Key features of σS required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme

Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other.-proteobacteria, the transcription factor Crl stimulates sigma(s) -dependent transcription during times of cellular stress by promoting the association of sigma(5) with core RNA polymerase. The molecular basis for specific recognition of sigma(5) by Crl, rather than the homologous and more abundant primary sigma factor sigma(70), is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoylphenylalanine cross-linking in vitro to define the features in s S responsible for specific recognition by Crl. We identify residues in s S conserved domain 2 (sigma(S) (2)) that are necessary and sufficient to allow recognition of sigma(70) conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of sigma(70.) We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on sigma(5). Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.