Cycling of the Sm-like Protein Hfq on the DsrA Small Regulatory RNA

Lease, Richard A.; Woodson, Sarah A.

doi:10.1016/j.jmb.2004.10.006

Cited by 158 publications

(336 citation statements)

References 56 publications

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“…The mechanism of cycling of RNAs on Hfq described here is fundamentally different from conventional (passive) cycling. Even though observations in line with ours have been published (e.g., Lease and Woodson 2004;Afonyushkin et al 2005), previous work did not consider that rapid cycling is promoted by RNA concentration-driven displacement.…”

Section: Competitor Rnas Readily Displace Hfq From Rna Duplexessupporting

confidence: 74%

“…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%

“…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.

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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Section: Competitor Rnas Readily Displace Hfq From Rna Duplexessupporting

confidence: 74%

Section: Hfq Binds Model Srnas and Mrnas With High Affinitymentioning

confidence: 99%

See 1 more Smart Citation

RNAs actively cycle on the Sm-like protein Hfq

Fender¹,

Elf²,

Hampel³

et al. 2010

Genes Dev.

142

197

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“…2B). Given the presence of at least 2 functionally important and distinct RNA binding surfaces, which can be filled simultaneously (8,53), Hfq is qualitatively similar to the small RNA quality control protein Ro, which also uses separate surfaces to bind its multiple RNA ligands (54). (A-R-N-N ) Tracts.…”

Section: Resultsmentioning

confidence: 99%

Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

Link

Valentin‐Hansen

Brennan

2009

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

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“…Previous biochemical experiments demonstrated that the ability of DsrA and RprA to anneal to rpoS mRNA is facilitated by Hfq (22). A long 5′ leader was found to be essential for Hfqdependent annealing of these sRNAs to rpoS mRNA (23,24).…”

mentioning

confidence: 95%

Positive regulation by small RNAs and the role of Hfq

Soper

Mandin

Majdalani

et al. 2010

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

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Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5′ leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an ðAANÞ 4 Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37°C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA • mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA • mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.Sigma 38 | translational control | Sm-like protein | RNA-protein interactions

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Cycling of the Sm-like Protein Hfq on the DsrA Small Regulatory RNA

Cited by 158 publications

References 56 publications

RNAs actively cycle on the Sm-like protein Hfq

RNAs actively cycle on the Sm-like protein Hfq

Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

Positive regulation by small RNAs and the role of Hfq

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