RNA chaperone activity and RNA-binding properties of the E. coli protein StpA

The E. coli protein StpA has RNA annealing and strand displacement activities and it promotes folding of RNAs by loosening their structures. To understand the mode of action of StpA, we analysed the relationship of its RNA chaperone activity to its RNA-binding properties. For acceleration of annealing of two short RNAs, StpA binds both molecules simultaneously, showing that annealing is promoted by crowding. StpA binds weakly to RNA with a preference for unstructured molecules. Binding of StpA to RNA is strongly dependent on the ionic strength, suggesting that the interactions are mainly electrostatic. A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity. This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA. We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites

Dissecting RNA chaperone activity

Many RNA-binding proteins help RNAs to fold via their RNA chaperone activity. This term has been used widely without accounting for the diversity of the observed reactions, which include complex events like restructuring of misfolded catalytic RNAs, promoting the assembly of RNA-protein complexes, and mediating RNA–RNA interactions. Proteins display very diverse activities depending on the assays used to measure RNA chaperone activity. To classify proteins with this activity, we compared three exemplary proteins from E. coli, host factor Hfq, ribosomal protein S1, and the histone-like protein StpA for their abilities to promote two simple reactions, RNA annealing and strand displacement. The results of a FRET-based assay show that S1 promotes only RNA strand displacement while Hfq solely enhances RNA annealing. StpA, in contrast, is active in both reactions. To test whether the two activities can be assigned to different domains of the bipartite-structured StpA, we assayed the purified N- and C- terminal domains separately. While both domains are unable to promote RNA annealing, we can attribute the RNA strand displacement activity of StpA to the C-terminal domain. Correlating with their RNA annealing activities, only Hfq and full-length StpA display simultaneous binding of two RNAs, suggesting a matchmaker-like model for this activity. For StpA, this “RNA crowding” requires protein–protein interactions, since a dimerization-deficient StpA mutant lost the ability to bind and anneal two RNAs. These results underline the difference between the two reaction types, making it necessary to distinguish and classify proteins according to their specific RNA chaperone activities

Coupling RNA annealing and strand displacement: a FRET-based microplate reader assay for RNA chaperone activity

Proteins with RNA chaperone activity help RNAs to obtain their native conformations, and many of them are active in the two basic reactions—RNA annealing and strand displacement. Therefore, we developed a time-saving in vitro assay that detects protein-facilitated annealing and strand displacement of fluorophore-labeled oligoribonucleotides in a microplate reader. The two reactions are followed by fluorescence resonance energy transfer (FRET) in real-time, and the effect of the proteins on the reaction constants can be quantified. The high-throughput property of the fluorescence microplate reader, the kinetic characterization, and the material-saving aspect of this assay enables a fast and convenient classification of proteins according to their RNA chaperone activity in annealing and strand displacement

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 (Hfq(65)) 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

Interactions of adenines in Hfq-nucleotide complexes.

<p>(<b>A</b>) Hydrogen bond <b>i</b>nteraction between ribose 2′-OH with the Gly-29 carbonyl atom, for which a specific main chain conformation is required. ATP is shown as balls and sticks with the following color code for atoms: C – yellow; N - blue; O - red; P – orange. Hfq₆₅ residues involved in the interaction are presented by a stick. (<b>B</b>) Superposition of Hfq_Bs-AGAGAG (<i>B. subtilis</i> Hfq), Hfq-ADP (<i>E. coli</i> Hfq), Hfq-polyA (<i>E. coli</i> Hfq), Hfq_Pa-ADPNP (<i>P. aeruginosa</i> Hfq) and Hfq₆₅-ATP complexes to highlight the spread of purine ring orientations around the normal to the ring. Blue and red circles enclose two main clusters: in the first the exocyclic N6 atom hydrogen bonds to the Gln-52′ side-chain, in the second to the Thr-61 side-chain. N6 atoms are depicted as spheres. The colour code for atoms in the first cluster is: C - cyan; N - blue; O - red; P – orange; in the second C - yellow, and in the outlier ligand of the Hfq_Pa-ADPNP complex C - green.</p