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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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Co-binding of rpoS and DsrA to Hfq. (A) Co-binding of Hfq to DsrA sRNA and rpoS mRNA is a possible mechanism of Hfq in mediating DsrA-dependent rpoS translation activation. The A-rich Hfq-binding sequence on rpoS, rpoS-AA [nucleotides 366–400, containing an (AAN)4 and an A6 element], is colored red. The fragment containing U-rich Hfq-binding site and stem loop II of DsrA, DsrAII (nucleotides 26–61, containing the AU6A U-rich Hfq-binding site), is shown in blue. Regions on both RNAs for base paring to each other is colored in green. In EMSA experiment using HfqFL and fluorescence-labeled RNAs, we have observed (B) a supershift to Hfq•rpoS-AA (rpoS-AA was labeled with fluorescent probe) complex on addition of DsrAII and (C) a supershift to DsrAII•Hfq (DsrAII was labeled with fluorescent probe) complex on addition of rpoS-AA, suggesting that a DsrAII•Hfq•rpoS-AA ternary complex may form. Unbound rpoS-AA RNA migrates as two bands (Supplementary Figure S4). Brightness, contrast and gamma adjustments were applied to the whole image. Full images of Figure 1B and C showed in Supplementary Figure S5.
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gkt276-F1: Co-binding of rpoS and DsrA to Hfq. (A) Co-binding of Hfq to DsrA sRNA and rpoS mRNA is a possible mechanism of Hfq in mediating DsrA-dependent rpoS translation activation. The A-rich Hfq-binding sequence on rpoS, rpoS-AA [nucleotides 366–400, containing an (AAN)4 and an A6 element], is colored red. The fragment containing U-rich Hfq-binding site and stem loop II of DsrA, DsrAII (nucleotides 26–61, containing the AU6A U-rich Hfq-binding site), is shown in blue. Regions on both RNAs for base paring to each other is colored in green. In EMSA experiment using HfqFL and fluorescence-labeled RNAs, we have observed (B) a supershift to Hfq•rpoS-AA (rpoS-AA was labeled with fluorescent probe) complex on addition of DsrAII and (C) a supershift to DsrAII•Hfq (DsrAII was labeled with fluorescent probe) complex on addition of rpoS-AA, suggesting that a DsrAII•Hfq•rpoS-AA ternary complex may form. Unbound rpoS-AA RNA migrates as two bands (Supplementary Figure S4). Brightness, contrast and gamma adjustments were applied to the whole image. Full images of Figure 1B and C showed in Supplementary Figure S5.

Mentions: In the process of Hfq-facilitated base pairing between DsrA and rpoS, an intermediate ternary complex in which Hfq simultaneously binds to DsrA and rpoS on proximal and distal sides, respectively, has been suggested crucial for the activity of Hfq (Figure 1A). Because Hfq cannot stably bridge DsrA and rpoS if the two RNAs are not base paired (3,19), to capture the transient ternary complex bridged by Hfq between DsrA and rpoS, we selected two non–base-paired RNA fragments, DsrAII and rpoS-AA, to represent DsrA and rpoS for further investigation. DsrAII, a 37-nt portion of DsrA, contains neither the A-rich sequence preceding AU6A nor the region for base pairing with rpoS (besides the few nucleotides required for Hfq binding, it also contains one additional G residue from the T7 promoter at the 5′ end). In contrast, rpoS-AA represents nucleotides 366–400 of rpoS, which contains the A-rich Hfq-binding tract but not the region recognized by DsrA (Figure 1A).Figure 1.


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)

Co-binding of rpoS and DsrA to Hfq. (A) Co-binding of Hfq to DsrA sRNA and rpoS mRNA is a possible mechanism of Hfq in mediating DsrA-dependent rpoS translation activation. The A-rich Hfq-binding sequence on rpoS, rpoS-AA [nucleotides 366–400, containing an (AAN)4 and an A6 element], is colored red. The fragment containing U-rich Hfq-binding site and stem loop II of DsrA, DsrAII (nucleotides 26–61, containing the AU6A U-rich Hfq-binding site), is shown in blue. Regions on both RNAs for base paring to each other is colored in green. In EMSA experiment using HfqFL and fluorescence-labeled RNAs, we have observed (B) a supershift to Hfq•rpoS-AA (rpoS-AA was labeled with fluorescent probe) complex on addition of DsrAII and (C) a supershift to DsrAII•Hfq (DsrAII was labeled with fluorescent probe) complex on addition of rpoS-AA, suggesting that a DsrAII•Hfq•rpoS-AA ternary complex may form. Unbound rpoS-AA RNA migrates as two bands (Supplementary Figure S4). Brightness, contrast and gamma adjustments were applied to the whole image. Full images of Figure 1B and C showed in Supplementary Figure S5.
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Related In: Results  -  Collection

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gkt276-F1: Co-binding of rpoS and DsrA to Hfq. (A) Co-binding of Hfq to DsrA sRNA and rpoS mRNA is a possible mechanism of Hfq in mediating DsrA-dependent rpoS translation activation. The A-rich Hfq-binding sequence on rpoS, rpoS-AA [nucleotides 366–400, containing an (AAN)4 and an A6 element], is colored red. The fragment containing U-rich Hfq-binding site and stem loop II of DsrA, DsrAII (nucleotides 26–61, containing the AU6A U-rich Hfq-binding site), is shown in blue. Regions on both RNAs for base paring to each other is colored in green. In EMSA experiment using HfqFL and fluorescence-labeled RNAs, we have observed (B) a supershift to Hfq•rpoS-AA (rpoS-AA was labeled with fluorescent probe) complex on addition of DsrAII and (C) a supershift to DsrAII•Hfq (DsrAII was labeled with fluorescent probe) complex on addition of rpoS-AA, suggesting that a DsrAII•Hfq•rpoS-AA ternary complex may form. Unbound rpoS-AA RNA migrates as two bands (Supplementary Figure S4). Brightness, contrast and gamma adjustments were applied to the whole image. Full images of Figure 1B and C showed in Supplementary Figure S5.
Mentions: In the process of Hfq-facilitated base pairing between DsrA and rpoS, an intermediate ternary complex in which Hfq simultaneously binds to DsrA and rpoS on proximal and distal sides, respectively, has been suggested crucial for the activity of Hfq (Figure 1A). Because Hfq cannot stably bridge DsrA and rpoS if the two RNAs are not base paired (3,19), to capture the transient ternary complex bridged by Hfq between DsrA and rpoS, we selected two non–base-paired RNA fragments, DsrAII and rpoS-AA, to represent DsrA and rpoS for further investigation. DsrAII, a 37-nt portion of DsrA, contains neither the A-rich sequence preceding AU6A nor the region for base pairing with rpoS (besides the few nucleotides required for Hfq binding, it also contains one additional G residue from the T7 promoter at the 5′ end). In contrast, rpoS-AA represents nucleotides 366–400 of rpoS, which contains the A-rich Hfq-binding tract but not the region recognized by DsrA (Figure 1A).Figure 1.

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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