The study of guanosine 5 '-diphosphate 3 '-diphosphate-mediated transcription regulation in vitro using a coupled transcription-translation system

The effects of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate (ppGp p) on regulation of the Salmonella typhimurium histindine operon and the Es cherichia coil tRNA(leu) operon were analyzed in vitro using a DNA-dependen t transcription-translation system, S-30. The expression of the hisG promot er is positively regulated by ppGpp, whereas that of the leuV promoter (of tRNA(leu)) is negatively regulated by ppGpp. In an attempt to understand th e global regulatory mechanism of ppGpp control, interrelationship between p pGpp-dependent activation and repression of gene expression was examined us ing these promoters as models. It has been traditionally supposed that the ppGpp-dependent regulation, at least for the activation, is by a passive mo de of control: the activation of gene expression by ppGpp is a consequence of the repression of stable RNA gene expression in the condition of RNA pol ymerase limiting. To test this model, the ppGpp-dependent regulations of bo th an activable promoter (hisGp) and a repressible promoter (leuVp) were de termined in vitro simultaneously using a mixed template setup. The rational e for this exercise was to see whether the ppGpp-dependent activation and r epression are inversely correlated in the in vitro condition in which RNA p olymerase is limiting. No correlation was observed. It was concluded that t he ppGpp-dependent activation is independent of the repression. Moreover, i t was proposed that ppGpp-dependent activation and repression are mediated by titratable factors, each of which operate independently.