Hfq proximity and orientation controls RNA annealing

Abstract

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.

Figure 1.

Positive regulation of gene expression by Hfq and sRNAs. Secondary structure in the mRNA leader (gray) masks the ribosome-binding site and prevents initiation of translation. Expression of a complementary sRNA (blue) up-regulates translation by opening the mRNA and exposing the RBS. Hfq is recruited to (ARN)_n motifs in the mRNA (green) through its distal face, facilitating sRNA binding through its proximal face and by restructuring the leader (31). Hfq cycles off the sRNA–mRNA duplex and may remain bound to the (ARN)_n motif or transfer to other sequences in the mRNA. In this scheme, the Hfq-binding site (green) is far from sRNA-binding site in the mRNA.

Figure 2.

A-rich Hfq-binding site stimulates RNA annealing. (a) Toy RNAs for Hfq-annealing assays. Base pairing of a molecular beacon (blue) with 16 nt oligo C target RNA (magenta) increases FAM fluorescence intensity. Oligo CA and oligo CU contain A₁₈ and U₆ Hfq-binding sites, respectively. (b) Rates of RNA annealing were measured by stopped-flow fluorescence in TNK buffer at 30°C, using 50 nM beacon (rMBDss), 50 nM target RNA and 5–5000 nM Hfq monomer. Observed rate constants from five independent experiments were averaged and plotted against Hfq concentration.

Figure 3.

Position of Hfq-binding site is important for RNA annealing. (a) Scheme of target RNAs listed in Supplementary Table S1. (b) Observed rate constants (k_obs) obtained from the binding kinetics between the molecular beacon and different RNA targets (left) in 1 µM Hfq monomer. Rate constants from five independent experiments were averaged and standard deviations indicated by error bars. See Figures S3–S5 for additional data.

Figure 4.

Hfq distal face interaction is required. Observed annealing rates for 50 nM molecular beacon and 50 nM oligo CA in Hfq. The Y25D mutation on the distal face (red) was more deleterious than the K56A mutation on the proximal face (blue). To test for complementation, Y25D and K56A Hfq subunits were mixed 1:1 (green). Total Hfq concentrations were 5 nM, 1 µM or 5 µM monomer.

Figure 5.

Close Hfq distal binding sites improve sRNA binding to rpoS mRNA. (a) Secondary structure of minimal rpoS138 leader sequence with and without A₁₈ insertions. (b) DsrA and rpoS binding at 30°C was measured using radiolabeled DsrA and 200 nM rpoS138-5′A18 RNA in 1 µM Hfq. Samples were loaded on a 6% native gel from 0.1 to 60 min; gels were run continuously during the experiment. Control reactions with DsrA only, DsrA plus 200 nM rpoS, DsrA plus 1 µM Hfq and DsrA plus rpoS plus Hfq were incubated at 30°C for 2 h and loaded as indicated in the figure. D, free DsrA; D•H1, DsrA bound to one Hfq hexamer; D•H2, DsrA with two Hfq hexamers; D•R, DsrA•rpoS; D•R•H, DsrA•rpoS•Hfq ternary complex, which persists when the mRNA contains an A-rich-binding site for Hfq. (c) Observed rate constants for annealing; no Hfq (D•R, gray); binary complexes with Hfq (D•R, red); ternary complexes with Hfq (D•R•H, blue). Error bars indicate the standard deviation from the average of three trials.

Figure 6.

Model for Hfq recruitment to sRNA complementary region. Binding of Hfq’s distal face to an (ARN)_n motif facilitates interactions between the target region and the proximal face of Hfq by looping around the hexamer (a) or possibly by direct transfer from one site to another (b). Stable interactions between the proximal face and U₆ inhibit annealing (c).