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Hfq-bridged ternary complex is important for translation activation of rpoS by DsrA.

Wang W, Wang L, Wu J, Gong Q, Shi Y - Nucleic Acids Res. (2013)

Bottom Line: Ternary complex has been further verified in solution by NMR.In vivo, activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex.This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA-mRNA annealing process.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
The rpoS mRNA, which encodes the master regulator σ(S) of general stress response, requires Hfq-facilitated base pairing with DsrA small RNA for efficient translation at low temperatures. It has recently been proposed that one mechanism underlying Hfq action is to bridge a transient ternary complex by simultaneously binding to rpoS and DsrA. However, no structural evidence of Hfq simultaneously bound to different RNAs has been reported. We detected simultaneous binding of Hfq to rpoS and DsrA fragments. Crystal structures of AU6A•Hfq•A7 and Hfq•A7 complexes were resolved using 1.8- and 1.9-Å resolution, respectively. Ternary complex has been further verified in solution by NMR. In vivo, activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex. This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA-mRNA annealing process.

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Global structures of AU6A•Hfq•A7 and Hfq•A7 complexes. In the AU6A•Hfq•A7 crystal, each asymmetric unit contained half of the Hfq hexamer. Biologically relevant assembly was generated according to crystallographic symmetry. (A) In the AU6A•Hfq•A7 structure, two A7 (red) molecules and one AU6A (blue) molecule are bound to each Hfq hexamer (gray) on distal and proximal sides, respectively. (B) Two A7 (red) molecules are bound to distal side of Hfq hexamer (gray) in the Hfq•A7 structure. (C) Clear density maps are observed for the two A7 molecules (red) in the AU6A•Hfq•A7 structure. (D) Part of AU6A in the AU6A•Hfq•A7 structure. (E) Electron densities for the two A7 molecules in the Hfq•A7 structure are also clearly observed. Difference maps Fo–Fc before inclusion of RNAs are shown as purple mesh (contoured at 2.0 σ), and 2Fo–Fc densities are shown as cyan mesh (contoured at 1.0 σ). The statistics of these two structures are shown in Supplementary Table S1.
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gkt276-F2: Global structures of AU6A•Hfq•A7 and Hfq•A7 complexes. In the AU6A•Hfq•A7 crystal, each asymmetric unit contained half of the Hfq hexamer. Biologically relevant assembly was generated according to crystallographic symmetry. (A) In the AU6A•Hfq•A7 structure, two A7 (red) molecules and one AU6A (blue) molecule are bound to each Hfq hexamer (gray) on distal and proximal sides, respectively. (B) Two A7 (red) molecules are bound to distal side of Hfq hexamer (gray) in the Hfq•A7 structure. (C) Clear density maps are observed for the two A7 molecules (red) in the AU6A•Hfq•A7 structure. (D) Part of AU6A in the AU6A•Hfq•A7 structure. (E) Electron densities for the two A7 molecules in the Hfq•A7 structure are also clearly observed. Difference maps Fo–Fc before inclusion of RNAs are shown as purple mesh (contoured at 2.0 σ), and 2Fo–Fc densities are shown as cyan mesh (contoured at 1.0 σ). The statistics of these two structures are shown in Supplementary Table S1.

Mentions: Hfq can bind to A-rich or U-rich ssRNA fragment using its distinct sides, indicating that Hfq is capable of simultaneously binding two types of short RNA strands. Ternary complex in which Hfq binds A-rich fragments on distal side and U-rich fragments on proximal side has been widely assumed (2,3,15,18,19,24,37–39). However no such kind of ternary complex structure has yet been reported. In the present research, we used Hfq65 to co-crystallize with a poly (A) fragment A7, or A7 together with AU6A ssRNA, and two high-resolution complex structures were obtained. The final structure model of the AU6A•Hfq•A7 ternary complex was refined to Rwork and Rfree values of 18.8 and 22.6%, respectively, at 1.8-Å resolution. The Hfq•A7 structure was refined to Rwork and Rfree values of 19.0 and 23.1%, respectively, at 1.9-Å resolution. The statistics of these two structures are shown in Supplementary Table S1. In AU6A•Hfq•A7 complex structure, each asymmetric unit contains three Hfq subunits, one A7 strand and 2 uridine nucleotides. The biological relevant assembly was generated according to crystallographic symmetry (Figure 2A). Two A7 strands are also observed bound on each Hfq hexamer in the Hfq•A7 structure (Figure 2B). Clear electron densities were observed for RNA fragments in both AU6A•Hfq•A7 and Hfq•A7 structures (Figure 2C–E).Figure 2.


Hfq-bridged ternary complex is important for translation activation of rpoS by DsrA.

Wang W, Wang L, Wu J, Gong Q, Shi Y - Nucleic Acids Res. (2013)

Global structures of AU6A•Hfq•A7 and Hfq•A7 complexes. In the AU6A•Hfq•A7 crystal, each asymmetric unit contained half of the Hfq hexamer. Biologically relevant assembly was generated according to crystallographic symmetry. (A) In the AU6A•Hfq•A7 structure, two A7 (red) molecules and one AU6A (blue) molecule are bound to each Hfq hexamer (gray) on distal and proximal sides, respectively. (B) Two A7 (red) molecules are bound to distal side of Hfq hexamer (gray) in the Hfq•A7 structure. (C) Clear density maps are observed for the two A7 molecules (red) in the AU6A•Hfq•A7 structure. (D) Part of AU6A in the AU6A•Hfq•A7 structure. (E) Electron densities for the two A7 molecules in the Hfq•A7 structure are also clearly observed. Difference maps Fo–Fc before inclusion of RNAs are shown as purple mesh (contoured at 2.0 σ), and 2Fo–Fc densities are shown as cyan mesh (contoured at 1.0 σ). The statistics of these two structures are shown in Supplementary Table S1.
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Related In: Results  -  Collection

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Show All Figures
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gkt276-F2: Global structures of AU6A•Hfq•A7 and Hfq•A7 complexes. In the AU6A•Hfq•A7 crystal, each asymmetric unit contained half of the Hfq hexamer. Biologically relevant assembly was generated according to crystallographic symmetry. (A) In the AU6A•Hfq•A7 structure, two A7 (red) molecules and one AU6A (blue) molecule are bound to each Hfq hexamer (gray) on distal and proximal sides, respectively. (B) Two A7 (red) molecules are bound to distal side of Hfq hexamer (gray) in the Hfq•A7 structure. (C) Clear density maps are observed for the two A7 molecules (red) in the AU6A•Hfq•A7 structure. (D) Part of AU6A in the AU6A•Hfq•A7 structure. (E) Electron densities for the two A7 molecules in the Hfq•A7 structure are also clearly observed. Difference maps Fo–Fc before inclusion of RNAs are shown as purple mesh (contoured at 2.0 σ), and 2Fo–Fc densities are shown as cyan mesh (contoured at 1.0 σ). The statistics of these two structures are shown in Supplementary Table S1.
Mentions: Hfq can bind to A-rich or U-rich ssRNA fragment using its distinct sides, indicating that Hfq is capable of simultaneously binding two types of short RNA strands. Ternary complex in which Hfq binds A-rich fragments on distal side and U-rich fragments on proximal side has been widely assumed (2,3,15,18,19,24,37–39). However no such kind of ternary complex structure has yet been reported. In the present research, we used Hfq65 to co-crystallize with a poly (A) fragment A7, or A7 together with AU6A ssRNA, and two high-resolution complex structures were obtained. The final structure model of the AU6A•Hfq•A7 ternary complex was refined to Rwork and Rfree values of 18.8 and 22.6%, respectively, at 1.8-Å resolution. The Hfq•A7 structure was refined to Rwork and Rfree values of 19.0 and 23.1%, respectively, at 1.9-Å resolution. The statistics of these two structures are shown in Supplementary Table S1. In AU6A•Hfq•A7 complex structure, each asymmetric unit contains three Hfq subunits, one A7 strand and 2 uridine nucleotides. The biological relevant assembly was generated according to crystallographic symmetry (Figure 2A). Two A7 strands are also observed bound on each Hfq hexamer in the Hfq•A7 structure (Figure 2B). Clear electron densities were observed for RNA fragments in both AU6A•Hfq•A7 and Hfq•A7 structures (Figure 2C–E).Figure 2.

Bottom Line: Ternary complex has been further verified in solution by NMR.In vivo, activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex.This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA-mRNA annealing process.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
The rpoS mRNA, which encodes the master regulator σ(S) of general stress response, requires Hfq-facilitated base pairing with DsrA small RNA for efficient translation at low temperatures. It has recently been proposed that one mechanism underlying Hfq action is to bridge a transient ternary complex by simultaneously binding to rpoS and DsrA. However, no structural evidence of Hfq simultaneously bound to different RNAs has been reported. We detected simultaneous binding of Hfq to rpoS and DsrA fragments. Crystal structures of AU6A•Hfq•A7 and Hfq•A7 complexes were resolved using 1.8- and 1.9-Å resolution, respectively. Ternary complex has been further verified in solution by NMR. In vivo, activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex. This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA-mRNA annealing process.

Show MeSH