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Disulfide cross-linking indicates that FlgM-bound and free σ28 adopt similar conformations

The dissociable σ subunit of bacterial RNA polymerase is required for the promoter-specific initiation of transcription. When bound to RNA polymerase, σ makes sequence-specific promoter contacts and plays a crucial role in DNA melting. In isolation, however, σ lacks significant promoter binding activity. In the crystal structure of the flagellar σ factor, σ28, bound to the anti-σ factor, FlgM, σ28 adopts a compact conformation in which the promoter binding surfaces are occluded by interdomain contacts. To test whether σ28 adopts this conformation in the absence of FlgM, we engineered a set of double cysteine mutants predicted to form interdomain disulfides in the conformation observed in the FlgM complex. We show that these disulfides form in both the presence and absence of FlgM. For two of the mutants, quantitative measurements of disulfide formation under equilibrium conditions suggest that the major solution conformation favors disulfide formation. The results indicate that the compact conformation of σ28 observed in the σ28/FlgM structure is similar to the predominant conformation of free σ28 in solution. This finding suggests that autoinhibition of DNA binding in free σ28 is accomplished by steric occlusion of the promoter binding surfaces by interdomain interactions within the σ factor as well as by a suboptimal distance between the promoter −10 and −35 element binding determinants in σ2 and σ4, respectively.