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) preQ₁-II ri

SUBMITTER: Dutta D 

PROVIDER: S-EPMC6643267 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature