Structural insights into the recognition of the internal A-rich linker from OxyS sRNA by Escherichia coli Hfq

Abstract

Small RNA OxyS is induced during oxidative stress in Escherichia coli and it is an Hfq-dependent negative regulator of mRNA translation. OxyS represses the translation of fhlA and rpoS mRNA, which encode the transcriptional activator and σ(s) subunit of RNA polymerase, respectively. However, little is known regarding how Hfq, an RNA chaperone, interacts with OxyS at the atomic level. Here, using fluorescence polarization and tryptophan fluorescence quenching assays, we verified that the A-rich linker region of OxyS sRNA binds Hfq at its distal side. We also report two crystal structures of Hfq in complex with A-rich RNA fragments from this linker region. Both of these RNA fragments bind to the distal side of Hfq and adopt a different conformation compared with those previously reported for the (A-R-N)n tripartite recognition motif. Furthermore, using fluorescence polarization, electrophoresis mobility shift assays and in vivo translation assays, we found that an Hfq mutant, N48A, increases the binding affinity of OxyS for Hfq in vitro but is defective in the negative regulation of fhlA translation in vivo, suggesting that the normal function of OxyS depends on the details of the interaction with Hfq that may be related to the rapid recycling of Hfq in the cell.

Figure 1.

The A-rich fragments from the linker region of OxyS bind to the distal side of Hfq. (A) A schematic diagram showing the secondary structure of OxySU8. This structure is based on the experimentally verified secondary structure of OxyS (4), which is in close agreement with the predicted secondary structure of OxySU8 RNA using Mfold (43). The two segments selected for cocrystallization with Hfq, namely A_us and A_ds, represent nucleotides 66–72 and nucleotides 68–74 of OxyS, respectively. OxySU8-A10dele (deletion of nucleotides 65–74) and OxySU8-A6U (A65U/A66U/A68U/A69U/A73U/A74U) mutants as indicated. Based on the Mfold prediction (43), the secondary structures of both mutants are similar to that of wild-type OxySU8. (B, C) Fluorescence polarization assay to determine the binding affinities of A_us and A_ds for wild-type Hfq and mutants. A mutation on the distal side, Y25A, dramatically decreased the binding affinity of both A_us and A_ds, whereas the proximal side mutation, F42S, did not exhibit a prominent effect. (D, E, F) TFQ experiments for Hfq Trp mutants by OxySU8, OxySU8-A10dele and OxySU8-A6U. F42W represents the proximal RNA binding site, and Y25W and K31W represent the distal RNA binding sites. The black bar represents the percent quenching by 1-μM RNA, whereas the gray bar above the black bar represents the quenching by 4-μM RNA.

Figure 2.

Global structures of the Hfq-A_us and Hfq-A_ds complexes. (A) Each Hfq65 hexamer (gray) binds to one A_us (orange) RNA fragment at the distal side in the structure of the Hfq65-A_us complex. (B) Clear electron density (difference maps 2F₀−F_c densities are shown as a cyan mesh contoured at 1.0σ) is observed for three of seven nucleotides (A68, A69 and C70; orange) in A_us. (C) Each Hfq hexamer binds to one A_ds (magenta) RNA fragment in the structure of the Hfq65-A_ds complex. (D) Electron density (difference maps 2F₀−F_c densities are shown as a cyan mesh contoured at 1.0σ) is observed for four of seven nucleotides of A_ds (U71, A72, A73 and A74; magenta). Three closely packed asymmetric units of Hfq65-A_us (E) and Hfq65-A_ds crystals (F) are shown. One hexamer is presented as gray carbon and the remaining two hexamers as blue or green Cα traces. A_us (orange) and A_ds (magenta) are represented as sticks. In the complex structures, C70 from A_us and A74 from A_ds both point outward from one Hfq distal side and bind at the ‘R-site’ on the distal side of a neighboring Hfq65 hexamer. Data collection and refinement statistics are summarized in Supplementary Table S1.

Figure 3.

A distinct RNA recognition mode in the Hfq-A_us and Hfq-A_ds complex structures compared with the Hfq-A₇ structure. Hfq is shown as a gray surface. (A) A_us and A_ds bind to Hfq in a similar conformation. A_us and A_ds are shown as orange and magenta sticks, respectively. A new nucleotide binding pocket close to the central pore of Hfq (formed near the interface of two adjacent Hfq subunits) is circled with a dotted line. (B) A_us and (C) A_ds binding at the distal side of Hfq differs from that of A₇ (blue sticks). Nucleotide U71 of A_ds binds to the adenosine selective site (‘A-site’) in the ‘A-R-N’ motif and is highlighted with a dotted circle in panel (C).

Figure 4.

Interactions with RNA on the distal face of Hfq. The carbon atoms of A_us (5′-AUAACUA-3′, nucleotides 66–72) and A_ds (5′-AACUAAA-3′ nucleotides 68–74) are colored orange and magenta, respectively. Hfq is shown as gray Cα traces except for the residues involved in RNA binding, which are shown as sticks. The carbon atoms of these residues are colored gray in one Hfq and yellow or green in two adjacent Hfq molecules. The prime sign ‘denotes residues from an adjacent Hfq65 subunit in the same hexamer. The superscripts ⁽²⁾ and ⁽³⁾ denote residues from the neighboring Hfq65 hexamers. (A, B) C70 of A_us and A74 of A_ds bind to the R-site of an adjacent Hfq hexamer. (C) A69 of A_us binds to a pocket on the distal side of Hfq close to the central pore. The adenosine ribosyl 2′-hydroxyl group of A69 interacts with the backbone amide of K31. The exocyclic N6 atom forms a hydrogen bond with the carbonyl oxygen groups of N28. (D) A68 of A_us binds to the R-site of Hfq. (E) U71 of A_ds binds to the A-site of Hfq via hydrogen bonds to Nδ of N48 and the backbone atom of Q33. (F) U71 is coordinated with A72 by two non-discriminating water-mediated hydrogen bonds.

Figure 5.

NMR chemical shift perturbations of Hfq by A_us and A_ds. Hfq65 R16A/R17A protein (0.1 mM) were titrated with A_us and A_ds, respectively. (A) Selected regions of ¹H-¹⁵N HSQC spectra of Hfq upon A_us (left) and A_ds (right) titration. (B) Chemical shift differences between the first and last titration points for A_us and A_ds are presented as green and red column bars, respectively. (C, E) Both A_us and A_ds binding to Hfq result in prominent chemical shift perturbations on the distal side of Hfq. (D, F) Residues Q8, Q41 and V43 on the proximal side of Hfq are also perturbed by A_us and A_ds titration. Hfq is colored according to chemical shift changes using a blue to red gradient. The resonances of F42 and H57, which disappeared upon A_us and A_ds titration, are colored purple. Unassigned residues are colored dark blue.

Figure 6.

The effects of Hfq mutations (N28A, K31A and N48A) on RNA binding affinities. (A) N28A (magenta), N48A (blue) and K31A (green) mutations dramatically decrease the binding affinity for A₇ to Hfq (compared with a K_d of ∼113 nM for wild-type Hfq; (19)). (B) The binding affinity of A_us RNA for N28A and K31A is both lower compared with that for wild type. In contrary, the N48A mutant exhibits higher affinity for A_us. (C) The N48A mutation dramatically increases the affinity for A_ds (∼16-fold) compared with wild type. (D) The binding affinity A_ds for the K31A mutant is significantly lower. The N28A mutation does not affect the binding of A_ds. In EMSA assays, compared to wild-type Hfq (E), the N48A mutant caused a prominent mobility shift of full-length OxySU8 at lower concentrations (F).

Figure 7.

An appropriate RNA binding affinity is important for Hfq regulation in vivo. (A) A schematic diagram of the reporter system used for in vivo translation assays. The DNA sequence encoding the fluorescent protein GFPuv is fused to the leader sequence of fhlA and is thus under the regulation of OxyS and Hfq. The level of GFPuv expression in this system will consequently reflect the translation level of the mRNA with this fhlA leader. (B) Deletion of hfq increases the GFPuv expression level (lane 1) compared with wild-type Hfq (lane 2). GFPuv expression in the presence of the Y25A mutant (lane 3) is similar to that in the hfq⁻ strain. Similarly, neither the N28A (lane 4) nor the K31A mutant (lane 5) was capable of suppressing GFPuv expression as wild-type Hfq. This finding is presumably due to the deficiency of these mutants in binding OxyS. The N48A mutant, which binds to OxyS more tightly than wild-type, also does not suppress the expression level of GFPuv (lane 6). GFPuv was stained with an anti-GFP antibody and GroEL was used as a loading control.