Promoter selectivity of Escherichia coli RNA polymerase sigma(F) holoenzyme involved in transcription of flagellar and chemotaxis genes

The rpoF gene of Escherichia coli codes for the RNA polymerase sigma(F) (or sigma(28)) subunit, which is involved in transcription of the flagellar and chemotaxis genes. Both sigma(F) and sigma(70) (the major sigma subunit in growing cells) were overexpressed, purified to homogeneity, and compared with respect to activity and specificity. The affinity of sigma(F) to core RNA polymerase (E) is higher than that of sigma(70), as measured by gel filtration high-pressure liquid chromatography. In an in vitro transcription system, the holoenzyme (E sigma(F)) containing sigma(F) selectively transcribed the flagellar and chemotaxis genes, all of which could not be transcribed by E sigma(70). This strict promoter recognition property of sigma(F) is similar to those of other stress response minor sigma subunits but different from those of the principal sigma subunits, sigma(70) and sigma(38). sigma(70)-dependent transcription in vitro is inhibited at high concentrations of all salts tested, shelving maximum activity at 50 mM. In contrast, sigma(F)-dependent transcription was maximum at 50 mM KCI and then decreased to negligible level at 300 mM; in the cases of potassium acetate and potassium glutamate, maximum transcription was between 200 and 300 mM. DNase I foot printing of the fliC and fliD promoters indicated that sigma(F) alone is unable to bind DNA, but E sigma(F) specifically recognizes -10 and -35 regions of the sigma(F)-dependent promoters with rather long upstream protection. Alteration of the promoter structure after binding of E sigma(F) was suggested.