Structural analysis of full-length Hfq from<i>Escherichia coli</i>

Beich-Frandsen, Mads; Večerek, Branislav; Sjöblom, Björn; Bläsi, Udo; Djinović-Carugo, Kristina

doi:10.1107/s174430911100786x

Cited by 29 publications

(47 citation statements)

References 47 publications

(51 reference statements)

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“…Hfq65 and Hfq102 had similar affinities for the unstructured RNA oligomers A18-FAM and D16-FAM (Table 1), which interact with the distal (A18) or the proximal (D16) surfaces of Hfq. Therefore, the CTD does not substantially alter recognition of U-and A-rich sequence motifs, consistent with its negligible effect on the structure of the Sm core (6)(7)(8)(9)(10)38).…”

Section: Resultssupporting

confidence: 54%

“…Surprisingly, minRCRB bound Hfq65 twofold more tightly than Hfq102 (Table 1). minRCRB primarily binds the rim of Hfq as intended, because the Hfq rim mutation R16A raised the K d for minRCRB above 170 nM for Hfq65 6 . This rim mutation also raised the K d 10-fold for synthetic molecular beacon (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Conversely, when Hfq65-Cy3·R16 was challenged with D16-FAM, the same initial increase in FRET was followed by an additional slow increase in FRET, again suggesting that Hfq65-Cy3 cannot release its dsRNA product. 6 -Cy3·R16] complex, forming a high-FRET ternary complex, followed by release of D16·R16 duplex from Hfq102 but not Hfq65. (D) Fluorescence anisotropy assay for RNA binding and release.…”

Section: Resultsmentioning

confidence: 99%

“…The anisotropy of D16-FAM was recorded for 200 s after each addition to the cuvette (details are provided in SI Materials and Methods). Black, Hfq storage buffer; blue, Hfq102 6 ; red, Hfq65 6 .…”

Section: Resultsmentioning

confidence: 99%

“…Escherichia coli Hfq comprises an Sm domain (amino acids 7-66) that assembles into a stable hexameric ring and an intrinsically disordered C-terminal domain (CTD) that projects from the rim of the hexamer (6)(7)(8)(9)(10). The Sm ring binds to both sRNAs and target mRNAs, stabilizing the sRNAs against turnover (11)(12)(13) and facilitating base pairing with complementary sequences in the mRNA (7,14,15).…”

mentioning

confidence: 99%

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C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

Santiago-Frangos

Kumari

Schu

et al. 2016

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

149

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The bacterial Sm protein and RNA chaperone Hfq stabilizes small noncoding RNAs (sRNAs) and facilitates their annealing to mRNA targets involved in stress tolerance and virulence. Although an arginine patch on the Sm core is needed for Hfq's RNA chaperone activity, the function of Hfq's intrinsically disordered C-terminal domain (CTD) has remained unclear. Here, we use stopped flow spectroscopy to show that the CTD of Escherichia coli Hfq is not needed to accelerate RNA base pairing but is required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and the kinetics of sRNA regulation in vivo. We propose that the Hfq CTD displaces sRNAs and annealed sRNA·mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA-mRNA interactions and rapidly cycle between competing targets in the cell.A member of the Sm protein family, Hfq was first identified as a host factor for phage Q beta. Hfq is found in at least 50% of sequenced bacterial genomes (1) and, in many bacteria, contributes to posttranscriptional regulation by small noncoding RNAs (sRNAs). Deletion of Hfq leads to pleiotropic effects, such as altered cellular morphology, slow growth, maladaptation to stress, and avirulence (2-5).Escherichia coli Hfq comprises an Sm domain (amino acids 7-66) that assembles into a stable hexameric ring and an intrinsically disordered C-terminal domain (CTD) that projects from the rim of the hexamer (6-10). The Sm ring binds to both sRNAs and target mRNAs, stabilizing the sRNAs against turnover (11-13) and facilitating base pairing with complementary sequences in the mRNA (7,14,15). The conserved "proximal" face of the Hfq hexamer interacts with uridines at the sRNA 3′ end (9, 16, 17), whereas the "distal" face of Hfq binds AAN triplet repeats (9, 16, 17) often found in the 5′ UTRs of target mRNAs. These sequence-specific interactions recruit sRNAs and mRNAs to Hfq, allowing arginine-rich patches along the rim of Hfq to catalyze base pairing between complementary strands (18).Although the functional importance of the Sm domain is established, the function of the disordered CTD has been unclear. The Hfq CTD varies greatly in length and sequence composition between bacterial families (1, 19), ranging from 7-residue stubs in Bacillaceae (20) to 100-residue tails in Moraxellaceae (21, 22) (Fig. S1). Previous studies reached conflicting conclusions about the importance of the Hfq CTD for sRNA regulation. In early studies, C-terminal deletions of hfq had no obvious phenotype in E. coli (23, 24) and little effect on sRNA binding (23,25). By contrast, later studies found that the CTD was required for in vitro annealing and proper regulation by sRNAs and normal binding to long RNAs, such as the rpoS mRNA (19,26,27). Moreover, Hfq from Pseudomonas aeruginosa and Clostridium difficile, which have much shor...

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Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

Santiago-Frangos

Kumari

Schu

et al. 2016

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

149

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Hfq chaperone brings speed dating to bacterial sRNA

2018

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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.

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Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly

Stanek

Mura

2018

Methods in Molecular Biology

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Hfq is a bacterial RNA-binding protein that plays key roles in the post-transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA∙∙∙RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways-including stress response, quorum sensing, and biofilm formation-has motivated genetic and "RNAomic" studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq∙∙∙RNA interactions (in vitro). Indeed, crystallographic and biophysical studies first established Hfq as a member of the phylogenetically conserved Sm superfamily. Crystallography and other biophysical methodologies enable the RNA-binding properties of Hfq to be elucidated in atomic detail, but such approaches have stringent sample requirements, viz.: reconstituting and characterizing an Hfq·RNA complex requires ample quantities of well-behaved (sufficient purity, homogeneity) specimens of Hfq and RNA (sRNA, mRNA fragments, short oligoribonucleotides, or even single nucleotides). The production of such materials is covered in this chapter, with a particular focus on recombinant Hfq proteins for crystallization experiments.

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Structural analysis of full-length Hfq fromEscherichia coli

Cited by 29 publications

References 47 publications

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

Hfq chaperone brings speed dating to bacterial sRNA

Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly

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