Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase Patricia Sanchez-Vazqueza, Colin N. Deweyb, Nicole Kittena, Wilma Rossa, and Richard L. Goursea,1 aDepartment of Bacteriology, University of Wisconsin–Madison, Madison, WI 53706; and bDepartment of Biostatistics and Medical Informatics, University of Wisconsin–Madison, Madison, WI 53706 Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved March 20, 2019 (received for review November 16, 2018) The second messenger nucleotide ppGpp dramatically alters gene Global transcriptional effects of ppGpp have been examined expression in bacteria to adjust cellular metabolism to nutrient previously, utilizing expression microarrays and strains containing or availability. ppGpp binds to two sites on RNA polymerase (RNAP) in lacking the genes for ppGpp synthesis (relA and spoT) following Escherichia coli,butithasalsobeenreportedtobindtomanyother starvation for serine (21) or isoleucine (22, 23). The transcriptomes proteins. To determine the role of the RNAP binding sites in the of wild-type, ΔrelA ΔspoT,andΔdksA strains were also compared genome-wide effects of ppGpp on transcription, we used RNA-seq in early stationary phase (24). Several hundred regulated genes were to analyze transcripts produced in response to elevated ppGpp levels identified, indicating that ppGpp affects the expression of many in strains with/without the ppGpp binding sites on RNAP. We exam- cellular products. However, these studies did not distinguish effects ined RNAs rapidly after ppGpp production without an accompanying on transcription resulting from ppGpp binding directly to RNAP nutrient starvation. This procedure enriched for direct effects of from effects of ppGpp binding to other targets or from secondary ppGpp on RNAP rather than for indirect effects on transcription events subsequent to the direct effects of ppGpp binding to RNAP. resulting from starvation-induced changes in metabolism or on sec- Distinguishing direct from indirect effects is necessary for un- ondary events from the initial effects on RNAP. The transcriptional derstanding the mechanisms underlying a regulatory network. responses of all 757 genes identified after 5 minutes of ppGpp in- High-throughput sequencing technologies (RNA-seq) that have duction depended on ppGpp binding to RNAP. Most (>75%) were not emerged since publication of the initial global studies are more reported in earlier studies. The regulated transcripts encode products sensitive, reproducible, and versatile than expression microarrays involved not only in translation but also in many other cellular pro- for genome-wide transcription regulation studies (25). To study the cesses. In vitro transcription analysis of more than 100 promoters global transcriptional effects of direct ppGpp binding to RNAP, we from the in vivo dataset identified a large collection of directly regu- used RNA-seq to identify regulated transcripts genome-wide pro- lated promoters, unambiguously demonstrated that most effects of duced before and after inducing synthesis of ppGpp in strains with ppGpp on transcription in vivo were direct, and allowed comparison or without the ppGpp binding sites on RNAP. Analysis of tran- of DNA sequences from inhibited, activated, and unaffected promoter scripts produced after only 5 min of ppGpp induction reduced classes. Our analysis greatly expands our understanding of the identification of products resulting from secondary events. Com- parison of the effects of ppGpp in strains with and without the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp. Significance stringent response | ppGpp | DksA | promoter | transcription Cells respond to nutritional challenges by dramatically reprog- he second messengers ppGpp and pppGpp (together abbre- ramming their transcriptomes. In bacteria, transcriptional regu- Tviated here as ppGpp) have long been known to alter gene lators are often pleiotropic, complicating identification of direct expression after starvation of Escherichia coli for a variety of versus indirect effects. Here, we present the genome-wide transcriptional profile resulting from RNA polymerase (RNAP) nutrients, down-regulating stable RNA (rRNA and tRNA) genes binding by ppGpp, a transcription regulator found throughout and up-regulating stress and starvation-related genes such as the bacterial domain of life. Most of the more than 750 tran- those for amino acid biosynthesis (1, 2). ppGpp regulates tran- scripts identified here were not identified as regulated by ppGpp scription initiation from specific promoters by binding directly to in previous studies. The excellent correlation between the genes RNA polymerase (RNAP) at two sites, generally conserved in identified as regulated by ppGpp in our RNA-seq analysis and proteobacteria (3–5). “Site 1” is at the interface of the ω subunit promoters regulated directly by ppGpp in vitro facilitated iden- and the β′ subunit (4), and “site 2” is at the interface of the tification of sequence features distinguishing inhibited, acti- transcription factor DksA and the secondary channel rim helices vated, and unregulated promoters from each other, providing of β′ (5). DksA is a small RNAP-binding protein present at tools for future mechanistic studies of ppGpp action. relatively constant concentrations in E. coli (6, 7). The combined effects of amino acid substitutions in β′, ω, and DksA within the Author contributions: P.S.-V., W.R., and R.L.G. designed research; P.S.-V. and N.K. per- formed research; P.S.-V., C.N.D., N.K., W.R., and R.L.G. analyzed data; and P.S.-V., W.R., two ppGpp binding sites render RNAP insensitive to effects of and R.L.G. wrote the paper. ppGpp on transcription initiation in vitro and in vivo (5). The authors declare no conflict of interest. ppGpp can also bind to and affect the activities of a large This article is a PNAS Direct Submission. number of other proteins in addition to RNAP (1, 2), thereby Published under the PNAS license. playing roles in processes as diverse as DNA replication (8, 9), – – Data deposition: The high-throughput sequencing data reported in this paper have been translation (10 12), nucleotide metabolism (2, 11 14), ribosome deposited in the National Center for Biotechnology Information Sequence Read Archive biogenesis (11, 12, 15, 16), amino acid decarboxylation (17), (SRA) (BioProject accession no. PRJNA504613). polyphosphate metabolism (18), and ppGpp metabolism itself 1To whom correspondence should be addressed. Email: [email protected]. (11, 19, 20). Our studies were designed to distinguish the effects This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of ppGpp on gene expression resulting from its binding to RNAP 1073/pnas.1819682116/-/DCSupplemental. versus other cellular targets. Published online April 10, 2019. 8310–8319 | PNAS | April 23, 2019 | vol. 116 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1819682116 Downloaded by guest on September 25, 2021 ppGpp binding sites on RNAP rather than in strains with and and B). Plasmid pALS14, which codes for an inactive RelA vari- without the ability to synthesize ppGpp eliminated potential effects ant, was used as a control for effects of IPTG induction. We from loss of ppGpp binding to other cellular targets. Furthermore, previously demonstrated that representative negatively (rRNA) or by producing ppGpp without nutrient starvation, we avoided iden- positively (iraP) regulated promoters were not regulated in the tification of effects resulting from changes in metabolism in- 1−2− strain in vivo following induction of ppGpp by amino acid dependent of ppGpp production. starvation with serine hydroxamate (5). We evaluated use of the The results show that all transcriptional responses to ppGpp under inducible relA plasmid system instead of treatment with serine these conditions result from binding of ppGpp to RNAP rather than hydroxamate, first by testing the response of the promoters for to transcription factors or other potential targets. The much larger transcription of rRNA and the iraP mRNA. number of genes and pathways identified here as regulated by ppGpp Unlike the mature rRNAs, the rRNA precursors are highly un- indicates that the transcriptional response to ppGpp is considerably stable, and the level of the leader RNA can serve as an estimate of broader than realized previously.Alargesubsetoftheregulated rRNA promoter activity. ppGpp induction inhibited rRNA leader synthesis approximately fourfold in the wild-type strain in the first promoters was analyzed by in vitro transcription, identifying DNA − − sequence features contributing to the promoter-specific effects of 5 min, but there was little or no effect in the 1 2 strain or in the ppGpp and thus to the mechanism of the stringent response. strains with the inactive ppGpp production plasmid (Fig. 1C). The positively regulated iraP mRNA increased approximately fourfold + + Results and Discussion within 5 min of ppGpp induction in the 1 2 strain but not in the − − relA 1 2 strain or in the strains with pALS14 (Fig. 1D). The fold effects Transcriptional Response to Synthesis of ppGpp by Induction of a of ppGpp were qualitatively consistent with, although somewhat Expression Plasmid. To study the transcriptional effects of ppGpp smaller than, the results obtained after treatment with serine binding to RNAP and to identify which of its effects are direct, hydroxamate