Regulation of factor competition by the alarmone ppGpp

Many regulons controlled by alternative factors, including S and , are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of -dependent promoters because underproduction of , specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti) restored expression from -dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type 70 to compete with 32 for core binding and the mutant 70 proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type . Similarly, an in vivo competition assay showed that the ability of both 32 and S to compete with 70 is diminished in cells lacking ppGpp. Consistently, the fraction of S and 32 bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of factors in accordance with cellular demands during physiological stress.