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Focus on Function: Single Molecule Enzymology of the VS ribozyme
Author(s) -
Walter Nils G,
Pereira Miguel J.B.,
Collins Richard A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.259.3
Subject(s) - ribozyme , hairpin ribozyme , rna , vs ribozyme , cleavage (geology) , biophysics , microbiology and biotechnology , chemistry , biology , nucleic acid structure , computational biology , genetics , gene , paleontology , fracture (geology)
Non‐coding RNAs of complex structure are involved in numerous aspects of the replication and processing of genetic information. Within the mitochondria of the bread mold Neurospora, the Varkud satellite (VS) RNA replicates parasitically by cycling between DNA and multimeric RNA forms. The RNA contains the VS ribozyme whose self‐cleavage is thought to be essential for replication. Models of VS ribozyme tertiary structure predict three parallel helical stacks built from two three‐way junctions and a kissing loop interaction. Whether or not such complex RNA architectures are dynamic and how catalytic activity emerges is ill‐understood. Here we exploit single molecule FRET to show that the VS ribozyme exhibits dynamic hierarchical folding. Reversible kissing loop formation involving the upstream substrate is required but only partially rate‐limiting for catalysis. Our results invoke a model whereby the VS ribozyme kinetically selects the distal downstream substrate for cleavage before the proximal upstream substrate, thus facilitating processing of the multimeric RNA replication intermediate. We are currently further testing this model.