Hfq-RNA Complex Research Articles

MD simulations reveal the basis for dynamic assembly of Hfq–RNA complexes

The conserved protein Hfq is a key factor in the RNA-mediated control of gene expression in most known bacteria. The transient intermediates Hfq forms with RNA support intricate and robust regulatory networks. In Pseudomonas, Hfq recognizes repeats of adenine–purine–any nucleotide (ARN) in target mRNAs via its distal binding side, and together with the catabolite repression control (Crc) protein, assembles into a translation–repression complex. Earlier experiments yielded static, ensemble-averaged structures of the complex, but details of its interface dynamics and assembly pathway remained elusive. Using explicit solvent atomistic molecular dynamics simulations, we modeled the extensive dynamics of the Hfq–RNA interface and found implications for the assembly of the complex. We predict that syn/anti flips of the adenine nucleotides in each ARN repeat contribute to a dynamic recognition mechanism between the Hfq distal side and mRNA targets. We identify a previously unknown binding pocket that can accept any nucleotide and propose that it may serve as a ‘status quo’ staging point, providing nonspecific binding affinity, until Crc engages the Hfq–RNA binary complex. The dynamical components of the Hfq–RNA recognition can speed up screening of the pool of the surrounding RNAs, participate in rapid accommodation of the RNA on the protein surface, and facilitate competition among different RNAs. The register of Crc in the ternary assembly could be defined by the recognition of a guanine-specific base–phosphate interaction between the first and last ARN repeats of the bound RNA. This dynamic substrate recognition provides structural rationale for the stepwise assembly of multicomponent ribonucleoprotein complexes nucleated by Hfq–RNA binding.

Intermolecular base stacking mediates RNA-RNA interaction in a crystal structure of the RNA chaperone Hfq

The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via their bound RNA molecules. The structure contains two Hfq6:A18 RNA assemblies positioned face-to-face, with the RNA molecules turned towards each other and connected via interdigitating base stacking interactions at the center. Biochemical data further confirm the observed interaction, and indicate that RNA-mediated contacts occur between Hfq-RNA complexes with various (ARN)X motif containing RNA sequences in vitro, including the stress response regulator OxyS and its target, fhlA. A systematic computational survey also shows that phylogenetically conserved (ARN)X motifs are present in a subset of sRNAs, some of which share similar modular architectures. We hypothesise that Hfq can co-opt RNA-RNA base stacking, an unanticipated structural trick, to promote the interaction of (ARN)X motif containing sRNAs with target mRNAs on a “speed-dating” fashion, thereby supporting their regulatory function.

Modulation of Sinorhizobium meliloti quorum sensing by Hfq-mediated post-transcriptional regulation of ExpR.

In Sinorhizobium meliloti, the timing of quorum sensing (QS)-dependent gene expression is controlled at multiple levels. RNA binding protein Hfq contributes to the regulation of QS signal production, and this regulation is exerted both in the manner that involves the acyl homoserine lactone receptor ExpR, and via expR-independent mechanisms. In the expR+ strain of S. meliloti, deletion of hfq resulted in the hyper-accumulation of QS signals at low population densities, increased diversity of the QS signals in mid-to-late exponential phase and then led to a sharp decrease in QS signal accumulation in stationary phase. Quantitative polymerase chain reaction revealed that the accumulation of expR and sinI (but not sinR) mRNA was increased in the late exponential phase in an hfq-dependent manner. A translational, but not transcriptional, expR-uidA reporter was controlled by hfq, while both transcriptional and translational sinI-uidA reporters were regulated in the hfq-dependent manner. In co-immunoprecipation experiments, expR mRNA was bound to and then released from Hfq, similar to the positive controls (small regulatory RNA SmrC9, SmrC15, SmrC16 and SmrC45). Neither sinI nor sinR transcripts were detected in the pool of RNA heat-released from Hfq-RNA complexes. Therefore, post-transcriptional regulator Hfq controls the production and perception of QS signals, and at higher population densities this control is mediated directly via interactions with expR.

Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5′-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.

RNA Type III Secretion Signals That Require Hfq

Salmonella virulence is largely mediated by two type III secretion systems (T3SS) that deliver effector proteins from the bacterium to a host cell; however, the secretion signal is poorly defined. Effector N termini are thought to contain the signal, but they lack homology, possess no identifiable motif, and adopt intrinsically disordered structures. Alternative studies suggest that RNA-encoded signals may also be recognized and that they can be located in the 5' untranslated leader sequence. We began our study by establishing the minimum sequence required for reporter translocation. Untranslated leader sequences predicted from 42 different Salmonella effector proteins were fused to the adenylate cyclase reporter (CyaA'), and each of them was tested for protein injection into J774 macrophages. RNA sequences derived from five effectors, gtgA, cigR, gogB, sseL, and steD, were sufficient for CyaA' translocation into host cells. To determine the mechanism of signal recognition, we identified proteins that bound specifically to the gtgA RNA. One of the unique proteins identified was Hfq. Hfq had no effect upon the translocation of full-length CigR and SteD, but injection of intact GtgA, GogB, and SseL was abolished in an hfq mutant, confirming the importance of Hfq. Our results demonstrated that the Salmonella pathogenicity island 2 (SPI-2) T3SS assembled into a functional apparatus independently of Hfq. Since particular effectors required Hfq for translocation, Hfq-RNA complexes may participate in signal recognition.

Structural and Biochemical Studies on ATP Binding and Hydrolysis by the Escherichia coli RNA Chaperone Hfq

In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs) and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A)15 and ADP were shown to bind to tripartite binding motifs (ARE) circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq65) in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions.

Rapid binding and release of Hfq from ternary complexes during RNA annealing

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼108 M−1s−1) observed with Cy3-Hfq was followed by a slow transition (0.5 s−1) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions.

Computational approach to ensure the stability of the favorable ATP binding site in E. coli Hfq

Bacterial Hfq is a highly conserved thermostable protein of about 10 kDa. The Hfq protein was discovered in 1968 as an E. coli host factor that was essential for replication of the bacteriophage Qβ. It is now clear that Hfq has many important physiological roles. In E. coli, Hfq mutants show a multiple stress response related phenotypes. Hfq is now known to regulate the translation of two major stress transcription factors RpoS and RpoE in Enterobacteria and mediates its plieotrophic effects through several mechanisms. It interacts with regulatory sRNA and facilitates their antisense interaction with their targets. It also acts independently to modulate mRNA decay and in addition acts as a repressor of mRNA translation. Recent paper from Arluison et al. [9] provided the first evidence indicating that Hfq is an ATP-binding protein. They determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. Experimental data suggest that the ATP-binding by the Hfq–RNA complex results in its significant destabilization of the protein and the result also proves important role of Tyr25 that flanks the cleft and stabilizes the adenine portion of ATP, possibly via aromatic stacking. In our study, the ATP molecule was docked into the predicted binding cleft using GOLD docking software. The binding nature of ATP and its effect on Hfq–RNA complex was studied using molecular dynamics simulations. Importance of Tyr25 residue was monitored and revealed using mutational study on the modeled systems. Our data and the corresponding results point to one of Hfq functional structural consequences due to ATP binding and Tyr25Ala mutation.

Molecular Dynamics Simulation Study for Ionic Strength Dependence of RNA-host factor Interaction in Staphylococcus aureus Hfq

The behavior of peptide or protein solutes in saline aqueous solution is a fundamental topic in physical chemistry. Addition of ions can strongly alter the thermodynamic and physical properties of peptide molecules in solution. In order to study the effects of added ionic salts on protein conformation and dynamics, we have used the molecular dynamics (MD) simulations to investigate the behavior of Staphylococcus aureus Hfq protein under two different ionic concentrations: 0.1 M NaCl and 1.0 M NaCl in presence and absence of RNA (a hepta-oligoribonucleotide AU5G). Hfq, a global regulator of gene expression is highly conserved and abundant RNA-binding protein. It is already reported that in vivo the increase of ionic strength results in a drastic reduction of Hfq affinity for <TEX>$Q{\beta}$</TEX> RNA and reduces the tendency of aggregation of Escherichia coli host factor hexamers. Our results revealed the crucial role of 0.1 M NaCl Hfq system on the bases with strong hydrogen bonding interactions and by stabilizing the aromatic stacking of Tyr42 residue of the adjacent subunits/monomers with the adenine and uridine nucleobases. An increase in RNA pore diameter and weakened compactness of the Hfq-RNA complex was clearly observed in 1.0 M NaCl Hfq system with bound RNA. Aggregation of monomers in Hfq and the interaction of Hfq with RNA are greatly affected due to the presence of high ionic strength. Higher the ionic concentration, weaker is the aggregation and interaction. Our results were compatible with the experimental data and this is the first theoretical report for the experimental study done in 1980 by Uhlenbeck group for the present system.

Molecular modeling study on the effect of residues distant from the nucleotide-binding portion on RNA binding in Staphylococcus aureus Hfq

Hfq is an abundant RNA-binding bacterial protein that was first identified in E. coli as a required host factor for phage Qβ RNA replication. The pleiotrophic phenotype resulting from the deletion of Hfq predicates the importance of this protein. Two RNA-binding sites have been characterized: the proximal site which binds sRNA and mRNA and the distal site which binds poly(A) tails. Previous studies mainly focused on the key residues in the proximal site of the protein. A recent mutation study in E. coli Hfq showed that a distal residue Val43 is important for the protein function. Interestingly, when we analyzed the sequence and structure of Staphylococcus aureus Hfq using the CONSEQ server, the results elicited that more functional residues were located far from the nucleotide-binding portion (NBP). From the analysis seven individual residues Asp9, Leu12, Glu13, Lys16, Gln31, Gly34 and Asp40 were selected to investigate the conformational changes in Hfq–RNA complex due to point mutation effect of those residues using molecular dynamics simulations. Results showed a significant effect on Asn28 which is an already known highly conserved functionally important residue. Mutants D9A, E13A and K16A depicted effects on base stacking along with increase in RNA pore diameter, which is required for the threading of RNA through the pore for the post-translational modification. Further, the result of protein stability analysis by the CUPSAT server showed destabilizing effect in the most mutants. From this study we characterized a series of important residues located far from the NBP and provide some clues that those residues may affect sRNA binding in Hfq.

Sm‐like protein Hfq: Location of the ATP‐binding site and the effect of ATP on Hfq–RNA complexes

Sm-like proteins are ubiquitous ring-shaped oligomers that exhibit a variety of nucleic acid-binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm-like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP-binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq-RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq-bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm-like proteins.

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).