title: The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts.
abstract: In response to intracellular signals in Gram--negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)-regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by 'bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Arlie J
authors.initials: AJ
authors.lastname: Rinaldi
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.|Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Lund
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Mario R
authors.initials: MR
authors.lastname: Blanco
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D058928:Riboswitch / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D046968:Thermoanaerobacter / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D058928:Riboswitch