Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

Mikulecky, Peter J.; Kaw, Meenakshi; Brescia, Cristin C; Takach, Jennifer C.; Sledjeski, Darren D.; Feig, Andrew L.

doi:10.1038/nsmb858

Cited by 223 publications

(425 citation statements)

References 58 publications

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“…S1). The K d values of RNA-Hfq complexes are often subnanomolar (e.g., Geissmann and Touati 2004;Sittka et al 2008) to mid-nanomolar (e.g., Lease and Woodson 2004;Mikulecky et al 2004). For MicA and ompA RNA, additional methods were used for comparison.…”

Section: Hfq Binds Model Srnas and Mrnas With High Affinitymentioning

confidence: 99%

“…Association and dissociation rates of RNA-Hfq pairs vary. Some RNAs have strong distal or proximal face preferences, which may result in simultaneous binding of two RNAs, rather than competition (de Haseth and Uhlenbeck 1980a; Mikulecky et al 2004;Sun and Wartell 2006;Rajkowitsch and Schroeder 2007;Vecerek et al 2008;Link et al 2009). In the model (Fig.…”

Section: Genes and Development 2623mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…Many RNAs bind tightly to Hfq, with K d values ranging from sub-to midnanomolar (Arluison et al 2004;Geissmann and Touati 2004;Lease and Woodson 2004;Mikulecky et al 2004;Sittka et al 2008;Soper and Woodson 2008;Hopkins et al 2009;Holmqvist et al 2010). Thus, if binding-competent RNAs were in molar excess, almost all Hfq would be bound to RNAs.…”

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confidence: 99%

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RNAs actively cycle on the Sm-like protein Hfq

Fender¹,

Elf²,

Hampel³

et al. 2010

Genes Dev.

141

196

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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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Section: Hfq Binds Model Srnas and Mrnas With High Affinitymentioning

confidence: 99%

Section: Genes and Development 2623mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

RNAs actively cycle on the Sm-like protein Hfq

Fender¹,

Elf²,

Hampel³

et al. 2010

Genes Dev.

141

196

View full text Add to dashboard Cite

show abstract

“…Two distinct RNA-binding sites have been described on opposite faces of the Hfq ring (17). The first, so-called distal binding site binds ARN repeats (A, adenine; R, purine; N, any nucleotide) and was cocrystallized with an oligo-ðAÞ 15 substrate.…”

mentioning

confidence: 99%

Structural basis for RNA 3′-end recognition by Hfq

Sauer

Weichenrieder

2011

Proc. Natl. Acad. Sci. U.S.A.

186

248

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The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq-RNA interaction that involves the direct recognition of a uridine-rich RNA 3′ end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3′ hydroxyl group. A free 3′ hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3′ end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation.RNA chaperone | regulation of translation | RNA degradation | prokaryotes H fq is an abundant and widely conserved RNA-binding protein in bacteria and a major player in the RNA-based regulation of gene expression, such as in the adaptive response to cell stress or in the induction of virulence (1, 2).Hfq was originally identified as a host factor in Escherichia coli for the replication of the Qβ phage (3), where it binds to the C-rich 3′ end of plus-strand viral RNA (4). Subsequently, physiological roles of Hfq were described in the regulation of mRNA translation and in mRNA degradation, where it modulates the processing of RNA 3′ ends (1,(5)(6)(7)(8). The most prominent function of Hfq, however, is its interaction with small regulatory RNAs (sRNAs) that act in trans and that are differentially expressed under various metabolic and environmental conditions (9, 10). They globally regulate gene expression via base-pairing to frequently entire sets of partially complementary mRNAs (11,12). Hfq stabilizes sRNAs in the absence of their targets (13). Hfq was also found to facilitate base-pairing to the mRNAs and help trigger subsequent steps, such as the repression of translation and/or the acceleration of decay, but also mRNA activation (11,14). Despite their structural diversity, the recognition of many sRNAs by Hfq is highly specific and even works across species barriers (15). It is an intriguing question how this selectivity is achieved.Crystal structures reveal that bacterial Hfq adopts an (L)Sm fold and forms homohexameric rings, whereas related (L)Sm proteins [Sm proteins and Sm-like (LSm) proteins] in archaea and eukaryotes are found to form homomeric or heteromeric heptamers, respectively (16). Two distinct RNA-binding sites have been described on opposite f...

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