Journal of Biological Chemistry
Volume 295, Issue 9, 28 February 2020, Pages 2555-2567
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Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation

https://doi.org/10.1074/jbc.RA119.010755Get rights and content
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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.

RNA structure
isothermal titration calorimetry (ITC)
gene expression
bacteria
mutant
A-minor motif
base triples
pseudoknot
riboswitch
Shine-Dalgarno sequence
gene regulation
preQ1 riboswitch

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This work was supported by National Institutes of Health Grant GM063162 (to J. E. W.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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This article contains Tables S1 and S2 and Figs. S1–S6.