Abstact

Late flagellar genes in Pseudomonas aeruginosa are transcribed by the group 3 sigma factor, FliA (sigma28). sigma28 drives the expression of flagellin, which assembles into the flagellar filament in this monoflagellated bacterium. This function is suppressed by the anti-sigma factor FlgM. Here, we present the 1.95A resolution crystal structure of sigma28-FlgM complex, along with a 3.4A structure of sigma28RNAP open promoter complex determined using single particle cryo-electron microscopy from P. aeruginosa. The sigma28 adopts a compact conformation upon binding to the anti-sigma factor FlgM, which contacts all three domains of the sigma factor. This conformation is neither conducive to interactions with RNA polymerase nor the promoter DNA. The cryo-EM structure reveals base-specific interactions of sigma28 domain 4 (sigma4) with -35 element, flipping of -11 base of the template strand, novel interactions of template strand with domain 2 (sigma2) and 3 (sigma3), and partial insertion of sigma finger into the active site cleft, offering unique features of group 3 sigma interactions with promoter DNA. Perturbation of key residues affects transcription in vitro and flagellar phenotypes as well as bacterial motility in vivo. Analysis of the structural data presented here reveals new insights into transcription regulation of late flagellar genes.