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Np4a Alarmones Function in Bacteria As Precursors to RNA Caps

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Np4a Alarmones Function in Bacteria As Precursors to RNA Caps Np4A alarmones function in bacteria as precursors to RNA caps Daniel J. Lucianoa,b and Joel G. Belascoa,b,1 aKimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, NY 10016; and bDepartment of Microbiology, New York University School of Medicine, New York, NY 10016 Edited by Gisela Storz, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, and approved January 2, 2020 (received for review August 15, 2019) Stresses that increase the cellular concentration of dinucleoside ΔapaH mutants, such caps appear to be present at a high level tetraphosphates (Np4Ns) have recently been shown to impact RNA on most primary transcripts whose 5′-terminal transcribed nu- degradation by inducing nucleoside tetraphosphate (Np4) capping cleotide is A, C, or U and at a lower level on transcripts that of bacterial transcripts. However, neither the mechanism by which beginwithG.Bycontrast,fewifanyNp4-capped transcripts are such caps are acquired nor the function of Np4Ns in bacteria is present in unstressed wild-type cells. These caps are removed in known. Here we report that promoter sequence changes upstream E. coli by either of two enzymes, ApaH or the RNA pyrophos- of the site of transcription initiation similarly affect both the effi- phohydrolase RppH, thereby triggering rapid RNA degradation ciency with which Escherichia coli RNA polymerase incorporates (21). Of the two, ApaH appears to play a greater role in decapping, dinucleoside polyphosphates at the 5′ end of nascent transcripts and its inactivation by inducers of disulfide stress helps to explain E. in vitro and the percentage of transcripts that are Np4-capped in the elevated concentration and prolonged lifetime of capped RNAs coli , clear evidence for Np4 cap acquisition by Np4N incorporation in cells subjected to this stress. The importance of ApaH for cap during transcription initiation in bacterial cells. E. coli RNA poly- removal and subsequent RNA decay raises the possibility that merase initiates transcription more efficiently with Np4As than the various phenotypes observed for mutants lacking this enzyme with ATP, particularly when the coding strand nucleotide that might result not from receptor-mediated signaling by Np4As, but immediately precedes the initiation site is a purine. Together, these rather from altered RNA lifetimes and consequent changes in findings indicate that Np4Ns function in bacteria as precursors to gene expression. BIOCHEMISTRY Np4 caps and that RNA polymerase has evolved a predilection for The mechanism by which Np4 caps are acquired by bacterial synthesizing capped RNA whenever such precursors are abundant. transcripts is unknown. In principle, they could be acquired at the moment of RNA synthesis if RNA polymerase initiates tran- diadenosine tetraphosphate | Ap A | Ap A | non-canonical initiating 4 3 scription with a dinucleoside tetraphosphate in place of a nucleo- nucleotide | ApaH side triphosphate. They could also be added posttranscriptionally, either by aminoacyl-tRNA synthetases if the aminoacyl-adenylate 1 4 he biological function of dinucleoside 5′,5′′′-P ,P-tetraphosphates intermediate reacts with the triphosphorylated 5′ end of cellular T(Np4Ns) has largely remained a mystery since they were first transcripts, or by some other enzyme. The feasibility of each of discovered more than 50 years ago (1, 2). Present in both eu- these capping mechanisms has been demonstrated in vitro with karyotes and prokaryotes (3, 4), they have long been thought to purified E. coli RNA polymerase and E. coli lysyl-tRNA synthetase function as second messengers despite a paucity of supporting (21, 22), but their relevance to Np4 cap acquisition in vivo has never evidence. Indeed, a pathway for signaling by Np4Ns has been been investigated. clearly delineated in only one cell type (5). The predominant dinucleoside tetraphosphates in bacteria are Significance Ap4A, Gp4A, Cp4A, and Up4A (Fig. 1) (6). Generated as byproducts of tRNA aminoacylation when the aminoacyl-adenylate intermediate Since their discovery more than 50 years ago, dinucleoside reacts with a nucleoside triphosphate (2, 3), these dinucleoside tetraphosphates have been found in all realms of life, at levels tetraphosphates are particularly abundant in bacterial cells expe- correlated with a variety of intriguing physiological changes. riencing stress, especially disulfide stress induced by treatment – However, their molecular mechanism of action has largely with cysteine cross-linkers, such as cadmium or diamide (6 8). For remained an enigma. Here we present evidence that in bacteria this reason, Np4As have been deemed alarmones, that is, second they are incorporated as an alternative chain-initiating nucle- messengers that signal stress (9). They are also present at ele- otide during transcription by RNA polymerase, generating vated concentrations in mutant strains lacking ApaH, the principal RNAs that bear a novel nucleoside tetraphosphate cap at the 5′ – proteobacterial Np4A hydrolase (10 13), whose absence results in a end. Their assimilation into nascent transcripts is remarkably variety of intriguing phenotypes that have been attributed to sig- efficient, up to nine times the efficiency of nucleoside triphosphates. naling by Np4As. These mutant phenotypes include diminished These and other findings suggest that dinucleoside tetraphosphates motility, increased sensitivity to heat shock and oxidative stress, influence bacterial physiology not by signaling via a hypo- greater vulnerability to aminoglycosides, reduced antibiotic tol- thetical receptor, but rather by modifying cellular RNAs and erance, diminished sporulation, impaired regulation of biofilm impacting their function. formation, disrupted coupling of DNA replication and cell di- vision, and a decreased ability to invade mammalian cells (11, 14– Author contributions: D.J.L. and J.G.B. designed research; D.J.L. performed research; D.J.L. and J.G.B. analyzed data; and D.J.L. and J.G.B. wrote the paper. 20). However, no receptor that mediates the influence of Np4As has ever been identified in bacteria (13). The authors declare no competing interest. The assumption that Np4As signal via a receptor has been This article is a PNAS Direct Submission. challenged by the recent discovery that, under conditions that Published under the PNAS license. elevate cellular Np4A concentrations, diverse Escherichia coli RNAs 1To whom correspondence may be addressed. Email: [email protected]. acquire a cognate nucleoside tetraphosphate (Np4) cap, in which This article contains supporting information online at https://www.pnas.org/lookup/suppl/ thecapnucleosidemaybeA,G,C,orU,amongothers(21).In doi:10.1073/pnas.1914229117/-/DCSupplemental. wild-type cells subjected to disulfide stress or in unstressed www.pnas.org/cgi/doi/10.1073/pnas.1914229117 PNAS Latest Articles | 1of8 Downloaded by guest on September 30, 2021 We have now tested whether Np4 caps are acquired during transcription in E. coli. Our data show that the promoter se- quence upstream of the site of transcription initiation influences both Np4A incorporation into nascent transcripts in vitro and levels of Np4 capping in vivo. In fact, E. coli RNA polymerase prefers Np4As over ATP as the chain-initiating nucleotide. These findings indicate that RNA polymerase makes a major contribution to Np4 capping in E. coli and that Np4As function as precursors to Np4 caps in bacterial cells. Results Effect of the Promoter Sequence on Cap Acquisition in E. coli. Np4- capped transcripts are abundant in E. coli cells that are experi- encing disulfide stress or that lack the principal Np4-decapping enzyme ApaH. By contrast, few Np3- or NAD-capped RNAs and almost no Np5-capped transcripts are present under those con- ditions (21, 23). The origin of Np4 caps in bacterial cells is unknown. Theo- retically, they might result from the incorporation of Np4As at the 5′ end of nascent transcripts during initiation of transcription by RNA polymerase. Alternatively, such caps might be added posttranscriptionally by aminoacyl-tRNA synthetases or other enzymes. To test the former possibility, we investigated whether mutating the promoter sequence upstream of the site of tran- scription initiation affects the percentage of transcripts that are Np4-capped in E. coli. We reasoned that sequence changes there might differentially influence the ease with which RNA poly- merase can initiate transcription with a dinucleoside tetraphos- phate vs. a nucleoside triphosphate by altering the structure of the open promoter complex to which the Np4A binds, but that such changes should not affect the ability of other enzymes to cap or decap the RNA 5′ end posttranscriptionally. As a model system for these studies, we chose the yeiP tran- script, which encodes a paralog of translation elongation factor EF-P and is among the most highly capped RNAs in E. coli under inducing conditions (76% capped in cadmium-treated cells and 36% capped in ΔapaH cells) (21). To determine whether the promoter affects levels of Np4 capping in vivo, we altered the DNA sequence at positions −3, −2, and −1 upstream of the transcription initiation site (position +1) of a plasmid-borne yeiP gene, in- troduced the resulting plasmid variants into an E. coli strain from which both the apaH gene and the chromosomal yeiP gene had Fig. 1. Major dinucleoside tetraphosphates in E. coli. The structures of the dinucleoside tetraphosphates Ap A, Gp A, Cp A, and Up
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