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Structural analysis of a class III preQ1riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

  • Author(s): Liberman, JA
  • Suddala, KC
  • Aytenfisu, A
  • Chan, D
  • Belashov, IA
  • Salim, M
  • Mathews, DH
  • Spitale, RC
  • Walter, NG
  • Wedekind, JE
  • et al.
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 aHLout-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 preQ1riboswitch 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 preQ1enhances 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.

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