ70, such as large movement of the α-helices, is not required for transcriptional activity; however, small conformational changes may affect stability of intermediate complexes.; An LRET-based homogeneous binding assay was used to examine the relative affinities of E. coli σ factors for RNA polymerase. Competition assays analyzing the ability of σ factors to compete for core RNA polymerase revealed that only σ32 can compete successfully for core binding in the presence of σ70 under conditions tested. Equilibrium dissociation constants were also obtained for σ70, σS, and σ 32. |
