Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA

National Heart, Lung and Blood Institute, 50 South Drive, MSC 8012, Bethesda, Maryland 20892-8012, USA.
Nature (Impact Factor: 41.46). 07/2013; 500(7462). DOI: 10.1038/nature12440
Source: PubMed


In Gram-positive bacteria, T-box riboswitches regulate the expression of aminoacyl-tRNA synthetases and other proteins in response to fluctuating transfer RNA aminoacylation levels under various nutritional states. T-boxes reside in the 5'-untranslated regions of the messenger RNAs they regulate, and consist of two conserved domains. Stem I contains the specifier trinucleotide that base pairs with the anticodon of cognate tRNA. 3' to stem I is the antiterminator domain, which base pairs with the tRNA acceptor end and evaluates its aminoacylation state. Despite high phylogenetic conservation and widespread occurrence in pathogens, the structural basis of tRNA recognition by this riboswitch remains ill defined. Here we demonstrate that the ∼100-nucleotide T-box stem I is necessary and sufficient for specific, high-affinity (dissociation constant (Kd) ∼150 nM) tRNA binding, and report the structure of Oceanobacillus iheyensis glyQ stem I in complex with its cognate tRNA at 3.2 Å resolution. Stem I recognizes the overall architecture of tRNA in addition to its anticodon, something accomplished by large ribonucleoproteins such as the ribosome, or proteins such as aminoacyl-tRNA synthetases, but is unprecedented for a compact mRNA domain. The C-shaped stem I cradles the L-shaped tRNA, forming an extended (1,604 Å(2)) intermolecular interface. In addition to the specifier-anticodon interaction, two interdigitated T-loops near the apex of stem I stack on the tRNA elbow in a manner analogous to those of the J11/12-J12/11 motif of RNase P and the L1 stalk of the ribosomal E-site. Because these ribonucleoproteins and T-boxes are unrelated, this strategy to recognize a universal tRNA feature probably evolved convergently. Mutually induced fit of stem I and the tRNA exploiting the intrinsic flexibility of tRNA and its conserved post-transcriptional modifications results in high shape complementarity, which in addition to providing specificity and affinity, globally organizes the T-box to orchestrate tRNA-dependent transcription regulation.

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Available from: Jinwei Zhang, Sep 11, 2014

  • Nature 07/2013; 500(7462). DOI:10.1038/nature12460 · 41.46 Impact Factor
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    ABSTRACT: In Gram-positive bacteria the tRNA-dependent T box riboswitch regulates the expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson-Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. Enhanced packing within the Specifier domain loop E motif may stabilize the complex and contribute to cognate tRNA selection.
    FEBS letters 09/2013; 587(21). DOI:10.1016/j.febslet.2013.09.003 · 3.17 Impact Factor
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    ABSTRACT: T box riboswitches are cis-acting RNA elements that bind to tRNA and sense its aminoacylation state to influence gene expression. Here, we present the 3.2 Å resolution X-ray crystal structures of the T box Stem I-tRNA complex and tRNA, in isolation. T box Stem I forms an arched conformation with extensive intermolecular contacts to two key points of tRNA, the anticodon and D/T-loops. Free and complexed tRNA exist in significantly different conformations, with the contacts stabilizing flexible D/T-loops and a rearrangement of the D-loop. Using a designed T box RNA/tRNA system, we demonstrate that the T box riboswitch monitors the length and orientation of two essential contacts. Length or orientation mismatches engineered into the T box riboswitch and tRNA disrupt the complex, whereas simultaneous insertion of full helical turns realigns the interfaces and restores interaction between artificially elongated T box riboswitch and tRNA molecules.
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