Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions

Li, Shuang; Su, Zhaoming; Lehmann, J.; Stamatopoulou, Vassiliki; Giarimoglou, Nikoleta; Henderson, Frances E.; Li, Fan; Pintilie, Grigore; Zhang, Kaiming; Chen, Muyuan; Ludtke, Steven J.; Wang, Yun-Xing; Stathopoulos, Constantinos; Chiu, Wah; Zhang, Jinwei

doi:10.1038/s41594-019-0326-7

Cited by 63 publications

(90 citation statements)

References 90 publications

(149 reference statements)

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“…17). In total, we examine four targeted mutations: Mutant 1 (G72T, G73T) targets the stability of the stem I domain, which is known to interact with the tRNA in order to ensure binding and recognition of the cognate tRNA ligand 41,44 ; Mutant 2 (ΔUGGU) is a deletion of the T-box sequence, which are the nucleotides where the uncharged tRNA binds and triggers the formation of the anti-terminator complex 45 ; Mutant 3 (C258U, G259U, U260C) targets conserved nucleotides of the anti-terminator conformation 43 , thereby affecting its stability; Mutant 4 is a 60 bp deletion (Δ306-365) in the terminator sequence, preventing the formation of the terminator hairpin.…”

Section: Resultsmentioning

confidence: 99%

A riboswitch gives rise to multi-generational phenotypic heterogeneity in an auxotrophic bacterium

2020

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Auxotrophy, the inability to produce an organic compound essential for growth, is widespread among bacteria. Auxotrophic bacteria rely on transporters to acquire these compounds from their environment. Here, we study the expression of both low-and high-affinity transporters of the costly amino acid methionine in an auxotrophic lactic acid bacterium, Lactococcus lactis. We show that the high-affinity transporter (Met-transporter) is heterogeneously expressed at low methionine concentrations, resulting in two isogenic subpopulations that sequester methionine in different ways: one subpopulation primarily relies on the high-affinity transporter (high expression of the Met-transporter) and the other subpopulation primarily relies on the low-affinity transporter (low expression of the Met-transporter). The phenotypic heterogeneity is remarkably stable, inherited for tens of generations, and apparent at the colony level. This heterogeneity results from a T-box riboswitch in the promoter region of the met operon encoding the high-affinity Met-transporter. We hypothesize that T-box riboswitches, which are commonly found in the Lactobacillales, may play as-yet unexplored roles in the predominantly auxotrophic lifestyle of these bacteria.

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Section: Resultsmentioning

confidence: 99%

A riboswitch gives rise to multi-generational phenotypic heterogeneity in an auxotrophic bacterium

2020

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“…Cryo-EM structure determination of RNA-only systems or macromolecules with molecular weights under 50 kDa has been accomplished only recently 7,[35][36][37][38] , and it was unclear whether cryo-EM might be applicable to an RNA as small as the SARS-CoV-2 FSE (28 kDa, 88 nucleotides; Fig. 1a).…”

Section: Cryo-em Resolves the 88-nt Fsementioning

confidence: 99%

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Zhang

Zheludev

Hagey

et al. 2020

Preprint

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135

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Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5′ end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

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“…The T-box riboswitches are modular RNA devices that consist of an obligate 5 0 Stem I domain which decodes the tRNA identity (Grigg & Ke, 2013;Lehmann, Jossinet, & Gautheret, 2013;Zhang & Ferré-D'Amaré, 2013), an optional intervening Stem II domain which reinforces Stem I-tRNA interactions (Battaglia, Grigg, & Ke, 2019;Suddala & Zhang, 2019a), and an essential 3 0 discriminator domain which senses tRNA aminoacylation and executes genetic switching ( Figure 1) (Battaglia et al, 2019;Li et al, 2019;Zhang & Ferré-D'Amaré, 2014). While the 3 0 -most discriminator domain employs a core architecture that is essentially identical among all known T-boxes, the Stem I and II domains are variable and exhibit interchangeability and characteristics of plug-and-play ( Figure 2; Gutierrez-Preciado et al, 2009;Vitreschak et al, 2008).…”

Section: Anatomy and Archetypes Of T-box Riboswitchesmentioning

confidence: 99%

“…As such, it lacks the specificity to engage without a prior contact (Zhang & Ferré-D'Amaré, 2013). The second distal interface between the tRNA termini and the T-box discriminator consists of four Watson-Crick base pairs, two instances of intermolecular coaxial helical stacking (involving the 5 0 tG1 and 3 0 tA76, respectively), six sets of minor groove interactions with a bipartite six-purine string, and finally three nucleobase-ribose packing contacts (Li et al, 2019). The individual affinities and specificities of the three binding sites follow the order of codon-anticodon > tRNA 3 0 -end-discriminator > IDTM-elbow.…”

Section: Lessons Learned From the T-boxesmentioning

confidence: 99%

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

Zhang

2020

WIREs RNA

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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 singlestranded 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.

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Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions

Cited by 63 publications

References 90 publications

A riboswitch gives rise to multi-generational phenotypic heterogeneity in an auxotrophic bacterium

A riboswitch gives rise to multi-generational phenotypic heterogeneity in an auxotrophic bacterium

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

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

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