title: Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.
abstract: PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.
authors.affiliation: Department of Biochemistry and Biophysics, and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642;
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Liberman
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109; Biophysics, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Krishna C
authors.initials: KC
authors.lastname: Suddala
authors.affiliation: Department of Biochemistry and Biophysics, and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642;
authors.forename: Asaminew
authors.initials: A
authors.lastname: Aytenfisu
authors.affiliation: Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697.
authors.forename: Dalen
authors.initials: D
authors.lastname: Chan
authors.affiliation: Department of Biochemistry and Biophysics, and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642;
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Belashov
authors.affiliation: Department of Biochemistry and Biophysics, and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642;
authors.forename: Mohammad
authors.initials: M
authors.lastname: Salim
authors.affiliation: Department of Biochemistry and Biophysics, and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642;
authors.forename: David H
authors.initials: DH
authors.lastname: Mathews
authors.affiliation: Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Spitale
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
authors.affiliation: Department of Biochemistry and Biophysics, and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; joseph.wedekind@rochester.edu.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Wedekind
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003013:Clostridium / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009704:Nucleoside Q / Q000737:chemistry / Q000378:metabolism; D011744:Pyrimidinones / Q000737:chemistry / Q000378:metabolism; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism; D059370:RNA Folding; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism; D058928:Riboswitch / Q000235:genetics*; D013816:Thermodynamics
chemical_list: C096876:7-(aminomethyl)-7-deazaguanine; D052157:Aptamers, Nucleotide; D011744:Pyrimidinones; D011758:Pyrroles; D012329:RNA, Bacterial; D058928:Riboswitch; D009704:Nucleoside Q
keywords: crystal structure; gene regulation; molecular dynamics; preQ1 riboswitch; single-molecule FRET