Regions of the Escherichia coli primary sigma factor σ70 that are involved in interaction with RNA polymerase core enzyme

Abstract

The σ factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation. Most σ factors share several regions with significant homology in their amino acid sequences (regions 1-4). Some primary σ factors carry a large nonconserved segment between regions 1 and 2. The binding of an σ factor to the core enzyme alters the structure and properties of the σ factor, but little is known about the binding mechanism and subsequent reactions. In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli σ70 by binding to core enzyme and promoter DNA. A segment between regions 1.1 and 1.2, and that in region 3.2, were preferentially cleaved by hydroxyl radicals. Upon binding to the core enzyme, segments in regions 1.1, 2, 3 and 4 were substantially protected, while cleavage at a small segment in region 4.2 was weakly enhanced. In a binary complex of holoenzyme and promoter DNA, additional segments in regions 2.4 and 4.2 were protected, while the protection at region 1.1 disappeared. The nonconserved acidic region of σ70 in the holoenzyme became hypersensitive upon binding to DNA. These results suggest that not only the conserved region 2, but also regions 1.1, 3 and 4 of the σ factor are involved in binding to the core enzyme. The nonconserved acidic region is likely to be more exposed by further binding of σ factor to promoter DNA. © Blackwell Science Limited.