Genome-wide effects onEscherichia colitranscription from ppGpp binding to its two sites on RNA polymerase

Abstract

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) inEscherichia 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.