Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis.
Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.
Pereira
Miguel J B
MJ
Department of Chemistry, Single Molecule Analysis Group, 930 N. University Ave., University of Michigan, Ann Arbor, MI 48109-1055, USA.
Nikolova
Evgenia N
EN
Hiley
Shawna L
SL
Jaikaran
Dominic
D
Collins
Richard A
RA
Walter
Nils G
NG
D001483:Base Sequence; D002384:Catalysis; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009491:Neurospora / Q000201:enzymology / Q000235:genetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D016337:RNA, Catalytic; D012331:RNA, Fungal; D022661:RNA, Untranslated; D004722:Endoribonucleases; C438195:varkud satellite ribozyme