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Unboxing the T‐box riboswitches—A glimpse into multivalent and multimodal RNA–RNA interactions

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Abstract The T‐box riboswitches are widespread bacterial noncoding RNAs that directly bind specific tRNAs, sense aminoacylation on bound tRNAs, and switch conformations to control amino‐acid metabolism and to maintain nutritional homeostasis. The core mechanisms of tRNA recognition, amino acid sensing, and conformational switching by the T‐boxes have been recently elucidated, providing a wealth of new insights into multivalent and multimodal RNA–RNA interactions. This review dissects the structures and tRNA‐recognition mechanisms by the Stem I, Stem II, and Discriminator domains, which collectively compose the T‐box riboswitches. It further compares and contrasts the two classes of T‐boxes that regulate transcription and translation, respectively, and integrates recent findings to derive general themes, trends, and insights into complex RNA–RNA interactions. Specifically, the T‐box paradigm reveals that noncoding RNAs can interact with each other through multiple coordinated contacts, concatenation of stacked helices, and mutually induced fit. Numerous tertiary contacts, especially those emanating from strings of single‐stranded purines, act in concert to reinforce long‐range base‐pairing and stacking interactions. These coordinated, mixed‐mode contacts allow the T‐box RNA to sterically sense aminoacylation on the tRNA using a bipartite steric sieve, and to couple this readout to a conformational switch mediated by tRNA‐T‐box stacking. Together, the insights gleaned from the T‐box riboswitches inform investigations into other complex RNA structures and assemblies, development of T‐box‐targeted antimicrobials, and may inspire design and engineering of novel RNA sensors, regulators, and interfaces. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Regulatory RNAs/RNAi/Riboswitches > Riboswitches
Two classes of T‐box riboswitches. (a) Transcriptional T‐boxes initially bind their cognate tRNAs co‐transcriptionally and bifurcate into two mutually exclusive conformations depending on the aminoacylation state of the bound tRNA. In starvation (upper right), an uncharged tRNA is enveloped by the T‐box mRNA through three distant contacts, producing a continuously stacked “central spine” that stabilizes the transcription antiterminator—allowing gene expression. In nutritional abundance (lower right), the aminoacyl moiety of charged tRNA creates steric conflict with the antiterminator, imploding the structure to form the terminator hairpin shuttering transcription. Red sticks represent intermolecular stacking. (b) Translational T‐boxes are conformationally bistable and can bind uncharged (left) or charged tRNA (right). The former stabilizes the anti‐sequestrator and permits access by the 30S ribosome to the Shine‐Dalgarno (SD) sequence—allowing translation initiation. When a charged tRNA binds, the steric conflict from the aminoacyl drives formation of the SD sequestrator—similar to its transcriptional counterpart—masking the SD sequence and disallowing translation initiation
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Molecular basis of RNA‐actuated sensing of tRNA aminoacylation and genetic switching. (a, b) Two views of the composite steric sieve against aminoacyl‐tRNAs. Modeled 2′‐ and 3′‐aminoacyl groups are shown in thick sticks. The steric barriers against 2′‐ and 3′‐aminoacyl are shown in surface representations and colored orange and red, respectively. (c) Cartoon scheme of the immobilization and steric selection of uncharged tRNA by a T‐box discriminator. The terminal nucleotide of tRNA, tA76, is colored based on surface burial by the discriminator. (d, e) Cryo‐EM Structure (d) and cartoon scheme (e) of a continuously stacked central spine formed by coaxial stacking of the upper half of tRNA with the Stem I IDTM and Discriminator. Three solid red lines indicate intermolecular coaxial stacking and dotted lines in the discriminator denote tertiary contacts. (f) Simple combination of three tRNA‐bound co‐crystal structures of T‐box Stem I (PDB: 4LCK), Stem II (PDB: 6UFM), and Discriminator (PDB: 6PMO) domains produce a feature‐complete T‐box‐tRNA complex model, which represents the most typical T‐box such as the original B. subtilis tyrS T‐box
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Stabilization of codon–anticodon interactions by Stem II. (a) Widened, irregular grooves of the S‐turn region of N. farcinica ileS T‐box stem II (PDB: 4UFM) compared to a model dsRNA (“ideal” dsRNA generated by Coot). (b) Three views of the S‐turn motif in the N. farcinica ileS T‐box Stem II. Red residues are those that face and latch the codon–anticodon duplex and form part of a 5‐purine string. (c) Anatomy of the Stem II S‐turn region. The 5‐purine string is shaded in green and the S‐turn boxed. (d) Stabilization of codon–anticodon interactions by the 5‐purine string. Other portions of Stem II are omitted for clarity. (e, f) Two views of the “anticodon pincer” formed by the Stem I codon and Stem II 5‐purine string. (g) Hydrogen bonds between the 5‐purine string and the minor groove of the codon–anticodon duplex at each of the three layers. A38 and A69 cross‐strand stack to form the inclined tandem A‐minor (ITAM) motif
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Decoding of tRNA anticodon by the T‐box Stem I. (a) Decoding of the tRNA anticodon by the T‐box specifier codon, illustrated by four co‐crystal structures. (b) Overlay of structures in (a) reveals a positional shift of the conserved purine immediately 3′ of the specifier codon (A90 and A19 in transcriptional and translational T‐boxes, respectively). (c) Scheme of anticodon‐decoding by the T‐box Stem I. (d) Recognition of the tRNA elbow by the Stem I IDTM (Interdigitated Double T‐loop Motif) through platform‐platform stacking interactions (Zhang & Ferré‐D'Amaré, 2013). Interfacial residues are numbered. (e) Anatomy and connectivity of the IDTM in (d). Each of the pentanucleotide T‐loops that interdigitate to form the IDTM is shaded in red and orange, respectively. Noncanonical base‐pairing interactions are illustrated using Leontis–Westhof symbols throughout (Leontis & Westhof, 2001)
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Gallery of T‐box riboswitch‐tRNA complex structures. (a) Transcriptional T‐boxes. Upper left: Oceanobacillus iheyensis glyQ T‐box Stem I—tRNAGlyBacillus subtilis YbxF ternary complex (PDB: 4LCK; Zhang & Ferré‐D'Amaré, 2013). Lower left: Geobacillus kaustophilus glyQ T‐box Stem I—tRNAGly complex (PDB: 4MGN; Grigg & Ke, 2013). Upper right: G. kaustophilus glyQ T‐box Discriminator—tRNAGly complex (PDB: 6PMO; Li et al., 2019). Lower right: B. subtilis glyQS full‐length T‐box—tRNAGly complex (PDB: 6POM; Li et al., 2019). T‐boxes and tRNAs are shown in blue and green, respectively, throughout, unless otherwise indicated. K‐turn‐binding protein YbxF is shown in yellow. (b) Translational T‐boxes. Upper: Nocardia farcinica ileS T‐box Stem I‐Stem II domains in complex with the cognate tRNAIle (PDB: 6UFM; Suddala & Zhang, 2019b). Lower: Mycobacterium tuberculosis ileS full‐length T‐box in complex with cognate tRNAIle (PDB: 6UFG; Battaglia et al., 2019)
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Regulatory RNAs/RNAi/Riboswitches > Riboswitches
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry

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