Despite Similar Binding to the Hfq Protein Regulatory RNAs Widely Differ in Their Competition Performance

Olejniczak, Mikołaj

doi:10.1021/bi102043f

Cited by 70 publications

(137 citation statements)

References 55 publications

(236 reference statements)

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“…Consistently, the sRNAs studied here do not contain ARN repeats (Fig. 1B), which direct other sRNAs to the distal face of Hfq (Olejniczak 2011;Małecka et al 2015;Schu et al 2015). Overall, these data suggest that Hfq could use the proximal surface of its ring to recruit RybB, SdsR, and MicC sRNAs toward the ompD mRNA coding sequence.…”

Section: Hfq Binds Tightly To the Ompd Mrnasupporting

confidence: 80%

“…The binding of Hfq to sRNAs RybB, SdsR, and MicC was mainly dependent on the proximal face contacts (Table 1). These sRNAs bound to wt Hfq with very tight affinities similar to those determined before for E. coli sRNAs (Olejniczak 2011). The Y25D mutation in the distal face of Hfq did not affect their binding, while the K56A mutation in the proximal face resulted in up to 100-fold weaker binding.…”

Section: Hfq Binds Tightly To the Ompd Mrnasupporting

confidence: 79%

“…To obtain templates for synthesis of mutated ompD mRNA fragments, the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and specific primers (Supplemental Table S3) were used to introduce mutations into the ompD sequence in the pGEM T-Easy plasmid. After transcription the RNA molecules were purified using denaturing gel electrophoresis as previously described (Olejniczak 2011). RNAs were 5 ′ -32 P-labeled using T4 polynucleotide kinase or 3 ′ end labeled with RNA ligase, which was followed by phenol-chloroform extraction, gel purification, and ethanol precipitation.…”

Section: Rna Preparationmentioning

confidence: 99%

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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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Section: Hfq Binds Tightly To the Ompd Mrnasupporting

confidence: 80%

Section: Hfq Binds Tightly To the Ompd Mrnasupporting

confidence: 79%

Section: Rna Preparationmentioning

confidence: 99%

See 1 more Smart Citation

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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“…2) and improves binding of structured RNAs to the rim (Table 1), we next asked whether the CTD influences competition between different sRNAs for Hfq. The competition of sRNAs for Hfq has been attributed to a combination of sRNA secondary structure and sequence (12,35,43), and the ability of one sRNA to outcompete another does not necessarily correspond to their relative binding affinities (35,44), consistent with active displacement of bound sRNAs by other sRNAs in solution (37).…”

Section: Resultsmentioning

confidence: 99%

“…3 A-C). The sRNA competitors included DsrA, a Class I sRNA that is a poor competitor (12,35,44), RyhB (Class I), RprA, which has an AAN sequence like Class II sRNAs but exhibits a mixed Class I/Class II phenotype in E. coli (31), and ChiX (Class II). The fraction of 32 P-ChiX bound to either Hfq102 (Fig.…”

Section: Resultsmentioning

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

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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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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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Despite Similar Binding to the Hfq Protein Regulatory RNAs Widely Differ in Their Competition Performance

Cited by 70 publications

References 55 publications

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

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

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

Hfq chaperone brings speed dating to bacterial sRNA

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