Coupling Green Fluorescent Protein Expression with Chemical Modification to Probe Functionally Relevant Riboswitch Conformations in Live Bacteria

Abstract

Noncoding RNAs engage in numerous biological activities including gene regulation. To fully understand RNA function it is necessary to probe biologically relevant conformations in living cells. To address this challenge, we coupled RNA-mediated regulation of the green fluorescent protein (GFP)uv-reporter gene to icSHAPE (in cell Selective 2'-Hydroxyl Acylation analyzed by Primer Extension). Our transcript-specific approach provides sensitive, fluorescence-based readout of the regulatory-RNA status as a means to coordinate chemical modification experiments. We chose a plasmid-based reporter compatible with Escherichia coli to allow use of knockout strains that eliminate endogenous effector biosynthesis. The approach was piloted using the Lactobacillus rhamnosus ( Lrh) preQ-II riboswitch, which senses the pyrrolopyrimidine metabolite preQ. Using an E. coli Δ queF strain incapable of preQ anabolism, the Lrh riboswitch yielded nearly one log unit of GFPuv-gene repression resulting from exogenously added preQ. We then subjected cells in gene "on" and "off" states to icSHAPE. The resulting differential analysis indicated reduction in Lrh riboswitch flexibility in the P3 helix of the pseudoknot, which comprises the ribosome-binding site (RBS) paired with the anti-RBS. Such expression platform modulation was not observed by in vitro chemical probing and demonstrates that the crowded cellular environment does not preclude detection of compact and loose RNA-regulatory conformations. Here we describe the design, methods, interpretation, and caveats of Reporter Coupled (ReCo) icSHAPE. We also describe mapping of the differential ReCo-icSHAPE results onto the Lrh riboswitch-preQ cocrystal structure. The approach should be readily applicable to functional RNAs triggered by effectors or environmental variations.

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

“…The preQ 1 -II riboswitch is a gene-regulatory ncRNA that attenuates translation by sensing cellular levels of preQ 1 (21,40,41). Translational regulation occurs by modulation of SDS accessibility-a common gene-regulation mechanism among bacterial riboswitches (1,9).…”

“…Despite the number of known riboswitch classes, only a handful of studies have sought to relate three-dimensional structure to in-cell gene-regulatory function (31, 36 -39, 47-49). To meet this challenge, we previously established that the co-crystal structure of the Lrh preQ 1 -II riboswitch bound to preQ 1 is a relevant gene-off conformation, based on chemical modification analysis of switched and unswitched states in live bacteria (41). This validation (Table S1).…”

“…Our results revealed that the Lrh preQ 1 -II riboswitch co-crystal structure in complex with preQ 1 agrees well with expectations for its gene-off conformational state in live bacteria. We further established that our method of folding the Lrh riboswitch for co-crystallization and binding analysis produces no appreciable difference in equilibrium binding to preQ 1 when compared with experiments in which the riboswitch was prepared by co-transcriptional folding and native purification (41).…”

“…The eluted RNA was precipitated in undiluted ethanol and resuspended before purification. Purification was performed by 8% denaturing PAGE using a 29:1 ratio of acrylamide to bisacrylamide, 1ϫ TBE buffer, and 7 M urea (41,68). The RNA was extracted from the gels and subjected to a second anion-exchange step.…”

“…1A) (40). Considering the breadth of in vitro work on the preQ 1 -II riboswitch (21,25,(27)(28)(29)(30) and the success of GFPreporter systems for its functional analysis, we adapted the E. coli-based reporter assay to couple Lrh preQ 1 -II riboswitch regulation with GFPuv fluorescence, accompanied by orthogonal analysis of gene-regulatory conformations using in-cell chemical modification (41). Our results revealed that the Lrh preQ 1 -II riboswitch co-crystal structure in complex with preQ 1 agrees well with expectations for its gene-off conformational state in live bacteria.…”