The key regulators of bacterial transcription initiation are the sigma factors, which direct promoter recognition and melting but only after binding to the core RNA polymerase to form the holoenzyme. X-ray crystal structures of the flagellar sigma, sigma(28), in complex with its anti-sigma, FlgM, explain the inhibition mechanism of FlgM, including its ability to attack and destabilize the sigma(28)-holoenzyme. The sigma domains (sigma(2), sigma(3), and sigma(4)) pack together in a compact unit with extensive interdomain interfaces that bury the promoter binding determinants, including the -35 element recognition helix of sigma(4), which fits in an acidic groove on the surface of sigma(3). The structure illustrates the large rearrangements that sigma(28) must undergo to form the holoenzyme and provides insights into the regulation of sigma(28) promoter binding activity that may extend, at least in principle, to other sigmas.

The dissociable subunit of bacterial RNA polymerase is required for the promoter-specific initiation of transcription. When bound to RNA polymerase, makes sequence-specific promoter contacts and plays a crucial role in DNA melting. In isolation, however, lacks significant promoter binding activity. In the crystal structure of the flagellar factor, 28 , bound to the anti-factor, FlgM, 28 adopts a compact conformation in which the promoter binding surfaces are occluded by interdomain contacts. To test whether 28 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 28 observed in the 28 ͞FlgM structure is similar to the predominant conformation of free 28 in solution. This finding suggests that autoinhibition of DNA binding in free 28 is accomplished by steric occlusion of the promoter binding surfaces by interdomain interactions within the factor as well as by a suboptimal distance between the promoter ؊10 and ؊35 element binding determinants in 2 and 4, respectively.protein conformation ͉ sigma factor ͉ transcription

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