Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

Robinson, Kirsten E.; Orans, Jillian; Kovach, Alexander R.; Link, Todd; Brennan, Richard G.

doi:10.1093/nar/gkt1171

Cited by 75 publications

(86 citation statements)

References 65 publications

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“…The CTD has been proposed to stabilize RNA·Hfq complexes via direct interactions with sRNAs (19,36,57) owing to the slightly weaker binding of sRNAs in the absence of the CTD (10, 25), which we have recapitulated (Table 1). However, the data presented in this paper do not support this stabilization model because the CTDs do not increase the affinity of RNA oligomers (A18-FAM and D16-FAM) ( Table 1) and actually decrease the affinity of RNA stem loops (beacon and minRCRB) ( Table 1).…”

Section: Discussionsupporting

confidence: 55%

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: Discussionsupporting

confidence: 55%

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

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“…It binds uridine-rich sRNAs using its proximal face (Mikulecky et al 2004;Sauer and Weichenrieder 2011;Panja et al 2015) and the outer rim (Sauer et al 2012), and it binds mRNAs containing (ARN) n sequence motifs using its distal face (de Haseth and Uhlenbeck 1980;Link et al 2009). However, recent studies have suggested that other modes of RNA interactions with Hfq are also possible, both in Escherichia coli (Zhang et al 2013;Małecka et al 2015;Schu et al 2015) and in Gram-positive bacteria Robinson et al 2014).…”

Section: Introductionmentioning

confidence: 99%

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

Wróblewska

Olejniczak

2016

RNA

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The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5 ′ -untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5 ′ -untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs.

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“…In contrast, the crystal structure of Escherichia coli (Ec) Hfq bound to A 15 RNA showed polyadenosine binding to the opposite or "distal face" (Link et al 2009). Unlike the proximal face, the distal face of Ec Hfq has three distinct nucleobase binding sites per protomer, originally called the A-R-N motif, where A is an adenine-only binding site, R is a purine-nucleotide specificity site with a strong preference for adenine (Robinson et al 2014), and N is a nondiscriminatory solvent-exposed nucleotide that is the likely distal-face RNA entry and exit location. As a consequence of this motif, a maximum of 18 nt can readily occupy the distal face of Ec Hfq.…”

Section: Introductionmentioning

confidence: 99%

“…Fluorescence polarization-based Hfq-RNA binding measurements also indicate that magnesium plays a role in RNA binding to the proximal face of the protein. Intriguingly, RNA-Hfq tryptophan fluorescence quenching assays (Robinson et al 2014) suggest strongly that Lm Hfq is a more promiscuous RNA-binding protein than Hfq proteins from other Gram-positive bacteria.…”

Section: Introductionmentioning

confidence: 99%

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes

Kovach

Hoff

Canty

et al. 2014

RNA

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Hfq is a post-transcriptional regulator that binds U-and A-rich regions of sRNAs and their target mRNAs to stimulate their annealing in order to effect translation regulation and, often, to alter their stability. The functional importance of Hfq and its RNA-binding properties are relatively well understood in Gram-negative bacteria, whereas less is known about the RNAbinding properties of this riboregulator in Gram-positive species. Here, we describe the structure of Hfq from the Grampositive pathogen Listeria monocytogenes in its RNA-free form and in complex with a U 6 oligoribonucleotide. As expected, the protein takes the canonical hexameric toroidal shape of all other known Hfq structures. The U 6 RNA binds on the "proximal face" in a pocket formed by conserved residues Q9, N42, F43, and K58. Additionally residues G5 and Q6 are involved in protein-nucleic and inter-subunit contacts that promote uracil specificity. Unlike Staphylococcus aureus (Sa) Hfq, Lm Hfq requires magnesium to bind U 6 with high affinity. In contrast, the longer oligo-uridine, U 16 , binds Lm Hfq tightly in the presence or absence of magnesium, thereby suggesting the importance of additional residues on the proximal face and possibly the lateral rim in RNA interaction. Intrinsic tryptophan fluorescence quenching (TFQ) studies reveal, surprisingly, that Lm Hfq can bind (GU) 3 G and U 6 on its proximal and distal faces, indicating a less stringent adenine-nucleotide specificity site on the distal face as compared to the Gram-positive Hfq proteins from Sa and Bacillus subtilis and suggesting as yet uncharacterized RNA-binding modes on both faces.

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Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

Cited by 75 publications

References 65 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 assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes

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