Regulation of <i>ompA</i> mRNA stability: the role of a small regulatory RNA in growth phase‐dependent control

Rasmussen, Anders Aamann; Eriksen, Maiken; Gilany, Kambiz; Udesen, Christina; Franch, Thomas; Petersen, Claus Leth; Valentin‐Hansen, Poul

doi:10.1111/j.1365-2958.2005.04911.x

Cited by 172 publications

(198 citation statements)

References 37 publications

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“…Accordingly, giving higher weight to deeply conserved regions in sRNAs is expected to improve the currently available target search algorithms. In support of this, several other regulatory sRNAs-e.g., MicA, MicC, and SgrS-exhibit a higher degree of conservation in their target interaction regions Vanderpool and Gottesman 2004;Rasmussen et al 2005;Udekwu et al 2005). Unlike these enterobacterial sRNAs, S. aureus RNAIII is not conserved in other species.…”

Section: Gcvb Targets Multiple Transporter Mrnas Genes and Development mentioning

confidence: 99%

A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites

Sharma¹,

Darfeuille²,

Plantinga³

et al. 2007

Genes Dev.

342

479

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The interactions of numerous regulatory small RNAs (sRNAs) with target mRNAs have been characterized, but how sRNAs can regulate multiple, structurally unrelated mRNAs is less understood. Here we show that Salmonella GcvB sRNA directly acts on seven target mRNAs that commonly encode periplasmic substrate-binding proteins of ABC uptake systems for amino acids and peptides. Alignment of GcvB homologs of distantly related bacteria revealed a conserved G/U-rich element that is strictly required for GcvB target recognition. Analysis of target gene fusion regulation in vivo, and in vitro structure probing and translation assays showed that GcvB represses its target mRNAs by binding to extended C/A-rich regions, which may also serve as translational enhancer elements. In some cases (oppA, dppA), GcvB repression can be explained by masking the ribosome-binding site (RBS) to prevent 30S subunit binding. However, GcvB can also effectively repress translation by binding to target mRNAs at upstream sites, outside the RBS. Specifically, GcvB represses gltI mRNA translation at the C/A-rich target site located at positions −57 to −45 relative to the start codon. Taken together, our study suggests highly conserved regions in sRNAs and mRNA regions distant from Shine-Dalgarno sequences as important elements for the identification of sRNA targets.[Keywords: Small RNA; riboregulator; post-transcriptional control; translation inhibition; ABC transporter; GcvB] Supplemental material is available at http://www.genesdev.org.

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Section: Gcvb Targets Multiple Transporter Mrnas Genes and Development mentioning

confidence: 99%

A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites

Sharma¹,

Darfeuille²,

Plantinga³

et al. 2007

Genes Dev.

342

479

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Section: Hfq Binds Model Srnas and Mrnas With High Affinitymentioning

confidence: 99%

“…These were MicA, MicC, and MicF, and their targets, ompA, ompC, and ompF mRNA. Their regulatory interactions are known, as is their Hfq dependence in vivo (Andersen and Delihas 1990;Chen et al 2004;Rasmussen et al 2005;Udekwu et al 2005).RNA binding at increasing concentrations of Hfq 6 (hexameric Hfq is Hfq 6 throughout this paper) at 37°C was assayed by gel-shift analysis. Supplemental Figure S1 indicates tight Hfq 6 -RNA binding, with K d values ranging from 0.9 to 4 nM ( Table 1).…”

mentioning

confidence: 99%

RNAs actively cycle on the Sm-like protein Hfq

Fender¹,

Elf²,

Hampel³

et al. 2010

Genes Dev.

142

197

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Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K d values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1-2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentrationdriven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are »1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the ''strong binding-high turnover'' paradox and permits efficient use of the Hfq pool. The homohexameric Hfq ring displays two faces: proximal and distal. Hfq-RNA interactions show a preference of U-rich for proximal and A-rich RNA sequences for distal face binding (de Haseth and Uhlenbeck 1980a;Mikulecky et al. 2004). Simultaneous binding may occur on both sides as well, which could facilitate intermolecular base-pairing and regulation (Rajkowitsch and Schroeder 2007).Structures of Hfq from E. coli, Staphylococcus aureus, and Pseudomonas aeroginosa have been determined by X-ray crystallography (Schumacher et al. 2002;Sauter et al. 2003;Nikulin et al. 2005). Two cocrystal structures support two distinct binding surfaces: In S. aureus Hfq, AU 5 G RNA is bound around the inner rim of the proximal face (Schumacher et al. 2002), and E. coli Hfq has oligo-A bound on the distal face (Link et al. 2009 Holmqvist et al. 2010). Thus, if binding-competent RNAs were in molar excess, almost all Hfq would be bound to RNAs. Hfq-RNA dissociation rate constants in vitro are too low to be compatible with a biologically relevant time scale; half-lives of complexes are in the range of a generation time. If newly induced sRNAs only could access free Hfq after its dissociation from bound RNAs, their activity should be severely delayed. Yet, the time frame from induction of an sRNA to a significant regulatory effect is short (1-2 min) (Massé et al. 2003), and hence sRNAs can acquire Hfq rapidly. This highlights a paradox, with Hfq being tightly sequestered by the intracellular pool of RNAs, contrasted by the need of new sRNAs to rapidly access Hfq. We considered here a conventional cycling model (dissociative/passive) (Fig. 1A) and associative/active cycling (Fig. 1B). In model A, newly synthesized RNA (Fig. 1A, in red) can only bind Hfq after the resident RNA (Fig. 1A, in blue) has dissociated; i.e., the rate of binding of the incoming RNA is limited by the Hfq-RNA dissociation rate constant and is not affected by the concentration of the free RNA. In model B, free RNA transiently binds the Hfq-RNA complex, whereupon one of the RNAs eventually dissociates. Thus, the dissociation rate of the bound RNA is a function of the concentration of the free RNA (Fig. 1B). This would render cycling much more rapidly, and the intracellular pool of binder RNAs would rapidly equilibrate on Hfq. The two models are distinguishable, since th...

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“…Most act by base pairing with transencoded mRNA targets to modulate translation and/or stability. Examples of sRNAs identified that regulate targets by this mechanism include SgrS, MicA, MicC, MicF, RhyB, OxyS and Spot42 (Argaman & Altuvia, 2000;Chen et al, 2004;Kawamoto et al, 2006;Masse & Gottesman, 2002;Moller et al, 2002;Rasmussen et al, 2005;Schmidt et al, 1995;Udekwu et al, 2005). In most cases, base pairing results in negative regulation of translational activity and greater instability of the target mRNA (Gottesman, 2004).…”

Section: Introductionmentioning

confidence: 99%

Role of the sRNA GcvB in regulation of cycA in Escherichia coli

Pulvermacher

Stauffer

2009

Microbiology

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In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

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Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase‐dependent control

Cited by 172 publications

References 37 publications

A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites

A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites

RNAs actively cycle on the Sm-like protein Hfq

Role of the sRNA GcvB in regulation of cycA in Escherichia coli

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