IDENTIFICATION OF SIGMA-FACTORS FOR GROWTH-PHASE-RELATED PROMOTER SELECTIVITY OF RNA-POLYMERASES FROM STREPTOMYCES-COELICOLOR-A3(2)

We examined the promoter selectivity of RNA polymerase (RNAP) from Str eptomyces coelicolor at two growth phases by in vitro transcription. D istinct sets of promoters were preferentially recognized by either exp onential or stationary phase RNAP. No change in molecular weight or ne t charge of the core subunits was observed, suggesting that the associ ated specificity factors determined phase-specific promoter selectivit y of the holoenzyme. Five different specificity factors and their cogn ate promoters were identified by in vitro holoenzyme reconstitution an d transcription assays, sigma(66) (sigma(hrdB)) and sigma(46) (sigma(h rdD)) recognized promoters (rrnDp2 and dagAp4 for sigma(66), actII-orf 4p and whiBp2 for sigma(46)) preferentially transcribed by the exponen tial phase RNAP sigma(52) recognized promoters (dagAp3 and actIIIpx1) preferentially transcribed by the stationary phase RNAP sigma(28) (sig ma(sigE)) recognized promoters (hrdDp1, whiBp1 and dagAp2) transcribed equally by both RNAPs. A novel 31 kDa specificity factor recognized a ctIIIpx2, glnRp2 and hrdDp2 promoters preferentially transcribed by th e stationary phase RNAP. This factor was isolated from the stationary phase RNAP and reconstituted holoenzyme in vitro as a sigma factor, Th e N-terminal sequence suggests that it is a novel factor, By examining phase-specific promoter recognition pattern we can predict that holoe nzyme E sigma(52) and E sigma(31) activities are higher in the station ary phase, whereas E sigma(66) and E sigma(46) activities are higher i n the exponential phase, Possible promoter sequences recognized by som e of these sigma factors were suggested.