Comparison of a PreQ1 Riboswitch Aptamer in Metabolite-bound and Free States with Implications for Gene Regulation

Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2011; 286(28):24626-37. DOI: 10.1074/jbc.M111.230375
Source: PubMed


Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity
aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility
to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity
for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression
signal accessibility. Here we present crystal structures of the preQ1 riboswitch from Thermoanaerobacter tengcongensis in the preQ1-bound and free states. Although the mode of preQ1 recognition is similar to that observed for preQ0, surface plasmon resonance revealed an apparent KD of 2.1 ± 0.3 nm for preQ1 but a value of 35.1 ± 6.1 nm for preQ0. This difference can be accounted for by interactions between the preQ1 methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer
structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ1-binding site, resulting in “closed” access to the metabolite while simultaneously increasing exposure of the ribosome-binding
site. Solution scattering data suggest that the free-state aptamer is compact, but the “closed” free-state crystal structure
is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ1 riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.

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    • "Apart from having sufficient amounts of pure receptor and ligand, it is helpful to know approximately how tightly the molecules will interact based on an educated guess. It is not unusual for riboswitches to bind their ligands with apparent K D values ranging from 2 nM to 2 μM ( Jenkins et al., 2011; Rieder, Kreutz, & Micura, 2010). There are examples of exceptionally tight binding riboswitches that bind with affinities of 100 pM (Nelson et al., 2013) to 10 pM (Smith et al., 2009). "
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    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.
    Methods in enzymology 11/2014; 549:435-50. DOI:10.1016/B978-0-12-801122-5.00018-0 · 2.09 Impact Factor
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    ABSTRACT: Understanding the nature of the free state of riboswitch aptamers is important for illuminating common themes in gene regulation by riboswitches. Prior evidence indicated the flavin mononucleotide (FMN)-binding riboswitch aptamer adopted a 'bound-like' structure in absence of FMN, suggesting only local conformational changes upon ligand binding. In the scope of pinpointing the general nature of such changes at the nucleotide level, we performed SHAPE mapping experiments using the aptamer domain of two phylogenetic variants, both in absence and in presence of FMN. We also solved the crystal structures of one of these domains both free (3.3 Å resolution) and bound to FMN (2.95 Å resolution). Our comparative study reveals that structural rearrangements occurring upon binding are restricted to a few of the joining regions that form the binding pocket in both RNAs. This type of binding event with minimal structural perturbations is reminiscent of binding events by conformational selection encountered in other riboswitches and various RNAs.
    Nucleic Acids Research 07/2011; 39(19):8586-98. DOI:10.1093/nar/gkr565 · 9.11 Impact Factor
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    ABSTRACT: Here I discuss findings that suggest a universal mechanism for proteins (and RNA) to recognize and interact with various binding partners by selectively binding to different conformations that pre-exist in the free protein's conformational ensemble. The tandem RNA recognition motif domains of splicing factor U2AF⁶⁵ fluctuate in solution between a predominately closed conformation in which the RNA binding site of one of the domains is blocked, and a lowly populated open conformation in which both RNA binding pockets are accessible. RNA binding to U2AF⁶⁵ may thus occur through the weakly populated open conformation, and the binding interaction stabilizes the open conformation. The conformational diversity observed in U2AF⁶⁵ might also facilitate binding to diverse RNA sequences as found in the polypyrimidine tracts that help define 3' splice sites. Similar binding pathways in other systems have important consequences in biological regulation, molecular evolution, and information storage.
    BioEssays 12/2011; 34(3):174-80. DOI:10.1002/bies.201100152 · 4.73 Impact Factor
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