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Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch

Nature volume 441, pages 1167–1171 (2006)Cite this article

Abstract

Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins1,2,3. Here we describe a 2.05 Å crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA4 that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B1, an essential participant in many protein-catalysed reactions5. Organisms from all three domains of life6,7,8,9, including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.

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Figure 1: Structural models of a TPP riboswitch and its ligands.
Figure 2: Structure and interactions in the TPP-binding pocket.
Figure 3: Tertiary interactions defining TPP riboswitch structure and accessibility to the binding pocket.
Figure 4: Structural probing of the TPP riboswitch and implications for TPP-mediated gene repression.

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Acknowledgements

We thank L. Malinina and Y.-R. Yuan for extensive discussions; A. Kazantsev and M. Yusupov for the gift of Os(NH3)6; and M. Becker for assistance with synchrotron data collection on beamline X25 at the Brookhaven National Laboratory, supported by the US Department of Energy. D.J.P. and R.R.B. were supported by funds from the NIH. Author Contributions A.S. was responsible for all structural and biochemical aspects of this project. A.P. participated in RNA preparation and crystallization trials, and A.T.P. participated in NMR experiments. D.J.P., A.S. and R.R.B. co-wrote the paper. All authors discussed the results and commented on the manuscript.

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Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, 10021, USA

    Alexander Serganov, Anna Polonskaia, Anh Tuân Phan & Dinshaw J. Patel

  2. Department of Molecular, Cellular and Developmental Biology and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, 06520, USA

    Ronald R. Breaker

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Correspondence to Alexander Serganov or Dinshaw J. Patel.

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Coordinates of the X-ray structure of the TPP-bound riboswitch have been deposited in the RCSB Protein Data Bank under accession code 2GDI. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

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This file contains the Supplementary Methods, Supplementary Figures 1–11 and Supplementary Table. (PDF 2910 kb)

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Serganov, A., Polonskaia, A., Phan, A. et al. Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch. Nature 441, 1167–1171 (2006). https://doi.org/10.1038/nature04740

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