The second messenger
nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability.
ppGpp binds to two sites on
RNA polymerase (RNAP) in Escherichia coli, but it has also been reported to bind to many other
proteins. To determine the role of the RNAP binding sites in the genome-wide effects of
ppGpp on transcription, we used
RNA-seq to analyze transcripts produced in response to elevated
ppGpp levels in strains with/without the
ppGpp binding sites on RNAP. We examined RNAs rapidly after
ppGpp production without an accompanying nutrient
starvation. This procedure enriched for direct effects of
ppGpp on RNAP rather than for indirect effects on transcription resulting from
starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of
ppGpp induction depended on
ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of
ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of
ppGpp.