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

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.

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation

“…Such compounds are not as effective in functional assays compared to preQ 1 , most likely because they interrupt the long-range stacking spine that links effector binding to the expression platform. Similar uncoupling was reported for specific mutants of the Lactobacillus rhamnosus preQ 1 -II riboswitch, which bound tightly to preQ 1 but exhibited poor gene regulation in live cells ( 2 ). The broader utility of stacking spines for interdomain communication is exemplified by their presence in the Vvu add riboswitch and the TLS-linker-UPD ribosome sensor within the TYMV 3′-UTR.…”

“…The principles of molecular recognition used by riboswitches to bind their cognate effectors are well understood ( 9 ). However, a knowledge gap exists in our understanding of how ligand binding leads to conformational changes that reprogram associated expression platforms that control gene expression ( 2 ). To address this challenge, we undertook local and global analyses of preQ 1 -dependent changes in nucleotide stacking, flexibility and base pairing.…”

“…An emerging theme among nonprotein-coding (nc)RNAs that interact with cognate effectors is their propensity to use structural communication conduits that relay the effector-binding status in one domain to a distal region that modulates biological activity through conformational changes ( 1 , 2 ). Identifying and understanding these networks remains a central challenge in the ncRNA field ( 1–4 ).…”

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

“…Following co-dialysis with RNA, protein samples were diluted in dialysis buffer to concentrations ∼10-fold higher than RNA. ITC measurements were conducted using a PEAQ-ITC (Malvern Panalytical) with protein in the syringe and RNA in the cell ( 75 , 76 , 77 ). Experiments were conducted at 20 °C unless noted.…”

Unraveling the structure and biological functions of RNA triple helices

“…Another consideration is that proteins could participate in the folding and conformational states of some riboswitches, although proteins are not required for ligand binding to the current experimentally validated riboswitches (Breaker, 2012). Importantly, the RNA triple helix can mediate both ligand binding and expression output for select riboswitches and the functional coupling is only beginning to be understood (Dutta & Wedekind, 2019).…”