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A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain

Nature Structural & Molecular Biology volume 14, pages 308–317 (2007)Cite this article

Abstract

A previous bioinformatics-based search for riboswitches yielded several candidate motifs in eubacteria. One of these motifs commonly resides in the 5′ untranslated regions of genes involved in the biosynthesis of queuosine (Q), a hypermodified nucleoside occupying the anticodon wobble position of certain transfer RNAs. Here we show that this structured RNA is part of a riboswitch selective for 7-aminomethyl-7-deazaguanine (preQ1), an intermediate in queuosine biosynthesis. Compared with other natural metabolite-binding RNAs, the preQ1 aptamer appears to have a simple structure, consisting of a single stem-loop and a short tail sequence that together are formed from as few as 34 nucleotides. Despite its small size, this aptamer is highly selective for its cognate ligand in vitro and has an affinity for preQ1 in the low nanomolar range. Relatively compact RNA structures can therefore serve effectively as metabolite receptors to regulate gene expression.

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Figure 1: Queuosine biosynthesis in eubacteria.
Figure 2: The 5′ UTR of the B. subtilis queCDEF mRNA undergoes structural modulation in response to preQ1.
Figure 3: Molecular discrimination by the B. subtilis queC 5′ UTR preQ1-binding region.
Figure 4: Determination of the minimal aptamer sequence required for binding of preQ1.
Figure 5: Evidence for a Watson-Crick pairing interaction between preQ1 and a conserved cytidyl residue of the aptamer.
Figure 6: Effects of variant preQ1 riboswitches on genetic control in vivo.

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Acknowledgements

We thank J.A. Collins for assistance in generating B. subtilis transformants and members of the Breaker laboratory for helpful discussions. This work was supported by grants from the US National Institutes of Health (R33 DK07027 and GM 068819) and the US National Science Foundation (EIA 0323510) to R.R.B. RNA research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute.

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Author notes

  1. Wade C Winkler & Jeffrey E Barrick

    Present address: Present addresses: Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA (W.C.W.) and Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, USA (J.E.B.).,

Authors and Affiliations

  1. Howard Hughes Medical Institute, Yale University, P.O. Box 208103, New Haven, 06520-8103, Connecticut, USA

    Adam Roth, Jeffrey E Barrick & Ronald R Breaker

  2. Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, 06520-8103, Connecticut, USA

    Wade C Winkler, Elizabeth E Regulski, Jinsoo Lim, Inbal Jona, Ankita Ritwik, Jane N Kim, Rüdiger Welz & Ronald R Breaker

  3. Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208103, New Haven, 06520-8103, Connecticut, USA

    Jeffrey E Barrick & Ronald R Breaker

  4. Department of Chemistry, Portland State University, P.O. Box 751, Portland, 97207, Oregon, USA

    Bobby W K Lee & Dirk Iwata-Reuyl

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Contributions

A. Roth, W.C.W., E.E.R., I.J., A. Ritwik, J.N.K. and R.W. carried out in vitro biochemical analyses of queC RNA constructs. E.E.R. conducted assays of reporter gene expression in vivo. B.W.K.L., J.L. and D.I.-R. synthesized preQ1 and related compounds. J.E.B. performed bioinformatics searches and prepared sequence alignments. A. Roth and R.R.B. prepared the manuscript.

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Correspondence to Ronald R Breaker.

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Competing interests

R.R.B. is a cofounder of BioRelix, a company interested in the use of riboswitches as drug targets. Although this publication is not directly related to the development of antimicrobials and thus does not currently constitute a competing financial interest, the discovery of novel riboswitches may ultimately lead to development of new classes of antibiotics.

Supplementary information

Supplementary Fig. 1

PreQ1 aptamer sequences. (PDF 128 kb)

Supplementary Fig. 2

PreQ1 riboswitch locations and associated genes. (PDF 164 kb)

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Roth, A., Winkler, W., Regulski, E. et al. A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain. Nat Struct Mol Biol 14, 308–317 (2007). https://doi.org/10.1038/nsmb1224

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