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Transcriptional Occlusion Caused by Overlapping Promoters

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Transcriptional Occlusion Caused by Overlapping Promoters Transcriptional occlusion caused by overlapping promoters M. Ammar Zafara,1, Valerie J. Carabettab,1, Mark J. Mandelc, and Thomas J. Silhavya,2 aDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; bPublic Health Research Institute at New Jersey Medical School, Rutgers University, Newark, NJ 07103; and cDepartment of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 Contributed by Thomas J. Silhavy, December 17, 2013 (sent for review November 6, 2013) RpoS (σ38) is required for cell survival under stress conditions, but has as its basis two overlapping promoters and a long noncoding it can inhibit growth if produced inappropriately and, consequently, RNA (lncRNA). Expression of the shorter transcript blocks ex- its production and activity are elaborately regulated. Crl, a transcrip- pression of the longer, but the former cannot be translated be- tional activator that does not bind DNA, enhances RpoS activity by cause it lacks a ribosome-binding site (rbs). This simple, but stimulating the interaction between RpoS and the core polymerase. economical, mechanism allows a near instantaneous reduction crl The gene has two overlapping promoters, a housekeeping, RpoD- in Crl synthesis without the need to produce any transacting σ70 σ54 ( ) dependent promoter, and an RpoN ( )promoterthatis regulatory factor. strongly up-regulated under nitrogen limitation. However, transcrip- tion from the RpoN promoter prevents transcription from the RpoD Results promoter, and the RpoN-dependent transcript lacks a ribosome- The Role of Crl Under Nitrogen-Stress Conditions. In rapidly growing binding site. Thus, activation of the RpoN promoter produces cells, RpoS is made but it is directed by the adaptor protein SprE a long noncoding RNA that silences crl gene expression simply by being made. This elegant and economical mechanism, which (RssB) to ClpXP where, in ATP-dependent fashion, it is un- allows a near-instantaneous reduction in Crl synthesis without the folded and rapidly degraded (15, 20, 21). Under carbon starva- need for transacting regulatory factors, restrains the activity of RpoS tion conditions, ATP levels drop, proteolysis ceases, and RpoS to allow faster growth under nitrogen-limiting conditions. levels increase 20-fold (22, 23). Under nitrogen-starvation con- ditions, ATP levels do not drop, proteolysis continues, and RpoS transcriptional repression | lncRNA levels increase only twofold (22). Nonetheless, transcription from the dps promoter, which strongly depends on RpoS, increases n Escherichia coli, RNA polymerase consists of a core, and a σ ∼30-fold under both conditions (Fig. 1A). Ifactor to provide specificity for promoter recognition. RpoD Because the effects of Crl on RpoS activity are greatest when (σ70) is the housekeeping σ factor and is essential for growth (1, RpoS levels are low (16), we wondered whether Crl might be 2). Six other σ factors compete for core polymerase with RpoD especially important under nitrogen-starvation conditions. To and regulate specific genes (3). One such factor is RpoS (σ38), test this possibility, we monitored transcription of the RpoS- the general stress response σ factor (4, 5); another is RpoN (σ54), dependent gene dps in the presence and absence of Crl. As the nitrogen-stress σ factor (6). shown in Fig. 1A, there is a significant reduction in dps tran- The RpoS regulon provides defense against harsh environ- scription in a crl null strain under nitrogen-starvation, but not mental conditions, and cells that lack RpoS are highly suscepti- carbon-starvation, conditions. Thus, Crl plays a significant ble to stress (7, 8). However, high levels of RpoS can inhibit role specifically under nitrogen-starvation conditions. It growth (9). For example, mutants lacking RpoS grow faster on should be noted that other effectors of σ factor competition, poor carbon sources like succinate (9, 10) and mutants with at- tenuated RpoS activity have a distinct fitness advantage known Significance as growth advantage in stationary phase (GASP; ref. 11). Bac- teria must achieve a fine balance between self-preservation and nutritional competence (SPANC; ref. 12) and for this reason, Under nitrogen-limiting conditions, the master regulator RpoS RpoS is polymorphic in wild and domesticated laboratory strains protects against stress, but slowed growth is the price cells pay (9, 13). Not surprisingly, given the functional duality of the for this protection. Under these conditions, transcription of the protein, RpoS is tightly regulated at all levels: transcription, gene for the RpoS activator Crl increases. Strikingly, we show translation, protein stability, and its activity (5). The regulatory that this increase in transcription actually decreases Crl protein mechanism described here regulates RpoS at yet an additional levels, thus decreasing RpoS activity and increasing growth MICROBIOLOGY level by controlling the synthesis of an RpoS activator, Crl. rate. This unique regulatory mechanism has as its basis two RpoS has the lowest affinity of all σ factors for core poly- overlapping promoters. Transcription from the promoter used merase (14). One of the ways that RpoS can overcome its lack of under nitrogen-limitation prevents transcription of the func- tional crl mRNA producing instead a long noncoding RNA that affinity for the core polymerase is by its interaction with its ac- has no function. Under conditions where protein synthesis is tivator, the prosigma factor Crl. Crl is a transcriptional activator compromised, this simple, but economical mechanism causes that does not bind DNA (15), ratheritbiasesthecompetitionbe- a near-instantaneous reduction in Crl levels and RpoS activity tween sigma factors by stimulating the interaction between RpoS without the need to synthesize a regulatory molecule. and core polymerase (16–19). Thus, Crl plays an important role in global gene regulation by enhancing RpoS activity (18, 19). Author contributions: M.A.Z., V.J.C., and T.J.S. designed research; M.A.Z., V.J.C., and M.J.M. Our efforts to understand how Escherichia coli controls the performed research; M.A.Z., V.J.C., and T.J.S. analyzed data; and M.A.Z. and T.J.S. activity of RpoS when protein synthesis is restricted by nitrogen wrote the paper. limitation led to the discovery that the crl gene produces less The authors declare no conflict of interest. protein despite the fact that it is transcribed at a substantially 1M.A.Z. and V.J.C. contributed equally to this work. increased rate under these conditions. Unraveling this paradox- 2To whom correspondence should be addressed. E-mail: [email protected]. ical result led to the elucidation of a completely unexpected and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. unique mechanism of transcriptional regulation. This mechanism 1073/pnas.1323413111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1323413111 PNAS | January 28, 2014 | vol. 111 | no. 4 | 1557–1561 Downloaded by guest on September 23, 2021 dps crl a steady state in media containing the poor nitrogen source argi- A + 50 40 nine. We denote this nitrogen-stress medium as M63 (Arg )in 40 contrast to the typical M63 medium that contains the preferred 30 ast 30 nitrogen source ammonia. Arginine is catabolized via the 20 20 pathway, and expression of these genes is induced by nitrogen stress Fold Induction Fold Induction in RpoN/NtrC-dependent fashion (25, 26). 10 10 + Under steady-state growth conditions in M63 (Arg ) media, 0 0 WT crl WT crl WT ntrC WT ntrC dps transcription increased to a similar level (∼30-fold) as ob- Nitrogen-Starved Carbon-Starved Nitrogen-Starved Carbon-Starved served under nitrogen starvation. This increase completely depended on RpoS, and it was significantly lowered in strains RpoD promoter Translation lacking Crl (Fig. 2A). Transcription of crl was also increased, B (RpoD promoter only) -35 -10 RpoD +1 A RBS Start Codon albeit more modestly, threefold (Fig. 2 ), than observed under *** * ** ** aaatttgctaaattttgccaatttggtaaaacagttgcatcacaacaggagatagcaATG nitrogen starvation. However, under nitrogen-limiting con- ** ** ditions, we could measure Crl protein levels under steady-state RpoN +1 -24 -12 growth conditions, a more meaningful measure of Crl protein RpoN promoter levels than was possible with starved, nongrowing cells. Surpris- ingly, although crl transcript levels are increased threefold, we ntrC C wild-type find that Crl protein levels are actually decreased threefold (Fig. 2B). Note that under these conditions, increased transcription of crl also is accompanied by decreased levels of Crl protein in LOG N- C- LOG N- C- strain MG1655, demonstrating that this surprising result is not strain specific (Fig. S3). D WT 1X 0.5 X 0.25X crl ntrC rpoN WT ntrC WT ntrC The Role of the RpoN-Dependent crl Transcript. As mentioned ear- crl B Crl lier, has two overlapping promoters (Fig. 1 ): One is RpoD dependent, and the other is RpoN dependent. RpoN is known to LOG N- C- bind to the crl promoter element (27, 28), and the transcription start sites (TSS) have been determined for both promoters. The Fig. 1. RpoS activity and crl expression under nitrogen-starvation conditions. (A) Induction of dps (Left)andcrl (Right) transcription measured under carbon- RpoD-dependent TSS is upstream from a conserved ribosome- starvation and nitrogen-starvation conditions relative to nonstarved cells. Error binding site (rbs; ref. 29), and the mRNA is translated efficiently bars indicate the
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