The alarmone guanosine tetraphosphate (ppGpp) acts as both a positive and a negative regulator of gene expression in the presence of DksA, but the underlying mechanisms of this differential control are unclear. Here, using uspA hybrid promoters, we show that an AT-rich discriminator region is crucial for positive control by ppGpp/DksA. The AT-rich discriminator makes the RNA polymerase-promoter complex extremely stable and therefore easily saturated with RNA polymerase. A more efficient transcription is achieved when the RNA polymerase-promoter complex is destabilized with ppGpp/DksA. We found that exchanging the AT-rich discriminator of uspA with the GC-rich rrnB-P1 discriminator made the uspA promoter negatively regulated by ppGpp/DksA both in vivo and in vitro. In addition, the GC-rich discriminator destabilized the RNA polymerase-promoter complex, and the effect of ppGpp/DksA on the kinetic properties of the promoter was reversed. We propose that the transcription initiation rate from promoters with GC-rich discriminators, in contrast to the uspA-promoter, is not limited by the stability of the open complex. The findings are discussed in view of models for both direct and indirect effects of ppGpp/DksA on transcriptional trade-offs.
Keywords: Bacterial Transcription; Discriminator; DksA; Gene Regulation; RNA Polymerase; Ribosomal RNA (rRNA); Stress Response; Stringent Control; ppGpp.