The σ factors are the key regulators of bacterial transcription. ECF (extracytoplasmic function) σ's are the largest and most divergent group of σ70 family members. ECF σ's are normally sequestered in an inactive complex by their specific anti-σ factor, which often spans the inner membrane. Here, we determined the 2 Å resolution crystal structure of the Escherichia coli ECF σ factor σE in an inhibitory complex with the cytoplasmic domain of its anti-σ, RseA. Despite extensive sequence variability, the two major domains of σE are virtually identical in structure to the corresponding domains of other σ70 family members. In combination with a model of the σE holoenzyme and biochemical data, the structure reveals that RseA functions by sterically occluding the two primary binding determinants on σE for core RNA polymerase.

ABSTRACT Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς 70 subunit. A motA positive control (pc) mutant, motA-pc1 , was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς 70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S -transferase fusions of the five mutant ς 70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς 70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. coli promoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

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.

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 β′ subunit (aa 262–309) induces σ70 to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the β′ coiled-coil and region 2.2 of σ70 promotes an allosteric transition that allows σ70 to selectively recognize the nontemplate strand. As the β′ 262–309 peptide can function with the previously crystallized portion of σ70, nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.