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Structure and mechanism of the T‐box riboswitches

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In most Gram‐positive bacteria, including many clinically devastating pathogens from genera such as Bacillus, Clostridium, Listeria, and Staphylococcus, T‐box riboswitches sense and regulate intracellular availability of amino acids through a multipartite messenger RNA (mRNA)–transfer RNA (tRNA) interaction. The T‐box mRNA leaders respond to nutrient starvation by specifically binding cognate tRNAs and sensing whether the bound tRNA is aminoacylated, as a proxy for amino acid availability. Based on this readout, T‐boxes direct a transcriptional or translational switch to control the expression of downstream genes involved in various aspects of amino acid metabolism: biosynthesis, transport, aminoacylation, transamidation, and so forth. Two decades after its discovery, the structural and mechanistic underpinnings of the T‐box riboswitch were recently elucidated, producing a wealth of insights into how two structured RNAs can recognize each other with robust affinity and exquisite selectivity. The T‐box paradigm exemplifies how natural noncoding RNAs can interact not just through sequence complementarity but can add molecular specificity by precisely juxtaposing RNA structural motifs, exploiting inherently flexible elements and the biophysical properties of post‐transcriptional modifications, ultimately achieving a high degree of shape complementarity through mutually induced fit. The T‐box also provides a proof‐of‐principle that compact RNA domains can recognize minute chemical changes (such as tRNA aminoacylation) on another RNA. The unveiling of the structure and mechanism of the T‐box system thus expands our appreciation of the range of capabilities and modes of action of structured noncoding RNAs, and hints at the existence of networks of noncoding RNAs that communicate through both, structural and sequence specificity. WIREs RNA 2015, 6:419–433. doi: 10.1002/wrna.1285 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
T‐box riboswitch senses and regulates intracellular amino acid availability. (a) Starvation for a particular amino acid (green circle) leads to reduced charging of its cognate tRNA (green). A cognate uncharged tRNA binds the T‐box and stabilizes the otherwise unstable antiterminator structure, allowing RNA polymerase to transcribe downstream genes. (b) The collective action of T‐box downstream genes in (a) carries out amino acid biosynthesis, import, and tRNA charging, thus restoring cellular amino acid and tRNA charging levels (>95% charged). Charged tRNAs are rejected sterically and are unable to stabilize the antiterminator. Thus, formation of the terminator prematurely terminates transcription.
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Mechanisms of sensing tRNA aminoacylation state and genetic switching by the T‐box riboswitch. (a) Starvation of a particular amino acid (green sphere) leads to deacylation of its carrier tRNA (green). The uncharged tRNA 3′ NCCA terminus base pairs with the antiterminator bulge, forming a 4‐bp intermolecular helix. This helix further coaxially stacks against the helix A1 of the antiterminator, thereby forming an extended continuous helical stack (29 layers) and stabilizing the antiterminator structure and permitting the RNA polymerase to proceed through into the downstream coding region. Thus, the uncharged tRNA is incorporated into the antiterminator assembly and dictates the outcome of the genetic switch. (b) When amino acids are replete, an aminoacylated tRNA 3′ terminus destabilizes the antiterminator structure by steric hindrance of the amino acid, causing the thermodynamically more stable transcription terminator (strong RNA hairpin followed by a track of uridines) to form. Transcription is therefore terminated and the downstream coding genes are shut off, completing a negative feedback loop that senses and maintains steady‐level supplies of amino acids for the ribosome in the form of aminoacylated tRNAs.
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Recognition of tRNA elbow through two interdigitated T‐loops. (a and b) Recognition of tRNA elbow by the Oceanobacillus iheyensis (marine blue, a) and Geobacillus kaustophilus (light orange, b) T‐box Stem I distal region, the core of which is formed by the interdigitation of two T‐loops (shown in red and light cyan). Interfacial nucleotides are labeled. (c and d) Depiction of how the isolated T‐loops approach each other to fill each other's stacking gap (dotted lines), arriving in an extensively paired (two base triples in the center; hydrogen bonds on each base triple are indicated by blue dashes) and heavily stacked structure, as shown in (d). (e and f) Detailed interaction between the T‐box base triple (C44•G63•A56) and the tRNA tertiary base pair (tG19•tC56) seen in the O. iheyensis (e) and G. kaustophilus (f) structures. The position and interaction of tU20 residue, albeit extrahelically flipped in both instances, differ in the two structures. The nucleobase of tU20 is seen forming a packing interaction (parallel lines) with the ribose of T‐box C64 in the O. iheyensis structure, whereas the same residue in the G. kaustophilus structure is shifted closer to and forms a base triple with the tRNA tG19•tC56 pair and appears to stack against T‐box G62. (g) Superimposition of the tRNA tG19•tC56 pairs (thin lines) of the two structures reveals appreciable variability in the precise location of the T‐box base triples.
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Variability in the T‐box‐tRNA interface adjacent to the codon–anticodon duplex. (a) Overlay of the Oceanobacillus iheyensis (marine blue) and Geobacillus kaustophilus (light orange) T‐box–tRNA complex structures by superimposing the tRNA anticodon trinucleotides (magenta). This leads to superimposition of the T‐box specifier codons (yellow) and reveals differences between the two structures in the region immediately distal to the codon–anticodon duplex. Backbone phosphates are shown in spheres and the spacer nucleotides in G. kaustophilus T‐box are shown in red. Nucleobases and riboses are omitted for clarity. (b) Compared to the O. iheyensis structure, the loop E motif (or bulged‐G or S‐turn motif) in the G. kaustophilus complex is translated more distally by 10 Å. (c) Upon translation, the two loop E motifs are superimposable. (d) Cartoon illustration of the T‐box–tRNA interaction adjacent to the codon–anticodon duplex. ‘n’ denotes the variable number of nucleotides that are inserted in between the codon trinucleotide and the loop E motif. n = 0 and 2 for O. iheyensis (e) and G. kaustophilus (f) T‐boxes, respectively. (e) In the O. iheyensis structure, the formation of the loop E motif immediately adjacent to the codon trinucleotide bends the Stem I backbone away from tA37, thus easily accommodating bulky hypermodifications at that position (dotted line). (f) In contrast, in the G. kaustophilus structure, where n = 2, the two inserted nucleotides, A85 and C86, shown in red, each makes a single hydrogen bond to the tRNA tG39 and tA37. Compared to the O. iheyensis structure, the G. kaustophilus T‐box is in closer proximity to the tRNA in the anticodon region (compare e and f), possibly offsetting its reduced shape complementarity and increased distance to the tRNA t26•t44 hinge region (compare Figure (b) and (c)). Further, the inserted nucleotides in the specifier loop may permit presentation and utilization of alternative codons, thus allowing a single T‐box riboswitch to respond to two or more tRNAs.
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A conserved geometric arrangement decodes tRNA anticodon using both structural and sequence determinants. (a and b) Structural arrangements of tRNA anticodon (magenta) decoding by the T‐box specifier (yellow) and neighboring residues observed in the Oceanobacillus iheyensis (a, marine blue) and Geobacillus kaustophilus (b, light orange) T‐boxes. (c) Structural arrangement that decodes the anticodon of the P‐site tRNA (PDB: 4V5D). The tRNA, mRNA, and rRNA are shown in light green, yellow, and marine blue, respectively. (d) Cartoon illustration of the geometric arrangement responsible for tRNA anticodon decoding by the T‐box riboswitch and the ribosome. Codon and anticodon are colored as in Figure .
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Two mutually exclusive secondary structures of a representative T‐box riboswitch and its base‐pairing interactions with its cognate tRNA. Unlike essentially all other T‐boxes, tRNAGly‐responsive T‐boxes (such as the Bacillus subtilis glyQS T‐box depicted here and the closely related Oceanobacillus iheyensis glyQ T‐box) do not contain the Stem II and Stem II A/B pseudoknot elements in the linker region. The mutually exclusive antiterminator and terminator conformations of the T‐box riboswitch are shown in (a) and (b), respectively. Sequence conservation across known T‐boxes is indicated in orange and yellow circles. Base‐pairing interactions between the Stem I specifier sequence in Stem I and the tRNA anticodon (magenta), and between the T‐box antiterminator domain and the tRNA acceptor end (green), are indicated by colored lines and boxes. The gray box indicates the functionally important K‐turn. The sequences of tRNAGly of B. subtilis and O. iheyensis are identical.
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Common and divergent features of the T‐box Stem I–tRNA complex structures. (a and b) Two orthogonal views of the overall structure of the Oceanobacillus iheyensis glyQ T‐box–tRNAGlyBacillus subtilis YbxF ternary complex (PDB: 4LCK), colored marine blue, forest green, and gray, respectively. The distal, specifier, and proximal regions of the Stem I are demarcated by the T‐box hinge at T‐box U30 and the K‐turn. The tRNA anticodon and T‐box specifier (codon) trinucleotides are colored magenta and yellow, respectively. (c) Overall structure of a truncated Geobacillus kaustophilus glyQ T‐box Stem I in complex with tRNAGly (PDB: 4MGN), colored light orange and cyan, respectively. (d–f) Structural superpositions of the O. iheyensis and G. kaustophilus Stem I–tRNA complex structures made by least‐squares alignment of the tRNAs (d), the Stem I (e), or the distal region of Stem I (f). It is evident that the tRNAs and the Stem Is cannot be superimposed simultaneously owing to structural differences between them. Superimposing the distal Stem I (f) automatically superimposes the tRNA elbow region, suggesting that the interaction between the T‐box distal region and the tRNA elbow is structurally constrained. It is also clear that the degree of swiveling of the tRNA anticodon stem loop relative to the rest of the tRNA about the t26•t44 hinge (indicated by the arrowhead) varies considerably between the two T‐box complex structures.
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