Abstract
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.
Highlights
Riboswitches are a class of nonprotein-coding RNAs that directly sense cellular metabolites to regulate gene expression
To benchmark riboswitch quality prior to the ensuing analysis, we showed that the WT Lrh preQ1-II riboswitch has strong heats of binding to preQ1 in an enthalpically driven process
The WT Lrh riboswitch remained intact after Mg2ϩ-dependent folding at 65 °C (Fig. S3A) and showed distinct CD features in the presence of Mg2ϩ that were absent when Mg2ϩ was excluded from the buffer (Fig. S3B)
Summary
To benchmark riboswitch quality prior to the ensuing analysis, we showed that the WT Lrh preQ1-II riboswitch has strong heats of binding to preQ1 in an enthalpically driven process CD spectroscopy was used to assess the folding of representative riboswitch mutants based on comparison with the WT spectrum. We chose to interrogate the inclined A-minor adenines due to their proximity to preQ1 in the binding pocket (see Fig. 2A). A second mutant, A70G, was designed to disrupt the hydrogen-bonding and van der Waals interactions with the pseudo-base triple, resulting from poor isostericity and reversal of hydrogen bond acceptor and donor groups on the A70G Watson–Crick face. This mutation was analyzed previously for the Spn riboswitch [27], where it resulted in a 280-fold loss of preQ1 binding affinity. Structure category: aSDS in P3 helix (expression platform) C37G/C38G C37U/C38U C37U C38U a Krel equals apparent KD of mutant divided by the apparent KD of WT. b NA, not applicable. c ND, not detected
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