Gene Regulation Gets in Tune : How Riboswitch Tertiary-Structure Networks Adapt to Meet the Needs of Their Transcription Units

Dutta, Debapratim; Wedekind, Joseph E.

doi:10.1016/j.jmb.2015.08.001

Cited by 3 publications

(1 citation statement)

References 32 publications

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“…A recent analysis corroborates this notion, wherein the EC 50 of the Spn riboswitch for cell reporter-gene regulation is 96 M (49), as compared with 19.5 nM for the Lrh riboswitch. Such differences in structure-activity relationships between riboswitches of the same class could be a strategy to fine-tune gene-regulatory functions to meet the distinct fitness needs of a specific organism (65,66).…”

Section: Editors' Pick: Uncoupling Riboswitch Sensing and Gene Controlmentioning

confidence: 99%

Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation

Dutta

Wedekind

2020

Journal of Biological Chemistry

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Riboswitches are a class of nonprotein-coding RNAs that directly sense cellular metabolites to regulate gene expression. They are model systems for analyzing RNA-ligand interactions and are established targets for antibacterial agents. Many studies have analyzed the ligand-binding properties of riboswitches, but this work has outpaced our understanding of the underlying chemical pathways that govern riboswitch-controlled gene expression. To address this knowledge gap, we prepared 15 mutants of the preQ1-II riboswitch—a structurally and biochemically well-characterized HLout pseudoknot that recognizes the metabolite prequeuosine1 (preQ1). The mutants span the preQ1-binding pocket through the adjoining Shine–Dalgarno sequence (SDS) and include A-minor motifs, pseudoknot-insertion helix P4, U·A-U base triples, and canonical G-C pairs in the anti-SDS. As predicted—and confirmed by in vitro isothermal titration calorimetry measurements—specific mutations ablated preQ1 binding, but most aberrant binding effects were corrected by compensatory mutations. In contrast, functional analysis in live bacteria using a riboswitch-controlled GFPuv-reporter assay revealed that each mutant had a deleterious effect on gene regulation, even when compensatory changes were included. Our results indicate that effector binding can be uncoupled from gene regulation. We attribute loss of function to defects in a chemical interaction network that links effector binding to distal regions of the fold that support the gene-off RNA conformation. Our findings differentiate effector binding from biological function, which has ramifications for riboswitch characterization. Our results are considered in the context of synthetic ligands and drugs that bind tightly to riboswitches without eliciting a biological response.

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Section: Editors' Pick: Uncoupling Riboswitch Sensing and Gene Controlmentioning

confidence: 99%

Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation

Dutta

Wedekind

2020

Journal of Biological Chemistry

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RNA in the Loop: Probing T-loop/T-loop Receptor Interactions as Mediators of Long-range RNA Contacts that Influence Gene Regulation

Wedekind

2023

Journal of Molecular Biology

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A riboswitch separated from its ribosome-binding site still regulates translation

Schroeder

Akinyemi

Malik

et al. 2023

Nucleic Acids Research

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Riboswitches regulate downstream gene expression by binding cellular metabolites. Regulation of translation initiation by riboswitches is posited to occur by metabolite-mediated sequestration of the Shine-Dalgarno sequence (SDS), causing bypass by the ribosome. Recently, we solved a co-crystal structure of a prequeuosine1-sensing riboswitch from Carnobacterium antarcticum that binds two metabolites in a single pocket. The structure revealed that the second nucleotide within the gene-regulatory SDS, G34, engages in a crystal contact, obscuring the molecular basis of gene regulation. Here, we report a co-crystal structure wherein C10 pairs with G34. However, molecular dynamics simulations reveal quick dissolution of the pair, which fails to reform. Functional and chemical probing assays inside live bacterial cells corroborate the dispensability of the C10–G34 pair in gene regulation, leading to the hypothesis that the compact pseudoknot fold is sufficient for translation attenuation. Remarkably, the C. antarcticum aptamer retained significant gene-regulatory activity when uncoupled from the SDS using unstructured spacers up to 10 nucleotides away from the riboswitch—akin to steric-blocking employed by sRNAs. Accordingly, our work reveals that the RNA fold regulates translation without SDS sequestration, expanding known riboswitch-mediated gene-regulatory mechanisms. The results infer that riboswitches exist wherein the SDS is not embedded inside a stable fold.

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Gene Regulation Gets in Tune : How Riboswitch Tertiary-Structure Networks Adapt to Meet the Needs of Their Transcription Units

Cited by 3 publications

References 32 publications

Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation

Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation

RNA in the Loop: Probing T-loop/T-loop Receptor Interactions as Mediators of Long-range RNA Contacts that Influence Gene Regulation

A riboswitch separated from its ribosome-binding site still regulates translation

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