Guanosine 3',5'-bis(diphosphate) (ppGpp)-dependent inhibition of transcription from stringently controlled Escherichia coli promoters can be explained by an altered initiation pathway that traps RNA polymerase.
An in vitro analysis was performed to investigate the inhibitory mechanism of the global regulatory substances guanosine 3',5'-bis(diphosphate) (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate (pppGpp) during initiation of transcription. Three promoters with well known differential ppGpp sensitivities in vivo were studied: the Escherichia coli rrnB P2 promoter that is only weakly ppGpp dependent; a P2 base change variant (P2F) that confers both stringent and growth rate regulation; and the completely unregulated PtacI promoter. The in vivo ppGpp dependency for all three promoters was verified in vitro in multiple round transcription reactions, reflecting a combination of the effects at initiation, promoter clearance, and elongation. In the main part of our study, we concentrated on the contribution of initiation complex formation to the overall inhibition of transcription. Kinetic measurements of complex association and dissociation revealed that at sensitive promoters (p)ppGpp triggered an alternative initiation pathway by RNA polymerase. This involved the stabilization of the initial closed complexes, and impeded open complex formation. Subsequently formed ternary complexes were structurally altered. Based on the above findings, we propose a model which suggests that ppGpp-altered RNA polymerases are preferentially bound and enter the alternative pathway. Thus, discrimination is obtained at early steps of initiation, which causes efficient inhibition at later steps of the transcription cycle probably involving promoter clearance and elongation.
Heinemann
M
M
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Wagner
R
R
D003101:Collodion; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D007700:Kinetics; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D006159:Guanosine Tetraphosphate; D003101:Collodion; D006493:Heparin; D012321:DNA-Directed RNA Polymerases