Structural basis of ligand binding by a c-di-GMP riboswitch.

Department of Chemistry, Yale University, New Haven, Connecticut, USA.
Nature Structural & Molecular Biology (Impact Factor: 11.63). 11/2009; 16(12):1218-23. DOI: 10.1038/nsmb.1702
Source: PubMed

ABSTRACT The second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 A resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Genetically encodable RNA devices that directly detect small molecules in the cellular environment are of increasing interest for a variety of applications including live cell imaging and synthetic biology. Riboswitches are naturally occurring sensors of intracellular metabolites, primarily found in the bacterial mRNA leaders and regulating their expression. These regulatory elements are generally composed of two domains: an aptamer that binds a specific effector molecule and an expression platform that informs the transcriptional or translational machinery. While it was long established that riboswitch aptamers are modular and portable, capable of directing different output domains including ribozymes, switches, and fluorophore-binding modules, the same has not been demonstrated until recently for expression platforms. We have engineered and validated a set of expression platforms that regulate transcription through a secondary structural switch that can host a variety of different aptamers, including those derived through in vitro selection methods, to create novel chimeric riboswitches. These synthetic switches are capable of a highly specific regulatory response both in vitro and in vivo. Here we present the methodology for the design and engineering of chimeric switches using biological expression platforms. © 2015 Elsevier Inc. All rights reserved.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cyclic bis(3'-5')diadenylic acid (c-di-AMP) (Chart I), recently identified as a second messenger monitoring DNA integrity during sporulation in the soil bacterium Bacillus subtilis, was synthesized on an 80 mu mol scale by a combination of the phosphoramidite and phosphotriester methods using a commercially available adenosine phosphoramidite as starting material. An artificial analog 2'-bis(tert-butyldimethylsilyl)-c-di-AMP was also obtained by our procedure.
    Chemistry Letters 10/2011; 40(10):1113-1114. DOI:10.1246/cl.2011.1113 · 1.30 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, KA, of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.