The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs.
ABSTRACT: MgrR is an Hfq-dependent sRNA, whose transcription is controlled by the level of Mg2+ ions in Escherichia coli MgrR belongs to Class II sRNAs because its stability in the cell is affected by mutations in Hfq differently than canonical, Class I sRNAs. Here, we examined the effect of mutations in RNA binding sites of Hfq on the kinetics of the annealing of MgrR to two different target mRNAs, eptB and ygdQ, by global data fitting of the reaction kinetics monitored by gel electrophoresis of intermediates and products. The data showed that the mutation on the rim of the Hfq ring trapped MgrR on Hfq preventing the annealing of MgrR to either mRNA. The mutation in the distal face slowed the ternary complex formation and affected the release of MgrR-mRNA complexes from Hfq, while the mutation in the proximal face weakened the MgrR binding to Hfq and in this way affected the annealing. Moreover, competition assays established that MgrR bound to both faces of Hfq and competed against other sRNAs. Further studies showed that uridine-rich sequences located in less structurally stable regions served as Hfq binding sites in each mRNA. Overall, the data show that the binding of MgrR sRNA to both faces of the Hfq ring enables it to efficiently anneal to target mRNAs. It also confers on MgrR a competitive advantage over other sRNAs, which could contribute to efficient cellular response to changes in magnesium homeostasis.
Project description: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.
Project description:The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity. Mutations in the arginine patch lower the ability of Hfq to act in sRNA regulation of rpoS translation and eliminate annealing of natural sRNAs or unstructured oligonucleotides, without preventing binding to either the proximal or distal face. Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines. RNAs bound to either face of Hfq quench the fluorescence of a tryptophan adjacent to the arginine patch, demonstrating that the rim can simultaneously engage two RNA strands. We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq's chaperone function.
Project description:Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.
Project description:Regulation of bacterial gene networks by small non-coding RNAs (sRNAs) requires base pairing with messenger RNA (mRNA) targets, which is facilitated by Hfq protein. Hfq is recruited to sRNAs and mRNAs through U-rich- and A-rich-binding sites, respectively, but their distance from the sRNA-mRNA complementary region varies widely among different genes. To determine whether distance and binding orientation affect Hfq's chaperone function, we engineered 'toy' RNAs containing strong Hfq-binding sites at defined distances from the complementary target site. We show that RNA annealing is fastest when the distal face of Hfq binds an A-rich sequence immediately 3' of the target. This recruitment advantage is lost when Hfq binds >20?nt away from the target, but is partially restored by secondary structure that shortens this distance. Although recruitment through Hfq's distal face accelerates RNA annealing, tight binding of six Us to Hfq's proximal face inhibits annealing. Finally, we show that ectopic A-rich motifs dramatically accelerate base pairing between DsrA sRNA and a minimal rpoS mRNA in the presence of Hfq, demonstrating that proximity and orientation predict the activity of Hfq on long RNAs.
Project description:The Escherichia coli stationary phase transcription factor RpoS is translated in response to small noncoding RNAs (sRNAs), which base pair with the rpoS mRNA leader. The bacterial Sm-like protein Hfq anneals sRNAs with their mRNA targets by simultaneously binding the mRNA and sRNA. Intriguingly, Hfq is recruited to the rpoS leader via AAN motifs far upstream of the sRNA. SHAPE (selective 2'-hydroxyl acylation and primer extension) chemical footprinting showed that the rpoS leader is divided into a far upstream domain, an Hfq binding domain, and a downstream inhibitory stem-loop containing the sRNA and ribosome binding sites. To investigate how Hfq promotes sRNA-mRNA base pairing from a distance, we deleted the natural AAN Hfq binding site, and we inserted artificial AAN binding sites at various positions in the rpoS leader. All the relocated AAN motifs restored tight Hfq binding in vitro, but only insertion at the natural position restored Hfq-dependent sRNA annealing in vitro and sRNA regulation of rpoS translation in vivo. Furthermore, U-rich motifs in the downstream inhibitory domain stabilized the rpoS mRNA-Hfq complex and contributed to regulation of rpoS expression. We propose that the natural Hfq binding domain is optimal for positive regulation because it recruits Hfq to the mRNA and allows it to act on incoming sRNAs without opening the inhibitory stem-loop when sRNA is absent.
Project description:In bacteria, small RNAs (sRNAs) silence or activate target genes through base pairing with the mRNA, thereby modulating its translation. A central player in this process is the RNA chaperone Hfq, which facilitates the annealing of sRNAs with their target mRNAs. Hfq has two RNA-binding surfaces that recognize A-rich and U-rich sequences, and is believed to bind an sRNA-mRNA pair simultaneously. However, how Hfq promotes annealing remains unclear. Here, the crystal structure of Escherichia coli Hfq is presented in complex with U6-RNA bound to its proximal binding site at 0.97?Å resolution, revealing the Hfq-RNA interaction in exceptional detail.
Project description:Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and translation. Hfq, a ring-shaped homohexamer, has multiple faces that can bind both sRNAs and their mRNA targets. We find that Hfq has at least two distinct ways in which it interacts with sRNAs; these different binding properties have strong effects on the stability of the sRNA in vivo and the sequence requirements of regulated mRNAs. Class I sRNAs depend on proximal and rim Hfq sites for stability and turn over rapidly. Class II sRNAs are more stable and depend on the proximal and distal Hfq sites for stabilization. Using deletions and chimeras, we find that while Class I sRNAs regulate mRNA targets with previously defined ARN repeats, Class II sRNAs regulate mRNAs carrying UA-rich rim-binding sites. We discuss how these different binding modes may correlate with different roles in the cell, with Class I sRNAs acting as emergency responders and Class II sRNAs acting as silencers.
Project description:A major class of small bacterial RNAs (sRNAs) regulate translation and mRNA stability by pairing with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq, related to the Lsm/Sm families of splicing proteins, binds the sRNAs and stabilizes them in vivo and stimulates pairing with mRNAs in vitro. Although Hfq is abundant, the sRNAs, when induced, are similarly abundant. Therefore, Hfq may be limiting for sRNA function. We find that, when overexpressed, a number of sRNAs competed with endogenous sRNAs for binding to Hfq. This correlated with lower accumulation of the sRNAs (presumably a reflection of the loss of Hfq binding), and lower activity of the sRNAs in regulating gene expression. Hfq was limiting for both positive and negative regulation by the sRNAs. In addition, deletion of the gene for an expressed and particularly effective competitor sRNA improved the regulation of genes by other sRNAs, suggesting that Hfq is limiting during normal growth conditions. These results support the existence of a hierarchy of sRNA competition for Hfq, modulating the function of some sRNAs.
Project description:Small RNAs (sRNAs) regulate diverse pathways, including stress responses, virulence, and metabolism in Escherichia coli. At the center of this large sRNA regulatory network is the Hfq protein. Hfq mediates the binding of sRNAs to their target mRNAs; without Hfq, most sRNAs cannot efficiently regulate target mRNA expression. Here, we show in vivo that Hfq can be a limiting factor for sRNA activity and that it can be easily depleted, causing disruption of the sRNA network. Depletion of the available Hfq can occur when sRNAs and target mRNAs are transcribed at high levels without their partners, resulting in the sequestration of Hfq into sRNA-Hfq and target mRNA-Hfq complexes. This can be avoided by coordinating the transcription of sRNAs with their target mRNAs so that they are turned on and off together to maximize duplex formation and minimize Hfq sequestration. Therefore, the limited availability of Hfq results in a highly interdependent sRNA network, wherein the activity of each sRNA depends on the activity of the other sRNAs and target mRNAs in the network.
Project description: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.