title: Atomic-scale characterization of conformational changes in the preQ₁ riboswitch aptamer upon ligand binding.
abstract: Riboswitches are mRNA structural elements that act as intracellular sensors of small-molecule metabolites. By undergoing conformational changes capable of modulating translation or terminating transcription, riboswitches are able to play a role in regulating the concentration of essential metabolites in the cell. Computer-guided fluorescence experiments were carried out to interrogate molecular dynamics and conformational changes in the minimal riboswitch aptamer that binds 7-aminomethyl-7-deazaguanine (preQ₁). Our combined experimental results and computational analysis suggest that the preQ₁ riboswitch apo form is structured but shows no evidence of a ligand-binding pocket. Simulations of the apo and bound forms indicate a large conformational change is triggered by the breaking of the Watson-Crick base pairing of nucleotides G11 and C31 upon preQ₁ removal, followed by collapse of the pocket due to interfering π-stacking. Computational predictions of local aptamer dynamics were validated by fluorescence experiments employing 2-aminopurine substitutions. In-line probing reactions confirmed that fluorophore-labeled riboswitches retain similar higher-order structural features as the unlabeled aptamer upon ligand binding, although their affinity for the ligand is reduced by the introduction of the fluorescent reporter.
authors.affiliation: Novartis Institutes for BioMedical Research, Inc., 100 Technology Square & 250 Massachusetts Ave. Cambridge, MA 02139, USA.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Petrone
authors.forename: Janetta
authors.initials: J
authors.lastname: Dewhurst
authors.forename: Ruben
authors.initials: R
authors.lastname: Tommasi
authors.forename: Lewis
authors.initials: L
authors.lastname: Whitehead
authors.forename: Andrea K
authors.initials: AK
authors.lastname: Pomerantz
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D011744:Pyrimidinones / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D058928:Riboswitch; D013050:Spectrometry, Fluorescence; D013499:Surface Properties
chemical_list: C096876:7-(aminomethyl)-7-deazaguanine; D052157:Aptamers, Nucleotide; D011744:Pyrimidinones; D011758:Pyrroles; D058928:Riboswitch; D015075:2-Aminopurine; D012313:RNA