Essential steps in the ppGpp-dependent regulation of bacterial ribosomal RNA promoters can be explained by substrate competition.
Transcription of stable RNA genes is known to be dramatically reduced in the presence of guanosine tetraphosphate (ppGpp), the mediator of the stringent response. Using in vitro transcription systems with ribosomal RNA P1 promoters, we have analyzed which step of the initiation cycle is inhibited by the effector ppGpp. We show that formation of the ternary transcription initiation complex consisting of RNA polymerase holoenzyme, the promoter DNA, and the first initiating nucleotide triphosphate is the major step at which ppGpp exerts its regulation. Neither primary binding of RNA polymerase to the promoter nor isomerization to the open binary complexes or the subsequent promoter clearance steps contributes notably to the observed inhibition. The effect of ppGpp-dependent inhibition in the formation of the ternary transcription initiation complex could be mimicked by nucleotide derivatives known to bind to the RNA polymerase active center. Using these model compounds, almost identical inhibition characteristics were observed as seen with ppGpp. The results support the previously published model, which suggests that ppGpp-dependent inhibition is based on competition between the inhibitor molecules and NTP substrates for access to the active center of RNA polymerase.
Jöres
Lars
L
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
Wagner
Rolf
R
D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D006159:Guanosine Tetraphosphate / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D015533:Transcriptional Activation
D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D006159:Guanosine Tetraphosphate; D012321:DNA-Directed RNA Polymerases