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Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA

Abstract

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5′-A/UCANGGANGU/A-3′ sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.

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Figure 1: Secondary structure of RsmE and genetic organization of the hcnA 5′ untranslated mRNA.
Figure 2: NMR solution structure of the RsmE–hcnA RNA complex.
Figure 3: Details of the RsmE–hcnA RNA complex NMR structure.
Figure 4: In vivo and in vitro functional studies of RsmE-RNA interaction.

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Protein Data Bank

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Acknowledgements

We thank C. Dominguez and R. Stefl for help with the structure calculation; K. Starke (Université de Lausanne) for supplying pME6624, pME6629 and pME6638; D. Witmer (ETH Zürich) for assistance in transcribing RNA and S. Auweter, C. Maris, M. Blatter, A. Clery, C. Reimmann and R. Glockshuber for helpful discussions. This investigation was supported by grants from the Swiss National Science Foundation to D.H. and F.H.-T.A., from the Structural Biology National Center of Competence in Research to F.H.-T.A. and I.J. and from the Roche Research Fund for Biology at the ETH Zurich to F.H.-T.A. F.H.-T.A. is a European Molecular Biology Organization Young Investigator.

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Protein and RNA samples for structural studies were prepared by M.S., O.D. and F.C.O. Functional experiments and cloning were carried out by K.L. and analyzed by K.L. and D.H. NMR data were analyzed by M.S., O.D. and F.H.-T.A. ITC measurements were performed by I.J. The structure calculation and refinement was carried out by M.S. The manuscript was written by M.S., D.H. and F.H.-T.A.

Corresponding authors

Correspondence to Dieter Haas or Frédéric H-T Allain.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2, Supplementary Methods (PDF 5757 kb)

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Schubert, M., Lapouge, K., Duss, O. et al. Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA. Nat Struct Mol Biol 14, 807–813 (2007). https://doi.org/10.1038/nsmb1285

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