title: Constitutive regulatory activity of an evolutionarily excluded riboswitch variant.
abstract: The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.
authors.affiliation: Groupe ARN/RNA Group, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec J1K 2R1, Canada.
authors.forename: Renaud
authors.initials: R
authors.lastname: Tremblay
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Lemay
authors.forename: Simon
authors.initials: S
authors.lastname: Blouin
authors.forename: Jérôme
authors.initials: J
authors.lastname: Mulhbacher
authors.forename: Éric
authors.initials: É
authors.lastname: Bonneau
authors.forename: Pascale
authors.initials: P
authors.lastname: Legault
authors.forename: Paul
authors.initials: P
authors.lastname: Dupont
authors.forename: J Carlos
authors.initials: JC
authors.lastname: Penedo
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Lafontaine
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D019143:Evolution, Molecular; D031541:Fluorescence Resonance Energy Transfer; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D052157:Aptamers, Nucleotide; D017931:DNA Primers; D012313:RNA; D001616:beta-Galactosidase