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 (ppGpp) on regulation of the Salmonella typhimurium histindine operon and the Escherichia coli tRNA(leu) operon were analyzed in vitro using a DNA-dependent transcription-translation system, S-30. The expression of the hisG promoter is positively regulated by ppGpp, whereas that of the leuV promoter (of tRNA(leu)) is negatively regulated by ppGpp. In an attempt to understand the global regulatory mechanism of ppGpp control, interrelationship between ppGpp-dependent activation and repression of gene expression was examined using these promoters as models. It has been traditionally supposed that the ppGpp-dependent regulation, at least for the activation, is by a passive mode 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 polymerase limiting. To test this model, the ppGpp-dependent regulations of both an activable promoter (hisGp) and a repressible promoter (leuVp) were determined in vitro simultaneously using a mixed template setup. The rationale for this exercise was to see whether the ppGpp-dependent activation and repression are inversely correlated in the in vitro condition in which RNA polymerase is limiting. No correlation was observed. It was concluded that the ppGpp-dependent activation is independent of the repression. Moreover, it was proposed that ppGpp-dependent activation and repression are mediated by titratable factors, each of which operate independently.