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...

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.

The RNA chaperone Hfq is an Sm protein that facilitates base pairing between bacterial small RNAs (sRNAs) and mRNAs involved in stress response and pathogenesis. Hfq possesses an intrinsically disordered C-terminal domain (CTD) that may tune the function of the Sm domain in different organisms. In Escherichia coli, the Hfq CTD increases kinetic competition between sRNAs and recycles Hfq from the sRNA-mRNA duplex. Here, de novo Rosetta modeling and competitive binding experiments show that the acidic tip of the E. coli Hfq CTD transiently binds the basic Sm core residues necessary for RNA annealing. The CTD tip competes against non-specific RNA binding, facilitates dsRNA release, and prevents indiscriminate DNA aggregation, suggesting that this acidic peptide mimics nucleic acid to auto-regulate RNA binding to the Sm ring. The mechanism of CTD auto-inhibition predicts the chaperone function of Hfq in bacterial genera and illuminates how Sm proteins may evolve new functions.

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Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic Graphical abstract Highlights d Sequence-specific recognition of RNA by CRISPR Csm complex activates Cas10 d Cas10 polymerizes ATP to make cyclic oligonucleotides, pyrophosphates, and protons d Cas10's rapidly amplified products are detectable in 1-30 min d RT-LAMP can be coupled to T7-Csm to rapidly and sensitively detect SARS-CoV-2 RNA