The sigma factors are the key regulators of bacterial transcription. ECF (extracytoplasmic function) sigma's are the largest and most divergent group of sigma(70) family members. ECF sigma's are normally sequestered in an inactive complex by their specific anti-sigma factor, which often spans the inner membrane. Here, we determined the 2 A resolution crystal structure of the Escherichia coli ECF sigma factor sigma(E) in an inhibitory complex with the cytoplasmic domain of its anti-sigma, RseA. Despite extensive sequence variability, the two major domains of sigma(E) are virtually identical in structure to the corresponding domains of other sigma(70) family members. In combination with a model of the sigma(E) holoenzyme and biochemical data, the structure reveals that RseA functions by sterically occluding the two primary binding determinants on sigma(E) for core RNA polymerase.

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The subunit of eubacterial RNA polymerase is required throughout initiation, but how it communicates with core polymerase (␣ 2 ␤␤) is poorly understood. The present work addresses the location and function of the interface of with core. Our studies suggest that this interface is extensive as mutations in six conserved regions of 70 hinder the ability of to bind core. Direct binding of one of these regions to core can be demonstrated using a peptide-based approach. The same regions, and even equivalent residues, in 32 and 70 alter core interaction, suggesting that 70 family members use homologous residues, at least in part, to interact with core. Finally, the regions of that we identify perform specialized functions, suggesting that different portions of the interface perform discrete roles during transcription initiation.

For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the -10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the beta' subunit (aa 262--309) induces sigma(70) to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the beta' coiled-coil and region 2.2 of sigma(70) promotes an allosteric transition that allows sigma(70) to selectively recognize the nontemplate strand. As the beta' 262--309 peptide can function with the previously crystallized portion of sigma(70), nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and sigma(70), the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between sigma(70) and specific domains of RNA polymerase and provide evidence that sigma(70) and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of sigma(70) with core RNA polymerase is associated with progression through the process of transcription initiation.