Talk abstracts
Talk on Friday 03:30-03:45pm submitted by Rajeev Yadav
Mechanics of divalent ion sensitivity of the manganese-sensing riboswitch measured by combined high-resolution optical tweezers and single-molecule fluorescence
Rajeev Yadav (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48824, USA. ), Shankar Mandal (Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA ), Shiba S Dandpat (Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA ), Nils G Walter (Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA ), Matthew J Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48824, USA)
Abstract:
Riboswitches are structured non-coding RNAs typically found in the 5' untranslated regions of bacterial mRNAs. They modulate gene expression through the binding of specific ligands that induce changes in the riboswitch structure, most often at the level of transcription and translation. The Mn2+-sensing yybP–ykoY RNA motif is one of the most widespread riboswitches across bacteria, including human and plant pathogens. It contains two divalent metal ion binding sites: one is specific for Mn2+ whereas the other is nonspecific for a divalent metal ion, including Mg2+. Previous crystal structures have shown that Mn2+ binds in between the two distal helical legs of a four-way junction and induces a large “docking” conformational change. It is unclear how competition between Mn2+ and Mg2+ for the two binding sites and the associated RNA conformational changes give rise to Mn2+ detection function and sensitivity. Therefore, we explored the global structural dynamics of the L. lactis Mn2+-sensing riboswitch in the presence of competing divalent cations using high-resolution optical tweezers simultaneous with confocal Förster resonance energy transfer (FRET) spectroscopy. Initial non-equilibrium force ramp measurements revealed force-dependent docking distributions, suggesting a highly selective docked state at sub-millimolar Mn2+ ion concentration. High-resolution equilibrium fixed-force measurements directly measured docking and undocking rate constants versus force to determine docking and undocking mechanics, including transition states and pathways, at varying Mn2+ versus Mg2+ concentrations. Simultaneous FRET measurements confirmed specific conformational transitions. Docking and undocking were symmetric processes with nearly equal force sensitivity and a transition state located midway between the docked and undocked states. Mn2+ sensitivity appears to be optimized at micromolar concentrations, whereas further increases in Mn2+ start competing with Mg2+ ions for the non-specific divalent metal ion binding site.
Keywords: Non-coding RNAs, Optical tweezers, FRET