In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS. Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.

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