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Hfq chaperone brings speed dating to bacterial sRNA

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Hfq is a ubiquitous, Sm‐like RNA binding protein found in most bacteria and some archaea. Hfq binds small regulatory RNAs (sRNAs), facilitates base pairing between sRNAs and their mRNA targets, and directly binds and regulates translation of certain mRNAs. Because sRNAs regulate many stress response pathways in bacteria, Hfq is essential for adaptation to different environments and growth conditions. The chaperone activities of Hfq arise from multipronged RNA binding by three different surfaces of the Hfq hexamer. The manner in which the structured Sm core of Hfq binds RNA has been well studied, but recent work shows that the intrinsically disordered C‐terminal domain of Hfq modulates sRNA binding, creating a kinetic hierarchy of RNA competition for Hfq and ensuring the release of double‐stranded sRNA–mRNA complexes. A combination of structural, biophysical, and genetic experiments reveals how Hfq recognizes its RNA substrates and plays matchmaker for sRNAs and mRNAs in the cell. The interplay between structured and disordered domains of Hfq optimizes sRNA‐mediated post‐transcriptional regulation, and is a common theme in RNA chaperones. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry
Structure and RNA binding surfaces of Hfq. (a) Secondary structure of E. coli Hfq, showing the ordered Sm core (cyan) with arginine patch (blue) necessary for annealing (Panja, Schu, & Woodson, ). Unstructured N‐terminal (NTD; dark violet) and C‐terminal (CTD; violet) domains are indicated schematically with the autoregulatory acidic CTD tip (Santiago‐Frangos, Jeliazkov, Gray, & Woodson, ) in red. A previously described set of 985 nonredundant bacterial Hfq sequences (40) were analyzed by DISOPRED (Jones & Cozzetto, ; Ward, Sodhi, McGuffin, Buxton, & Jones, ) to estimate the range of NTD (0–49 aa) and CTD (0–185 aa) lengths. (b)–(d) Superposition of crystal structures of Hfq bound to RNA oligomers. (b) Proximal face U‐rich RNA (yellow) bound to inner pore (3RER; Wang et al., ). (c) Distal face bound to A18 RNA (green) (3GIB; Link, Valentin‐Hansen, & Brennan, ). (d) View of lateral rim (4V2S; Dimastrogiovanni et al., ) with A/U‐rich RNA (orange) bound to the outer edge of the proximal side
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Hfq springloads rpoS mRNA for sRNA entry. (b) The long (572 nt) leader of rpoS mRNA contains Hfq binding motifs upstream (green) and downstream (gold) of the sRNA binding site (tan), which are necessary for sRNA regulation of rpoS translation (Peng et al., ; Soper & Woodson, ). (b) SHAPE footprinting and SAXS showed that rpoS mRNA contacts every RNA binding surface of Hfq, wrapping the RNA into a compact conformation that partially unwinds the sRNA target site and facilitates base‐pairing with complementary sRNAs (Peng et al., ). Hfq NTDs and CTDs are omitted for clarity
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Binding of CTD to rim drives kinetic competition between sRNAs. (a) Model of Class I sRNA RydC (red cartoon) bound to full‐length Hfq. Crystallographic structure of RydC–Hfq complex (4VQS; Dimastrogiovanni et al., ) was superimposed on a ROSETTA model of full‐length Hfq (Santiago‐Frangos et al., ). Clashes introduced by the superposition were alleviated by gradient‐based energy minimization and side‐chain repacking with backbone coordinates kept constant (Conway, Tyka, DiMaio, Konerding, & Baker, ). Shown is only one possible model for the flexible CTDs (violet shades), which can also sample the space on the distal side of the Hfq ring. Basic residues in the core and acidic residues in the CTD tip are colored blue and red, respectively. Some arginine patches are inaccessible due to electrostatic interactions with acidic CTD tips (Santiago‐Frangos et al., ). The U‐tail of RydC (gold) binds the inner proximal pore, while an A/U‐rich motif upstream of the terminal stem‐loop interacts with the arginine‐rich patch (Dimastrogiovanni et al., ; Ishikawa et al., ; Sauer et al., ). The body of RydC sRNA may weakly interact with the CTDs (Dimastrogiovanni et al., ). (b) Class II sRNAs outcompete Class I sRNAs for access to Hfq, despite having similar binding affinities (Malecka et al., ; Schu et al., ). Class I sRNAs bind the proximal face and rim (Schu et al., ), and are readily displaced by acidic CTD tips (Santiago‐Frangos et al., ; Santiago‐Frangos et al., ). Class II sRNAs contain an AAN motif (green) that binds the distal face (Zhang et al., ) and resists displacement by CTDs, leading to a hierarchy of sRNA competition for binding to Hfq (Santiago‐Frangos et al., ; Santiago‐Frangos et al., )
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Cycle of RNA binding, annealing and release from Hfq. A working model for RNA annealing by Escherichia coli Hfq. (1) sRNA (red) and mRNA (green and gray) rapidly bind the Hfq hexamer in random order to form ternary complexes. Short RNAs bind near the diffusion‐controlled limit (Hopkins, Panja, & Woodson, ); longer RNAs that change structure bind more slowly. Noncognate ternary complexes are unstable due to active cycling of excess Class I sRNAs on the proximal face of the hexamer (Wagner, ), until (2) a cognate sRNA eventually binds to form a cognate ternary complex. (3) In the slow step of the reaction, nucleation of a helix between complementary regions of the sRNA and mRNA is facilitated by arginine‐rich patches on the rim of the hexamer (blue). (4) Remaining base pairs rapidly zipper into a fully annealed sRNA–mRNA pair (Panja, Paul, Greenberg, & Woodson, ). CTDs efficiently displace dsRNA from the arginine‐rich patches, preventing destabilization of the annealed segment (Santiago‐Frangos et al., ; Santiago‐Frangos et al., ). (5) sRNA cycling, recruitment of a new mRNA or class II sRNA, or RNase E turnover, may assist complete dissociation of annealed sRNA–mRNA complex from Hfq core. Hfq NTDs and CTDs are omitted from the pictograms for clarity
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