Transcriptional pausing of RNA polymerase in the presence of guanosine tetraphosphate depends on the promoter and gene sequence.

We have studied the response of the effector molecule guanosine 3',5'-bisdiphosphate (ppGpp) on RNA polymerase pausing during in vitro transcription elongation. Pausing was followed during single round extension of stalled ternary complexes excluding possible ppGpp effects on initiation. The ppGpp dependences of early pausing sites within different transcription systems controlled by promoters with known response to enhanced ppGpp levels in vivo were quantitatively characterized. Transcription of stable RNAs and mRNA genes were analyzed. In addition, the in vitro pausing behavior of two promoter variants directing the same sequence but differing in their in vivo ppGpp sensitivity were compared. In the presence of ppGpp we noted a slight general enhancement of specific pauses in all transcription systems. However, genes known to be under stringent or growth rate control in vivo revealed a notably stronger pausing enhancement. The sites of pausing are not changed by the presence of ppGpp but appear to be sequence-specific. The effect of ppGpp on the extent of pausing depends on the particular promoter and closely adjacent sequences that the RNA polymerase has passed during initiation. Pausing enhancement requires the presence of ppGpp during elongation but not during initiation. The results underline the importance of pausing for transcription regulation and offer a plausible explanation for inhibition of stable RNA expression under conditions of elevated concentrations of ppGpp.