Single VS Ribozyme Molecules Reveal Dynamic and Hierarchical Folding Toward Catalysis

• Published in: Journal of molecular biology Vol. 382; no. 2; pp. 496 - 509
• Main Authors: Pereira, Miguel J.B., Nikolova, Evgenia N., Hiley, Shawna L., Jaikaran, Dominic, Collins, Richard A., Walter, Nils G.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 03.10.2008
• Subjects: Base Sequence; Catalysis; catalytic RNA; Endoribonucleases - chemistry; Endoribonucleases - genetics; Endoribonucleases - metabolism; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurospora; Neurospora - enzymology; Neurospora - genetics; non-coding RNA folding; Nucleic Acid Conformation; RNA dynamics; RNA, Catalytic - chemistry; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; RNA, Fungal - chemistry; RNA, Fungal - genetics; RNA, Fungal - metabolism; RNA, Untranslated - chemistry; RNA, Untranslated - genetics; RNA, Untranslated - metabolism; single molecule fluorescence resonance energy transfer; RNA dynamics; single molecule fluorescence resonance energy transfer; catalytic RNA; Neurospora; non-coding RNA folding; FRET; WT
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.07.020

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.