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Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch

Nature Structural & Molecular Biology volume 16, pages 1212–1217 (2009)Cite this article

Abstract

The cyclic diguanylate (bis-(3′-5′)-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

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Figure 1: Overall structure of the c-di-GMP riboswitch.
Figure 2: Specific binding of c-di-GMP.
Figure 3: c-di-GMP- and Mg2+-induced size and shape changes of the riboswitch, monitored by SAXS.
Figure 4: Global rearrangement of the riboswitch induced by c-di-GMP binding.

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Acknowledgements

We thank the staff of ALS beamline 8.2.2 and J. Bolduc for assistance with synchrotron and home laboratory single-crystal diffraction data collection, respectively, L. Guo from BioCAT at the Advanced Photon Source (APS) for assistance with SAXS data collection and T. Hamma, J. Pitt, J. Posakony, A. Roll-Mecak and H. Suga for discussions. Use of the APS was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract No. W-31-109-ENG-38. BioCAT is a US National Institutes of Health–supported Research Center (RR-08630). This work was supported by the Howard Hughes Medical Institute (HHMI) and the W.M. Keck Foundation. A.R.F.-D. is an Investigator of the HHMI.

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Author notes

  1. Nadia Kulshina and Nathan J Baird: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, USA

    Nadia Kulshina & Adrian R Ferré-D'Amaré

  2. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Nadia Kulshina, Nathan J Baird & Adrian R Ferré-D'Amaré

  3. Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Nathan J Baird & Adrian R Ferré-D'Amaré

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  1. Nadia Kulshina
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Contributions

N.K. designed and prepared RNA constructs, analyzed ligand binding, obtained crystals, carried out diffraction data collection and participated in structure determination and in SAXS data collection. N.J.B. participated in SAXS data collection, analyzed the SAXS data and designed, performed and analyzed the nuclease probing experiments. A.R.F.-D. participated in diffraction data collection, structure determination and refinement. All authors contributed to manuscript preparation.

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Correspondence to Adrian R Ferré-D'Amaré.

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Kulshina, N., Baird, N. & Ferré-D'Amaré, A. Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch. Nat Struct Mol Biol 16, 1212–1217 (2009). https://doi.org/10.1038/nsmb.1701

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