Effect of salt and RNA structure on annealing and strand displacement by Hfq

Hopkins, Julia F.; Panja, Subrata; McNeil, Stephanie A. N.; Woodson, Sarah A.

doi:10.1093/nar/gkp646

Cited by 41 publications

(73 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results show that, at least in the conditions used in this study, the strength of the final sRNA • rpoS complex is a better predictor of whether translation will occur than the rate of complex formation. Many studies have examined the role of Hfq in facilitating RNA • RNA binding (32), and Hfq is clearly capable of increasing the rate of duplex formation. However, the results presented above suggest that Hfq's effect on the rate of RNA • RNA binding does not drive its in vivo effects on sRNA activity.…”

Section: Resultsmentioning

confidence: 99%

Positive regulation by small RNAs and the role of Hfq

Soper

Mandin

Majdalani

et al. 2010

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

257

305

View full text Add to dashboard Cite

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

show abstract

Section: Resultsmentioning

confidence: 99%

Positive regulation by small RNAs and the role of Hfq

Soper

Mandin

Majdalani

et al. 2010

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

257

305

View full text Add to dashboard Cite

show abstract

“…The affinities of Hfq102 and Hfq65 for 32 P-labeled sRNAs at 30°C were measured by native gel mobility shift as previously described (15) and fit to a partition function for two independent sites (15). Binding constants for D16-FAM, A18-FAM, molecular beacon, or Cy3-minRCRB (5 nM) were measured in TNK buffer (10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 50 mM KCl) at 30°C by FAM fluorescence anisotropy as described before (65).…”

Section: Methodsmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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...

show abstract

“…Fluorescence experiments show that Hfq binds and releases oligonucleotide substrates very rapidly (in a second or less), such that the resulting ternary complexes are short-lived (Hopkins et al 2011). In general, the rapid dissociation of Hfq from the RNA correlates with increased annealing activity (Rajkowitsch and Schroeder 2007;Hopkins et al 2009). Recently, Fender et al (2010 proposed that RNAs are actively displaced from the Hfq hexamer by invasion of a second RNA strand.…”

Section: Contributions From Transient Co-bindingmentioning

confidence: 99%

“…The proximal face pore preferentially binds U-rich RNA (1 nucleotide [nt] per subunit) (Schumacher et al 2002), while the distal face binds A-rich RNA (3 nt per subunit) (Mikulecky et al 2004;Link et al 2009). Hfq accelerates annealing and exchange of complementary oligonucleotides (Moll et al 2003;Arluison et al 2007;Rajkowitsch and Schroeder 2007;Hopkins et al 2009), in part by transiently engaging both strands in a ternary complex (Rajkowitsch and Schroeder 2007;Fender et al 2010). Natural sRNAs and mRNAs are larger and more structured.…”

Section: Introductionmentioning

confidence: 99%

Major role for mRNA binding and restructuring in sRNA recruitment by Hfq

Soper

Doxzen

Woodson³

2011

RNA

102

View full text Add to dashboard Cite

Bacterial small RNAs (sRNAs) modulate gene expression by base-pairing with target mRNAs. Many sRNAs require the Sm-like RNA binding protein Hfq as a cofactor. Well-characterized interactions between DsrA sRNA and the rpoS mRNA leader were used to understand how Hfq stimulates sRNA pairing with target mRNAs. DsrA annealing stimulates expression of rpoS by disrupting a secondary structure in the rpoS leader, which otherwise prevents translation. Both RNAs bind Hfq with similar affinity but interact with opposite faces of the Hfq hexamer. Using mutations that block interactions between two of the three components, we demonstrate that Hfq binding to a functionally critical (AAN) 4 motif in rpoS mRNA rescues DsrA binding to a hyperstable rpoS mutant. We also show that Hfq cannot stably bridge the RNAs. Persistent ternary complexes only form when the two RNAs are complementary. Thus, Hfq mainly acts by binding and restructuring the rpoS mRNA. However, Hfq binding to DsrA is needed for maximum annealing in vitro, indicating that transient interactions with both RNAs contribute to the regulatory mechanism.

show abstract

Effect of salt and RNA structure on annealing and strand displacement by Hfq

Cited by 41 publications

References 44 publications

Positive regulation by small RNAs and the role of Hfq

Positive regulation by small RNAs and the role of Hfq

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

Major role for mRNA binding and restructuring in sRNA recruitment by Hfq

Contact Info

Product

Resources

About