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 fl agellar and chemotaxis genes. Both sigma(F) and sigma(70) (the major s igma subunit in growing cells) were overexpressed, purified to homogen eity, and compared with respect to activity and specificity. The affin ity of sigma(F) to core RNA polymerase (E) is higher than that of sigm a(70), as measured by gel filtration high-pressure liquid chromatograp hy. In an in vitro transcription system, the holoenzyme (E sigma(F)) c ontaining sigma(F) selectively transcribed the flagellar and chemotaxi s genes, all of which could not be transcribed by E sigma(70). This st rict 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)-depe ndent transcription in vitro is inhibited at high concentrations of al l salts tested, shelving maximum activity at 50 mM. In contrast, sigma (F)-dependent transcription was maximum at 50 mM KCI and then decrease d 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 reco gnizes -10 and -35 regions of the sigma(F)-dependent promoters with ra ther long upstream protection. Alteration of the promoter structure af ter binding of E sigma(F) was suggested.