Proceedings of the National Academy of Sciences | 2019
Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase
Abstract
Significance Cells respond to nutritional challenges by dramatically reprogramming their transcriptomes. In bacteria, transcriptional regulators are often pleiotropic, complicating identification of direct versus indirect effects. Here, we present the genome-wide transcriptional profile resulting from RNA polymerase (RNAP) binding by ppGpp, a transcription regulator found throughout the bacterial domain of life. Most of the more than 750 transcripts identified here were not identified as regulated by ppGpp in previous studies. The excellent correlation between the genes identified as regulated by ppGpp in our RNA-seq analysis and promoters regulated directly by ppGpp in vitro facilitated identification of sequence features distinguishing inhibited, activated, and unregulated promoters from each other, providing tools for future mechanistic studies of ppGpp action. 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.